Carol L Dieckmann

Interests

Research

The general focus in this lab is on understanding the coordination of organelle/host cell interactions. Two different systems under study arenuclear control of mitochondrial mRNA expression in yeast mitochondria, and eyespot assembly in Chlamydomonas. Mitochondrial tRNA processing is inhibited in yeast strains defective in fatty acid biosynthesis in the organelle. We are trying to understand why gene expression and fatty acid metabolism intersect. Mitochondrial fatty acid synthesis precedes the synthesis of lipoic acid, the swinging arm cofactor of pyruvate dehydrogenase and two other enzyme complexes. We are studying how lipoic acid is synthesized and attached to target proteins in mitochondria, and how this process is controlled. The eyespot in Chlamydomonas is made up of components in the chloroplast and in the plasma membrane of the cell. We are interested in how the cell controls the assembly of these components into a functional light-sensing structure. Originally isolated as part of a collection of 168 phototaxis-defective mutants, 28 mutants define four genes required for eyespot assembly. eye2 and eye3 mutants are eyeless. min1 mutants have small eyespots and mlt1 mutants have multiple eyespots. We are trying to understand how these mutations affect eyespot assembly and placement relative to the cytoskeleton through a combination of molecular genetics and light, fluorescence and electron microscopy.

Teaching

Molecular basis of life MCB 301 is the first of three core courses in the MCB major. This course explores the foundational concepts of molecular biology in depth: DNA replication and transcription, RNA processing, modification and translation. Biochemical principles governing molecular interactions are emphasized. Honors students demonstrate their understanding of nucleic acid - protein interaction through mastery of molecular graphics.

Courses

Scholarly Contributions

Chapters

• Thompson, M. D., Mittelmeier, T. M., & Dieckmann, C. L. (2017). Chlamydomonas: The Eyespot. In Chlamydomonas: Molecular Genetics and Physiology(pp 257-281). Springer International Publications. doi:DOI 10.1007/978-3-319-66365-4
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  Full-lenth review on the Chlamydomonas eyespot.

Journals/Publications

• Pietikainen, L. P., Rahman, M. T., Hiltunen, J. K., Dieckmann, C. L., & Kastaniotis, A. J. (2021). Genetic dissection of the mitochondrial lipoylation pathway in yeast. BMC Biology, 19(1), 14. doi:https://doi.org/10.1186/s12915-021-00951-3
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  We show that the requirement for mitochondrial fatty acid synthesis to provide substrates for lipoylation of the 2-ketoacid dehydrogenases can be bypassed by supplying the cells with free lipoic acid (LA) or octanoic acid(C8) and a mitochondrially targeted fatty acyl/lipoyl activating enzyme. We also provide evidence that the S. cerevisiae lipoyl transferase Lip3, in addition to transferring LA from the glycine cleavage system H protein to the pyruvate dehydrogenase (PDH) and α-ketoglutarate dehydrogenase (KGD) E2 subunits, can transfer this cofactor from the PDH complex to the KGD complex. In support of yeast as a model system for human metabolism, we demonstrate that the human octanoyl/lipoyl transferases can substitute for their counterparts in yeast to support respiratory growth and protein lipoylation. Like the wild-type yeast enzyme, the human lipoyl transferase LIPT1 responds to LA supplementation in the presence of the activating enzyme LplA.
• Bailey, P. S., Hiltunen, J. K., Dieckmann, C. L., Kastaniotis, A. J., & Nathan, J. A. (2018). Different opinion on the reported role of Poldip2 and ACSM1 in a mammalian lipoic acid salvage pathway controlling HIF-1 activation. PNAS, 115(32), E7458–E7459. doi:doi: 10.1073/pnas.1804041115
• Dieckmann, C. L., Mittelmeier, T. M., & Dieckmann, C. L. (2016). Phototaxis: Life in focus.. eLife, 5. doi:10.7554/elife.14169
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  Single-celled photosynthetic bacteria determine the direction of incoming light by acting as lenses.
• Mittelmeier, T. M., Thompson, M. D., Lamb, M. R., Lin, H., & Dieckmann, C. L. (2015). MLT1 links cytoskeletal asymmetry to organelle placement in chlamydomonas. Cytoskeleton (Hoboken, N.J.), 72(3), 113-23.
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  Asymmetric placement of the photosensory eyespot organelle in Chlamydomonas is patterned by mother-daughter differences between the two basal bodies, which template the anterior flagella. Each basal body is associated with two bundled microtubule rootlets, one with two microtubules and one with four, forming a cruciate pattern. In wild-type cells, the single eyespot is positioned at the equator in close proximity to the plus end of the daughter rootlet comprising four microtubules, the D4. Here we identify mutations in two linked loci, MLT1 and MLT2, which cause multiple eyespots. Antiserum raised against MLT1 localized the protein along the D4 rootlet microtubules, from the basal bodies to the eyespot. MLT1 associates immediately with the new D4 as it extends during cell division, before microtubule acetylation. MLT1 is a low-complexity protein of over 300,000 Daltons. The expression or stability of MLT1 is dependent on MLT2, predicted to encode a second large, low-complexity protein. MLT1 was not restricted to the D4 rootlet in cells with the vfl2-220 mutation in the gene encoding the basal body-associated protein centrin. The cumulative data highlight the role of mother-daughter basal body differences in establishing asymmetry in associated rootlets, and suggest that eyespot components are directed to the correct location by MLT1 on the D4 microtubules.
• Baker, P. R., Friederich, M. W., Swanson, M. A., Shaikh, T., Bhattacharya, K., Scharer, G. H., Aicher, J., Creadon-Swindell, G., Geiger, E., Maclean, K. N., Lee, W., Deshpande, C., Freckmann, M., Shih, L., Wasserstein, M., Rasmussen, M. B., Lund, A. M., Procopis, P., Cameron, J. M., , Robinson, B. H., et al. (2014). Variant non ketotic hyperglycinemia is caused by mutations in LIAS, BOLA3 and the novel gene GLRX5. Brain, 137(2), 366-379.
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  Abstract: Patients with nonketotic hyperglycinemia and deficient glycine cleavage enzyme activity, but without mutations in AMT, GLDC or GCSH, the genes encoding its constituent proteins, constitute a clinical group which we call 'variant nonketotic hyperglycinemia'. We hypothesize that in some patients the aetiology involves genetic mutations that result in a deficiency of the cofactor lipoate, and sequenced genes involved in lipoate synthesis and iron-sulphur cluster biogenesis. Of 11 individuals identified with variant nonketotic hyperglycinemia, we were able to determine the genetic aetiology in eight patients and delineate the clinical and biochemical phenotypes. Mutations were identified in the genes for lipoate synthase (LIAS), BolA type 3 (BOLA3), and a novel gene glutaredoxin 5 (GLRX5). Patients with GLRX5-associated variant nonketotic hyperglycinemia had normal development with childhood-onset spastic paraplegia, spinal lesion, and optic atrophy. Clinical features of BOLA3-associated variant nonketotic hyperglycinemia include severe neurodegeneration after a period of normal development. Additional features include leukodystrophy, cardiomyopathy and optic atrophy. Patients with lipoate synthase-deficient variant nonketotic hyperglycinemia varied in severity from mild static encephalopathy to Leigh disease and cortical involvement. All patients had high serum and borderline elevated cerebrospinal fluid glycine and cerebrospinal fluid:plasma glycine ratio, and deficient glycine cleavage enzyme activity. They had low pyruvate dehydrogenase enzyme activity but most did not have lactic acidosis. Patients were deficient in lipoylation of mitochondrial proteins. There were minimal and inconsistent changes in cellular iron handling, and respiratory chain activity was unaffected. Identified mutations were phylogenetically conserved, and transfection with native genes corrected the biochemical deficiency proving pathogenicity. Treatments of cells with lipoate and with mitochondrially-targeted lipoate were unsuccessful at correcting the deficiency. The recognition of variant nonketotic hyperglycinemia is important for physicians evaluating patients with abnormalities in glycine as this will affect the genetic causation and genetic counselling, and provide prognostic information on the expected phenotypic course. © 2013 The Author (2013).
• A., V., Pietikäinen, L. P., Fontanesi, F., Aaltonen, M. J., Suomi, F., Nair, R. R., Schonauer, M. S., Dieckmann, C. L., Barrientos, A., Hiltunen, J. K., & Kastaniotis, A. J. (2013). Defects in mitochondrial fatty acid synthesis result in failure of multiple aspects of mitochondrial biogenesis in Saccharomyces cerevisiae. Molecular Microbiology, 90(4), 824-840.
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  PMID: 24102902;Abstract: Mitochondrial fatty acid synthesis (mtFAS) shares acetyl-CoA with the Krebs cycle as a common substrate and is required for the production of octanoic acid (C8) precursors of lipoic acid (LA) in mitochondria. MtFAS is a conserved pathway essential for respiration. In a genetic screen in Saccharomyces cerevisiae designed to further elucidate the physiological role of mtFAS, we isolated mutants with defects in mitochondrial post-translational gene expression processes, indicating a novel link to mitochondrial gene expression and respiratory chain biogenesis. In our ensuing analysis, we show that mtFAS, but not lipoylation per se, is required for respiratory competence. We demonstrate that mtFAS is required for mRNA splicing, mitochondrial translation and respiratory complex assembly, and provide evidence that not LAper se, but fatty acids longer than C8 play a role in these processes. We also show that mtFAS- and LA-deficient strains suffer from a mild haem deficiency that may contribute to the respiratory complex assembly defect. Based on our data and previously published information, we propose a model implicating mtFAS as a sensor for mitochondrial acetyl-CoA availability and a co-ordinator of nuclear and mitochondrial gene expression by adapting the mitochondrial compartment to changes in the metabolic status of the cell. © 2013 John Wiley & Sons Ltd.
• Mittelmeier, T. M., Thompson, M. D., Öztürk, E., & Dieckmann, C. L. (2013). Independent localization of plasma membrane and chloroplast components during eyespot assembly. Eukaryotic Cell, 12(9), 1258-1270.
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  PMID: 23873865;PMCID: PMC3811559;Abstract: Like many algae, Chlamydomonas reinhardtii is phototactic, using two anterior flagella to swim toward light optimal for photosynthesis. The flagella are responsive to signals initiated at the photosensory eyespot, which comprises photoreceptors in the plasma membrane and layers of pigment granules in the chloroplast. Phototaxis depends on placement of the eyespot at a specific asymmetric location relative to the flagella, basal bodies, and bundles of two or four highly acetylated microtubules, termed rootlets, which extend from the basal bodies toward the posterior of the cell. Previous work has shown that the eyespot is disassembled prior to cell division, and new eyespots are assembled in daughter cells adjacent to the nascent four-membered rootlet associated with the daughter basal body (D4), but the chronology of these assembly events has not been determined. Here we use immunofluorescence microscopy to follow assembly and acetylation of the D4 rootlet, localization of individual eyespot components in the plasma membrane or chloroplast envelope, and flagellar emergence during and immediately following cell division. We find that the D4 rootlet is assembled before the initiation of eyespot assembly, which occurs within the same time frame as rootlet acetylation and flagellar outgrowth. Photoreceptors in the plasma membrane are correctly localized in eyespot mutant cells lacking pigment granule layers, and chloroplast components of the eyespot assemble in mutant cells in which photoreceptor localization is retarded. The data suggest that plasma membrane and chloroplast components of the eyespot are independently responsive to a cytoskeletal positioning cue. © 2013, American Society for Microbiology. All Rights Reserved.
• Boyd, J. S., Gray, M. M., Thompson, M. D., Horst, C. J., & Dieckmann, C. L. (2011). The daughter four-membered microtubule rootlet determines anterior-posterior positioning of the eyespot in Chlamydomonas reinhardtii. Cytoskeleton, 68(8), 459-469.
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  PMID: 21766471;PMCID: PMC3201734;Abstract: The characteristic geometry of the unicellular chlorophyte Chlamydomonas reinhardtii has contributed to its adoption as a model system for cellular asymmetry and organelle positioning. The eyespot, a photosensitive organelle, is localized asymmetrically in the cell at a precisely defined position relative to the flagella and cytoskeletal microtubule rootlets. We have isolated a mutant, named pey1 for posterior eyespot, with variable microtubule rootlet lengths. The length of the acetylated daughter four-membered (D4) microtubule rootlet correlates with the position of the eyespot, which appears in a posterior position in the majority of cells. The correlation of rootlet length with eyespot positioning was also observed in the cmu1 mutant, which has longer acetylated microtubules, and the mlt1 mutant, in which the rootlet microtubules are shorter. Observation of eyespot positioning after depolymerization of rootlet microtubules indicated that eyespot position is fixed early in eyespot development and becomes independent of the rootlet. Our data demonstrate that the length of the D4 rootlet is the major determinant of eyespot positioning on the anterior-posterior axis and are suggestive that the gene product of the PEY1 locus is a novel regulator of acetylated microtubule length. © 2011 Wiley-Liss, Inc.
• Boyd, J. S., Lamb, M. R., & Dieckmann, C. L. (2011). Miniature-and multiple-eyespot loci in chlamydomonas reinhardtii define new modulators of eyespot photoreception and assembly. G3: Genes, Genomes, Genetics, 1(6), 489-498.
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  PMID: 22384359;PMCID: PMC3276157;Abstract: The photosensory eyespot of the green alga Chlamydomonas reinhardtii is a model system for the study of organelle biogenesis and placement. Eyespot assembly and positioning are governed by several genetic loci that have been identified in forward genetic screens for phototaxis-defective mutants. These include the previously described miniature-eyespot mutant min1, the multiple-eyespot mutant mlt1, the eyeless mutants eye2 and eye3, and two previously uncharacterized eyespot mutants, min2 and mlt2. In this study, effects of miniature-and multiple-eyespot mutations and their combinations on the localization and expression levels of the rhodopsin photoreceptor channelrhodopsin-1 (ChR1) and the localization of the eyespot-assembly proteins EYE2 and EYE3 were examined. min2 mutants assemble a properly organized, albeit nonfunctional, eyespot that is slightly smaller than wild-type; however, combination of the min2 and mlt1 mutations resulted in drastic reduction of photoreceptor levels. Both stationary-phase mlt1 and mlt2 cells have supernumerary, mislocalized eyespots that exhibit partial or total dissociation of the eyespot layers. In these mutant strains, photoreceptor patches in the plasma membrane were never associated with pigment granule arrays in the chloroplast stroma unless EYE2 was present in the intervening envelope. The data suggest that MIN2 is required for the photoreceptive ability of the eyespot and that MLT2 plays a major role in regulating eyespot number, placement, and integrity. © 2011 Boyd et al.
• Boyd, J. S., Mittelmeier, T. M., Lamb, M. R., & Dieckmann, C. L. (2011). Thioredoxin-family protein EYE2 and Ser/Thr kinase EYE3 play interdependent roles in eyespot assembly. Molecular Biology of the Cell, 22(9), 1421-1428.
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  PMID: 21372178;PMCID: PMC3084665;Abstract: The eyespot of the biflagellate unicellular green alga Chlamydomonas reinhardtii is a complex organelle that facilitates directional responses of the cell to environmental light stimuli. The eyespot, which assembles de novo after every cell division and is associated with the daughter four-membered (D4) microtubule rootlet, comprises an elliptical patch of rhodopsin photoreceptors on the plasma membrane and stacks of carotenoid-rich pigment granule arrays in the chloroplast. Two loci, EYE2 and EYE3, define factors involved in the formation and organization of the eyespot pigment granule arrays. Whereas EYE3, a serine/threonine kinase of the ABC1 family, localizes to pigment granules, EYE2 localization corresponds to an area of the chloroplast envelope in the eyespot. EYE2 is positioned along, and adjacent to, the D4 rootlet in the absence of pigment granules. The eyespot pigment granule array is required for maintenance of the elliptical shape of both the overlying EYE2 and channelrhodopsin-1 photoreceptor patches. We propose a model of eyespot assembly wherein rootlet and photoreceptor direct EYE2 to an area of the chloroplast envelope, where it acts to facilitate assembly of pigment granule arrays, and EYE3 plays a role in the biogenesis of the pigment granules. © 2011 Boyd et al.
• Dieckmann, C. L., Mittelmeier, T. M., Dieckmann, C. L., & Boyd, J. S. (2011). New insights into eyespot placement and assembly in Chlamydomonas.. Bioarchitecture, 1(4), 196-199. doi:10.4161/bioa.1.4.17697
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  Aspects of cellular architecture, such as cytoskeletal asymmetry cues, play critical roles in directing the placement of organelles and establishing the sites of their formation. In the model green alga Chlamydomonas, the photosensory eyespot occupies a defined position in relation to the flagella and microtubule cytoskeleton. Investigations into the cellular mechanisms of eyespot placement and assembly have aided our understanding of the interplay between cytoskeletal and plastid components of the cell. The eyespot, which must be assembled anew after each cell division, is a multi-layered organelle consisting of stacks of carotenoid-filled pigment granules in the chloroplast and rhodopsin photoreceptors in the plasma membrane. Placement of the eyespot is determined on both the latitudinal and longitudinal axes of the cell by the daughter four-membered (D4) microtubule rootlet. Recent findings have contributed to the hypothesis that the eyespot photoreceptor molecules are directed from the Golgi to the daughter hemisphere of the cell and trafficked along the D4 microtubule rootlet. EYE2, a chloroplast-envelope protein, forms an elliptical patch together with the photoreceptors and establishes the site for assembly of the pigment granule arrays in the chloroplast, connecting the positioning information of the cytoskeleton to assembly of the pigment granule arrays in the chloroplast.
• Dieckmann, C., Boyd, J. S., Gray, M. M., Thompson, M. D., Horst, C. J., & Dieckmann, C. L. (2011). The daughter four-membered microtubule rootlet determines anterior-posterior positioning of the eyespot in Chlamydomonas reinhardtii. Cytoskeleton (Hoboken, N.J.), 68(8).
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  The characteristic geometry of the unicellular chlorophyte Chlamydomonas reinhardtii has contributed to its adoption as a model system for cellular asymmetry and organelle positioning. The eyespot, a photosensitive organelle, is localized asymmetrically in the cell at a precisely defined position relative to the flagella and cytoskeletal microtubule rootlets. We have isolated a mutant, named pey1 for posterior eyespot, with variable microtubule rootlet lengths. The length of the acetylated daughter four-membered (D4) microtubule rootlet correlates with the position of the eyespot, which appears in a posterior position in the majority of cells. The correlation of rootlet length with eyespot positioning was also observed in the cmu1 mutant, which has longer acetylated microtubules, and the mlt1 mutant, in which the rootlet microtubules are shorter. Observation of eyespot positioning after depolymerization of rootlet microtubules indicated that eyespot position is fixed early in eyespot development and becomes independent of the rootlet. Our data demonstrate that the length of the D4 rootlet is the major determinant of eyespot positioning on the anterior-posterior axis and are suggestive that the gene product of the PEY1 locus is a novel regulator of acetylated microtubule length.
• Mittelmeier, T. M., Boyd, J. S., Lamb, M. R., & Dieckmann, C. L. (2011). Asymmetric properties of the Chlamydomonas reinhardtii cytoskeleton direct rhodopsin photoreceptor localization. Journal of Cell Biology, 193(4), 741-753.
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  PMID: 21555459;PMCID: PMC3166873;Abstract: The eyespot of the unicellular green alga Chlamydomonas reinhardtii is a photoreceptive organelle required for phototaxis. Relative to the anterior flagella, the eyespot is asymmetrically positioned adjacent to the daughter four-membered rootlet (D4), a unique bundle of acetylated microtubules extending from the daughter basal body toward the posterior of the cell. Here, we detail the relationship between the rhodopsin eyespot photoreceptor Channelrhodopsin 1 (ChR1) and acetylated microtubules. In wild-type cells, ChR1 was observed in an equatorial patch adjacent to D4 near the end of the acetylated microtubules and along the D4 rootlet. In cells with cytoskeletal protein mutations, supernumerary ChR1 patches remained adjacent to acetylated microtubules. In mlt1 (multieyed) mutant cells, supernumerary photoreceptor patches were not restricted to the D4 rootlet, and more anterior eyespots correlated with shorter acetylated microtubule rootlets. The data suggest a model in which photoreceptor localization is dependent on microtubule-based trafficking selective for the D4 rootlet, which is perturbed in mlt1 mutant cells. © 2011 Mittelmeier et al.
• Dieckmann, C. L. (2010). The 2010 Thomas Hunt Morgan Medal: Alexander Tzagoloff. Genetics, 184(4), 865-867. doi:10.1534/genetics.110.114405
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  The Genetics Society of America annually honors members who have made outstanding contributions to genetics. The Thomas Hunt Morgan Medal recognizes a lifetime contribution to the science of genetics. The Genetics Society of America Medal recognizes particularly outstanding contributions to the
• Hiltunen, J. K., Autio, K. J., Schonauer, M. S., Kursu, V. S., Dieckmann, C. L., & Kastaniotis, A. J. (2010). Mitochondrial fatty acid synthesis and respiration. Biochimica et Biophysica Acta - Bioenergetics, 1797(6-7), 1195-1202.
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  PMID: 20226757;Abstract: Recent studies have revealed that mitochondria are able to synthesize fatty acids in a malonyl-CoA/acyl carrier protein (ACP)-dependent manner. This pathway resembles bacterial fatty acid synthesis (FAS) type II, which uses discrete, nuclearly encoded proteins. Experimental evidence, obtained mainly through using yeast as a model system, indicates that this pathway is essential for mitochondrial respiratory function. Curiously, the deficiency in mitochondrial FAS cannot be complemented by inclusion of fatty acids in the culture medium or by products of the cytosolic FAS complex. Defects in mitochondrial FAS in yeast result in the inability to grow on nonfermentable carbon sources, the loss of mitochondrial cytochromes a/a3 and b, mitochondrial RNA processing defects, and loss of cellular lipoic acid. Eukaryotic FAS II generates octanoyl-ACP, a substrate for mitochondrial lipoic acid synthase. Endogenous lipoic acid synthesis challenges the hypothesis that lipoic acid can be provided as an exogenously supplied vitamin. Purified eukaryotic FAS II enzymes are catalytically active in vitro using substrates with an acyl chain length of up to 16 carbon atoms. However, with the exception of 3-hydroxymyristoyl-ACP, a component of respiratory complex I in higher eukaryotes, the fate of long-chain fatty acids synthesized by the mitochondrial FAS pathway remains an enigma. The linkage of FAS II genes to published animal models for human disease supports the hypothesis that mitochondrial FAS dysfunction leads to the development of disorders in mammals. © 2010 Elsevier B.V.
• Hiltunen, J. K., Schonauer, M. S., Autio, K. J., Mittelmeier, T. M., Kastaniotis, A. J., & Dieckmann, C. L. (2009). Mitochondrial fatty acid synthesis type II: More than just fatty acids. Journal of Biological Chemistry, 284(14), 9011-9015.
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  PMID: 19028688;PMCID: PMC2666548;Abstract: Eukaryotes harbor a highly conserved mitochondrial pathway for fatty acid synthesis (FAS), which is completely independent of the eukaryotic cytosolic FAS apparatus. The activities of the mitochondrial FAS system are catalyzed by soluble enzymes, and the pathway thus resembles its prokaryotic counterparts. Except for octanoic acid,whichis the direct precursor for lipoic acid synthesis, other end products and functions of the mitochondrial FAS pathway are still largely enigmatic. In addition to low cellular levels of lipoic acid, disruption of genes encoding mitochondrial FAS enzymes in yeast results in a respiratory-deficient phenotype and small rudimentary mitochondria. Recently, two distinct links between mitochondrial FAS and RNA processing have been discovered in vertebrates and yeast, respectively. In vertebrates, the mitochondrial 3-hydroxyacyl-acyl carrier protein dehydratase and the RPP14 subunit of RNase P are encoded by the same bicistronic transcript in an evolutionarily conserved arrangement that is unusual for eukaryotes. In yeast, defects in mitochondrial FAS result in inefficient RNase P cleavage in the organelle. The intersection of mitochondrial FAS and RNA metabolism in both systems provides a novel mechanism for the coordination of intermediary metabolism in eukaryotic cells. © 2009 by The American Society for Biochemistry and Molecular Biology, Inc.
• Schonauer, M. S., Kastaniotis, A. J., Kursu, V. S., Hiltunen, J. K., & Dieckmann, C. L. (2009). Lipoic acid synthesis and attachment in yeast mitochondria. Journal of Biological Chemistry, 284(35), 23234-23242.
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  PMID: 19570983;PMCID: PMC2749097;Abstract: Lipoic acid is a sulfur-containing cofactor required for the function of several multienzyme complexes involved in the oxidative decarboxylation of α-keto acids and glycine. Mechanistic details of lipoic acid metabolism are unclear in eukaryotes, despite two well defined pathways for synthesis and covalent attachment of lipoic acid in prokaryotes. We report here the involvement of four genes in the synthesis and attachment of lipoic acid in Saccharomyces cerevisiae. LIP2 and LIP5 are required for lipoylation of all three mitochondrial target proteins: Lat1 and Kgd2, the respective E2 subunits of pyruvate dehydrogenase and α-ketoglutarate dehydrogenase, and Gcv3, the H protein of the glycine cleavage enzyme. LIP3, which encodes a lipoate-protein ligase homolog, is necessary for lipoylation of Lat1 and Kgd2, and the enzymatic activity of Lip3 is essential for this function. Finally, GCV3, encoding the H protein target of lipoylation, is itself absolutely required for lipoylation of Lat1 and Kgd2. We show that lipoylated Gcv3, and not glycine cleavage activity per se, is responsible for this function. Demonstration that a target of lipoylation is required for lipoylation is a novel result. Through analysis of the role of these genes in protein lipoylation, we conclude that only one pathway for de novo synthesis and attachment of lipoic acid exists in yeast. We propose a model for protein lipoylation in which Lip2, Lip3, Lip5, and Gcv3 function in a complex, which may be regulated by the availability of acetyl-CoA, and which in turn may regulate mitochondrial gene expression. © 2009 by The American Society for Biochemistry and Molecular Biology, Inc.
• Autio, K. J., Kastaniotis, A. J., Pospiech, H., Miinalainen, I. J., Schonauer, M. S., Dieckmann, C. L., & Hiltunen, J. K. (2008). An ancient genetic link between vertebrate mitochondrial fatty acid synthesis and RNA processing. FASEB Journal, 22(2), 569-578.
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  PMID: 17898086;Abstract: In bacteria, functionally related gene products are often encoded by a common transcript. Such polycistronic transcripts are rare in eukaryotes. Here we isolated several clones from human cDNA libraries, which rescued the respiratory-deficient phenotype of a yeast mitochondrial 3-hydroxyacyl thioester dehydratase 2 (htd2) mutant strain. All complementing cDNAs were derived from the RPP14 transcript previously described to encode the RPP14 subunit of the human ribonuclease P (RNase P) complex. We identified a second, 3′ open reading frame (ORF) on the RPP14 transcript encoding a protein showing similarity to known dehydratases and hydratase 2 enzymes. The protein was localized in mitochondria, and the recombinant enzyme exhibited (3R)-specific hydratase 2 activity. Based on our results, we named the protein human 3-hydroxyacyl-thioester dehydratase 2 (HsHTD2), which is involved in mitochondrial fatty acid synthesis. The bicistronic arrangement of RPP14 and HsHTD2, as well as the general exon structure of the gene, is conserved in vertebrates from fish to humans, indicating a genetic link conserved for 400 million years between RNA processing and mitochondrial fatty acid synthesis. © FASEB.
• Dieckmann, C., Dieckmann, C. L., Schonauer, M. S., Kastaniotis, A. J., & Hiltunen, J. K. (2008). Intersection of RNA processing and the type II fatty acid synthesis pathway in yeast mitochondria. Molecular and Cellular biology, 28(21).
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  Distinct metabolic pathways can intersect in ways that allow hierarchical or reciprocal regulation. In a screen of respiration-deficient Saccharomyces cerevisiae gene deletion strains for defects in mitochondrial RNA processing, we found that lack of any enzyme in the mitochondrial fatty acid type II biosynthetic pathway (FAS II) led to inefficient 5' processing of mitochondrial precursor tRNAs by RNase P. In particular, the precursor containing both RNase P RNA (RPM1) and tRNA(Pro) accumulated dramatically. Subsequent Pet127-driven 5' processing of RPM1 was blocked. The FAS II pathway defects resulted in the loss of lipoic acid attachment to subunits of three key mitochondrial enzymes, which suggests that the octanoic acid produced by the pathway is the sole precursor for lipoic acid synthesis and attachment. The protein component of yeast mitochondrial RNase P, Rpm2, is not modified by lipoic acid in the wild-type strain, and it is imported in FAS II mutant strains. Thus, a product of the FAS II pathway is required for RNase P RNA maturation, which positively affects RNase P activity. In addition, a product is required for lipoic acid production, which is needed for the activity of pyruvate dehydrogenase, which feeds acetyl-coenzyme A into the FAS II pathway. These two positive feedback cycles may provide switch-like control of mitochondrial gene expression in response to the metabolic state of the cell.
• Dieckmann, C., Mittelmeier, T. M., Berthold, P., Danon, A., Lamb, M. R., Levitan, A., Rice, M. E., & Dieckmann, C. L. (2008). C2 domain protein MIN1 promotes eyespot organization in Chlamydomonas reinhardtii. Eukaryotic cell, 7(12).
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  Assembly and asymmetric localization of the photosensory eyespot in the biflagellate, unicellular green alga Chlamydomonas reinhardtii requires coordinated organization of photoreceptors in the plasma membrane and pigment granule/thylakoid membrane layers in the chloroplast. min1 (mini-eyed) mutant cells contain abnormally small, disorganized eyespots in which the chloroplast envelope and plasma membrane are no longer apposed. The MIN1 gene, identified here by phenotypic rescue, encodes a protein with an N-terminal C2 domain and a C-terminal LysM domain separated by a transmembrane sequence. This novel domain architecture led to the hypothesis that MIN1 is in the plasma membrane or the chloroplast envelope, where membrane association of the C2 domain promotes proper eyespot organization. Mutation of conserved C2 domain loop residues disrupted association of the MIN1 C2 domain with the chloroplast envelope in moss cells but did not abolish eyespot assembly in Chlamydomonas. In min1 null cells, channelrhodopsin-1 (ChR1) photoreceptor levels were reduced, indicating a role for MIN1 in ChR1 expression and/or stability. However, ChR1 localization was only minimally disturbed during photoautotrophic growth of min1 cells, conditions under which the pigment granule layers are disorganized. The data are consistent with the hypothesis that neither MIN1 nor proper organization of the plastidic components of the eyespot is essential for localization of ChR1.
• Fekete, Z., Ellis, T. P., Schonauer, M. S., & Dieckmann, C. L. (2008). Pet127 governs a 5′ →3′-exonuclease important in maturation of apocytochrome b mRNA in Saccharomyces cerevisiae. Journal of Biological Chemistry, 283(7), 3767-3772.
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  PMID: 18086665;Abstract: The details of mRNA maturation in Saccharomyces mitochondria are not well understood. All seven mRNAs are transcribed as part of multigenic units. The mRNAs are processed at a common 3′-dodecamer sequence, but the 5′-ends have seven different sequences. To investigate whether apocytochrome b (COB) mRNAis processed at the 5′-end from a longer precursor by an endonuclease or an exonuclease, a 64-nucleotide sequence, which is required for the protection of COB mRNA by the Cbp1 protein and is found at the 5′-end of the processed COB mRNA,was duplicated in tandem. The wild-type 64-nucleotide element functioned in either the upstream or downstream position when paired with a mutant element. In the tandem wild-type strain, the 5′-end of them RNA was at the 5′-end of the upstream unit, demonstrating that them RNA is processed by an exonuclease. Accumulation of precursor COB RNA in single and double element strains with a deletion of PET127 demonstrated that the encoded protein governs the 5′-exonuclease responsible for processing the precursor to the mature form. © 2008 by The American Society for Biochemistry and Molecular Biology, Inc.
• Mittelmeier, T. M., Berthold, P., Danon, A., Lamb, M. R., Levitan, A., Rice, M. E., & Dieckmann, C. L. (2008). C2 domain protein MIN1 promotes eyespot organization in Chlamydomonas reinhardtii. Eukaryotic Cell, 7(12), 2100-2112.
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  PMID: 18849467;PMCID: PMC2593190;Abstract: Assembly and asymmetric localization of the photosensory eyespot in the biflagellate, unicellular green alga Chlamydomonas reinhardtii requires coordinated organization of photoreceptors in the plasma membrane and pigment granule/thylakoid membrane layers in the chloroplast. min1 (mini-eyed) mutant cells contain abnormally small, disorganized eyespots in which the chloroplast envelope and plasma membrane are no longer apposed. The MIN1 gene, identified here by phenotypic rescue, encodes a protein with an N-terminal C2 domain and a C-terminal LysM domain separated by a transmembrane sequence. This novel domain architecture led to the hypothesis that MIN1 is in the plasma membrane or the chloroplast envelope, where membrane association of the C2 domain promotes proper eyespot organization. Mutation of conserved C2 domain loop residues disrupted association of the MIN1 C2 domain with the chloroplast envelope in moss cells but did not abolish eyespot assembly in Chlamydomonas. In min1 null cells, channelrhodopsin-1 (ChR1) photoreceptor levels were reduced, indicating a role for MIN1 in ChR1 expression and/or stability. However, ChR1 localization was only minimally disturbed during photoautotrophic growth of min1 cells, conditions under which the pigment granule layers are disorganized. The data are consistent with the hypothesis that neither MIN1 nor proper organization of the plastidic components of the eyespot is essential for localization of ChR1. Copyright © 2008, American Society for Microbiology. All Rights Reserved.
• Schonauer, M. S., Kastaniotis, A. J., Hiltunen, J. K., & Dieckmann, C. L. (2008). Intersection of RNA processing and the type II fatty acid synthesis pathway in yeast mitochondria. Molecular and Cellular Biology, 28(21), 6646-6657.
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  PMID: 18779316;PMCID: PMC2573234;Abstract: Distinct metabolic pathways can intersect in ways that allow hierarchical or reciprocal regulation. In a screen of respiration-deficient Saccharomyces cerevisiae gene deletion strains for defects in mitochondrial RNA processing, we found that lack of any enzyme in the mitochondrial fatty acid type II biosynthetic pathway (FAS II) led to inefficient 5′ processing of mitochondrial precursor tRNAs by RNase P. In particular, the precursor containing both RNase P RNA (RPM1) and tRNAPro accumulated dramatically. Subsequent Pet127-driven 5′ processing of RPM1 was blocked. The FAS II pathway defects resulted in the loss of lipoic acid attachment to subunits of three key mitochondrial enzymes, which suggests that the octanoic acid produced by the pathway is the sole precursor for lipoic acid synthesis and attachment. The protein component of yeast mitochondrial RNase P, Rpm2, is not modified by lipoic acid in the wild-type strain, and it is imported in FAS II mutant strains. Thus, a product of the FAS II pathway is required for RNase P RNA maturation, which positively affects RNase P activity. In addition, a product is required for lipoic acid production, which is needed for the activity of pyruvate dehydrogenase, which feeds acetyl-coenzyme A into the FAS II pathway. These two positive feedback cycles may provide switch-like control of mitochondrial gene expression in response to the metabolic state of the cell. Copyright © 2008, American Society for Microbiology. All Rights Reserved.
• Merchant, S. S., Prochnik, S. E., Vallon, O., Harris, E. H., Karpowicz, S. J., Witman, G. B., Terry, A., Salamov, A., Fritz-Laylin, L. K., Maréchal-Drouard, L., Marshall, W. F., Qu, L., Nelson, D. R., Sanderfoot, A. A., Spalding, M. H., Kapitonov, V. V., Ren, Q., Ferris, P., Lindquist, E., , Shapiro, H., et al. (2007). The Chlamydomonas genome reveals the evolution of key animal and plant functions. Science, 318(5848), 245-251.
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  PMID: 17932292;PMCID: PMC2875087;Abstract: Chlamydomonas reinhardtii is a unicellular green alga whose lineage diverged from land plants over 1 billion years ago. It is a model system for studying chloroplast-based photosynthesis, as well as the structure, assembly, and function of eukaryotic flagella (cilia), which were inherited from the common ancestor of plants and animals, but lost in land plants. We sequenced the ∼120-megabase nuclear genome of Chlamydomonas and performed comparative phylogenomic analyses, identifying genes encoding uncharacterized proteins that are likely associated with the function and biogenesis of chloroplasts or eukaryotic flagella. Analyses of the Chlamydomonas genome advance our understanding of the ancestral eukaryotic cell, reveal previously unknown genes associated with photosynthetic and flagellar functions, and establish links between ciliopathy and the composition and function of flagella.
• Schonauer, M. S., Schonauer, M. S., Pospiech, H., Pospiech, H., Miinalainen, I., Miinalainen, I., Kastaniotis, A. J., Kastaniotis, A. J., Hiltunen, K., Hiltunen, K., Dieckmann, C. L., Dieckmann, C. L., Autio, K. J., & Autio, K. J. (2007). An ancient genetic link between mitochondrial fatty acid synthesis and RNA processing. Chemistry and Physics of Lipids, 149, S21. doi:10.1016/j.chemphyslip.2007.06.046
• Renninger, S., Dieckmann, C. L., & Kreimer, G. (2006). Toward a protein map of the green algal eyespot: Analysis of eyespot globule-associated proteins. Phycologia, 45(2), 199-212.
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  Abstract: Despite the well-established biophysical function of the green algal eyespot as a combined absorption/reflection screen for the retinal-based photoreceptors, only little is known about structural eyespot proteins and proteins involved in its assembly. A recently developed method for preparation of eyespot globules from Spermatozopsis similis (Chlorophyceae) has allowed now a detailed characterisation of this specific subset of eyespot proteins. Five antisera were raised against eyespot globule-associated proteins (GAPs). Two-dimensional gel electrophoresis and subsequent immunoblotting were combined to analyse the GAPs in detail. Approximately 180 spots were resolved in silver-stained two-dimensional gels, indicating the complexity of proteins needed for formation and maintenance of the highly ordered eyespot globule plate and its interactions with the chloroplast envelope and thylakoid. Topological analyses identified 19 proteins possessing domains hidden in the globule matrix or otherwise not susceptible to thermolysin treatment. These GAPs might be involved in globule stabilisation. Antibody screening of a cDNA expression library resulted in the isolation of a full-length clone coding for a novel basic proline- and alanine-rich protein of 727 amino acids. The deduced amino acid sequence revealed the presence of a putative N-terminal signal peptide, followed by a hydrophobic region and four TPR motifs in the C terminal region. This protein, termed GAP1, might thus be involved in mediating protein-protein interactions in a protein scaffold around the hydrophobic globule core. This study paves the way for future molecular identification of GAPs by systematic proteomic approaches.
• Dieckmann, C., Ellis, T. P., Schonauer, M. S., & Dieckmann, C. L. (2005). CBT1 interacts genetically with CBP1 and the mitochondrially encoded cytochrome b gene and is required to stabilize the mature cytochrome b mRNA of Saccharomyces cerevisiae. Genetics, 171(3).
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  Mutation of a CCG sequence in the 5'-untranslated region of the mitochondrially encoded cytochrome b mRNA in Saccharomyces cerevisiae results in destabilization of the message and respiratory deficiency of the mutant strain. This phenotype mimics that of a mutation in the nuclear CBP1 gene. Here it is shown that overexpression of the nuclear CBT1 gene, due to a transposon insertion in the 5'-untranslated region, rescues the respiratory defects resulting from mutating the CCG sequence to ACG. Overexpressing alleles of CBT1 are allelic to soc1, a previously isolated suppressor of cbp1ts-induced temperature sensitivity of respiratory growth. Quantitative primer extension analysis indicated that cbt1 null strains have defects in 5'-end processing of precursor cytochrome b mRNA to the mature form. Cbt1p is also required for stabilizing the mature cytochrome b mRNA after 5' processing.
• Dieckmann, C., Krause, K., & Dieckmann, C. L. (2004). Analysis of transcription asymmetries along the tRNAE-COB operon: evidence for transcription attenuation and rapid RNA degradation between coding sequences. Nucleic acids research, 32(21).
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  Mitochondrial gene expression in yeast is believed to be regulated predominantly at the post-transcriptional level. However, the contribution of mitochondrial transcription and RNA-turnover rates to differential gene regulation is still largely unknown. Mitochondrial run-on transcription and hybrid selection assays showed that some of the multigenic transcription units of the mitochondrial genome are transcribed evenly, whereas others are transcribed asymmetrically, with higher transcription rates for promoter-proximal genes, than for promoter-distal genes. The tRNA(E)-cytochrome b (COB) operon was analyzed in detail to investigate the mechanisms underlying transcription rate asymmetries in yeast mitochondria. We showed that a drop in transcription rates occurs in a particular region between the coding sequences and is independent of the coding sequence of the downstream COB gene. Deletion of the region between tRNA(E) and COB coding sequences decreases the drop in transcription rates. Deletion of the nuclear gene encoding the Pet 127 protein, which is involved in mitochondrial RNA 5' processing and degradation, also partially relieves transcriptional asymmetry. Therefore, asymmetry is probably due to a combination of attenuated transcription at specific sites between the coding sequences and very rapid RNA degradation.
• Dieckmann, C., Krause, K., Lopes de Souza, R., Roberts, D. G., & Dieckmann, C. L. (2004). The mitochondrial message-specific mRNA protectors Cbp1 and Pet309 are associated in a high-molecular weight complex. Molecular biology of the cell, 15(6).
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  In Saccharomyces cerevisiae, the nuclear-encoded protein Cbp1 promotes stability and translation of mitochondrial cytochrome b transcripts through interaction with the 5' untranslated region. Fusion of a biotin binding peptide tag to the C terminus of Cbp1 has now allowed detection in mitochondrial extracts by using peroxidase-coupled avidin. Cbp1 is associated with the mitochondrial membranes when high ionic strength extraction conditions are used. However, the protein is easily solubilized by omitting salt from the extraction buffer, which suggests Cbp1 is loosely associated with the membrane through weak hydrophobic interactions. Gel filtration analysis and blue native PAGE showed that Cbp1 is part of a single 900,000-Da complex. The complex was purified using the biotin tag and a sequence-specific protease cleavage site. In addition to Cbp1, the complex contains several polypeptides of molecular weights between 113 and 40 kDa. Among these, we identified another message-specific factor, Pet309, which promotes the stability and translation of mitochondrial cytochrome oxidase subunit I mRNA. A hypothesis is presented in which the Cbp1-Pet309 complex contains several message-specific RNA binding proteins and links transcription to translation of the mRNAs at the membrane.
• Ellis, T. P., Helfenbein, K. G., Tzagoloff, A., & Dieckmann, C. L. (2004). Aep3p Stabilizes the Mitochondrial Bicistronic mRNA Encoding Subunits 6 and 8 of the H⁺-translocating ATP Synthase of Saccharomyces cerevisiae. Journal of Biological Chemistry, 279(16), 15728-15733.
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  PMID: 14742425;Abstract: Subunits 6 and 8 of the mitochondrial ATPase in Saccharomyces cerevisiae are encoded by the mitochondrial genome and translated from bicistronic mRNAs containing both reading frames. The stability of the two major species of ATP8/ 6 mRNA, which differ in the length of the 5′-untranslated region, depends on the expression of several nuclear-encoded factors. In the present study, the product of the gene designated AEP3 (open reading frame YPL005W) is shown to be required for stabilization and/or processing of both ATP8/6 mRNA species. In an aep3-disruptant strain, the shorter ATP8/6 mRNA was undetectable, and the level of the longer mRNA was reduced to ∼35% that of wild type. Localization of a hemagglutinin-tagged version of Aep3p showed that the protein is an extrinsic constituent of the mitochondrial inner membrane facing the matrix.
• Krause, K., & Dieckmann, C. L. (2004). Analysis of transcription asymmetries along the tRNA^E-COB operon: Evidence for transcription attenuation and rapid RNA degradation between coding sequences. Nucleic Acids Research, 32(21), 6276-6283.
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  PMID: 15576354;PMCID: PMC535675;Abstract: Mitochondrial gene expression in yeast is believed to be regulated predominantly at the post-transcriptional level. However, the contribution of mitochondrial transcription and RNA-turnover rates to differential gene regulation is still largely unknown. Mitochondrial run-on transcription and hybrid selection assays showed that some of the multigenic transcription units of the mitochondrial genome are transcribed evenly, whereas others are transcribed asymmetrically, with higher transcription rates for promoter-proximal genes, than for promoter-distal genes. The tRNAE-cytochrome b (COB) operon was analyzed in detail to investigate the mechanisms underlying transcription rate asymmetries in yeast mitochondria. We showed that a drop in transcription rates occurs in a particular region between the coding sequences and is independent of the coding sequence of the downstream COB gene. Deletion of the region between tRNAE and COB coding sequences decreases the drop in transcription rates. Deletion of the nuclear gene encoding the Pet 127 protein, which is involved in mitochondrial RNA 5′ processing and degradation, also partially relieves transcriptional asymmetry. Therefore, asymmetry is probably due to a combination of attenuated transcription at specific sites between the coding sequences and very rapid RNA degradation. © Oxford University Press 2004; all rights reserved.
• Krause, K., Lopes, R., Roberts, D. G., & Dieckmann, C. L. (2004). The mitochondrial message-specific mRNA protectors Cbp1 and Pet309 are associated in a high-molecular weight complex. Molecular Biology of the Cell, 15(6), 2674-2683.
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  PMID: 15047869;PMCID: PMC420092;Abstract: In Saccharomyces cerevisiae, the nuclear-encoded protein Cbp1 promotes stability and translation of mitochondrial cytochrome b transcripts through interaction with the 5′ untranslated region. Fusion of a biotin binding peptide tag to the C terminus of Cbp1 has now allowed detection in mitochondrial extracts by using peroxidase-coupled avidin. Cbp1 is associated with the mitochondrial membranes when high ionic strength extraction conditions are used. However, the protein is easily solubilized by omitting salt from the extraction buffer, which suggests Cbp1 is loosely associated with the membrane through weak hydrophobic interactions. Gel filtration analysis and blue native PAGE showed that Cbp1 is part of a single 900,000-Da complex. The complex was purified using the biotin tag and a sequence-specific protease cleavage site. In addition to Cbp1, the complex contains several polypeptides of molecular weights between 113 and 40 kDa. Among these, we identified another message-specific factor, Pet309, which promotes the stability and translation of mitochondrial cytochrome oxidase subunit I mRNA. A hypothesis is presented in which the Cbp1-Pet309 complex contains several message-specific RNA binding proteins and links transcription to translation of the mRNAs at the membrane.
• Schonauer, M. S., & Dieckmann, C. L. (2004). Mitochondrial genomics and proteomics. Current Genomics, 5(7), 575-588.
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  Abstract: Factors involved in mitochondrial biogenesis and function have been studied classically via mutagenesis screens and subsequent genetic and biochemical analyses. The recent advent of high-throughput technologies has provided a wealth of information regarding mitochondrial function, morphology, gene regulation, protein complexes, and disease in a fraction of the time. This review will describe past and present genomic and proteomic methods used to study mitochondria both in yeast and mammalian cells, their advantages and limitations, and the current knowledge of the number of genes and proteins that are required for proper functioning of the organelle. © 2004 Bentham Science Publishers Ltd.
• Dieckmann, C. L. (2003). Eyespot placement and assembly in the green alga Chlamydomonas. BioEssays, 25(4), 410-416.
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  PMID: 12655648;Abstract: The eyespot organelle of the green alga Chlamydomonas allows the cell to phototax toward (or away) from light to maximize the light intensity for photosynthesis and minimize photo-damage. At cytokinesis, the eyespot is resorbed at the cleavage furrow and two new eyespots form in the daughter cells 180° from each other. The eyespots are positioned asymmetrically with respect to the microtubule cytoskeleton. Eyespots are assembled from all three chloroplast membranes and carotenoid-filled granules, which form a sandwich structure overlaid by the tightly apposed plasma membrane. This review describes (1) my interest in cellular asymmetry and organelle biology, (2) isolation of mutations that describe four genes governing eyespot placement and assembly, (3) the characterization of the EYE2 gene, which encodes a thioredoxin superfamily member, and (4) the characterization of the MIN1 gene, which is required for the layered organization of granules and membranes in the eyespot. © 2003 Wiley Periodicals, Inc.
• Dieckmann, C., Islas-Osuna, M. A., Ellis, T. P., Mittelmeier, T. M., & Dieckmann, C. L. (2003). Suppressor mutations define two regions in the Cbp1 protein important for mitochondrial cytochrome b mRNA stability in Saccharomyces cerevisiae. Current genetics, 43(5).
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  Nuclear-encoded Cbp1 stabilizes and promotes translation of mitochondrial cytochrome b (COB) mRNA. A CCG triplet within the 5'UTR of COB mRNA is essential for Cbp1-dependent stability. Like cbp1 mutations, mutation of any nucleotide in CCG results in degradation of COB transcripts. In this study, CBP1-linked pseudorevertants of the temperature-sensitive CCU strain were isolated. The suppressors are missense mutations within a central cluster or a carboxyl cluster in the linear sequence of Cbp1. Strains with mutations in the carboxyl half of the central cluster or the carboxyl cluster respire better than those with mutations in the amino half of the central cluster. COB mRNA levels in the suppressor strains were increased compared with that in the CCU strain and were positively correlated with respiratory capability. This correlation supports a model in which the primary role of Cbp1 is to protect COB mRNAs and deliver them to the mitochondrial translational apparatus.
• Helfenbein, K. G., Ellis, T. P., Dieckmann, C. L., & Tzagoloff, A. (2003). ATP22, a nuclear gene required for expression of the F₀ sector of mitochondrial ATPase in Saccharomyces cerevisiae. Journal of Biological Chemistry, 278(22), 19751-19756.
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  PMID: 12646576;Abstract: Expression of the mitochondrial proton-translocating ATPase of Saccharomyces cerevisiae has been shown to depend on chaperones that target the F1 and F0 sectors of this inner membrane complex. Here we report a new gene, designated ATP22 (reading frame YDR350C on chromosome IV), that provides an essential function in the assembly of F0. ATP22 was cloned by transformation of C208/L2, a strain previously assigned to complementation group G99 of a collection of respiration-defective nuclear pet mutants. C208/L2 and the other atp22 mutants have oligomycin-insensitive F1-ATPase, suggesting that the lesion is confined to F0. This is supported by the sedimentation properties of the mutant ATPase and results of immunochemical analysis of F0 subunit polypeptides. Northern analysis of ATPase transcripts and in vivo pulse labeling of the mitochondrial translation products in the mutant indicate normal expression of subunits 6, 8, and 9, the three mitochondrial gene products of F0. Atp22p therefore functions at a posttranslational stage in assembly of F0. Localization studies indicate Atp22p to be a component of the mitochondrial inner membrane. Protease protection experiments further indicate that Atp22p faces the matrix side of the membrane where most of the ATPase proteins are located and assembled.
• Islas-Osuna, M. A., Ellis, T. P., Mittelmeier, T. M., & Dieckmann, C. L. (2003). Suppressor mutations define two regions in the Cbp1 protein important for mitochondrial cytochrome b mRNA stability in Saccharomyces cerevisiae. Current Genetics, 43(5), 327-336.
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  PMID: 12764667;Abstract: Nuclear-encoded Cbp1 stabilizes and promotes translation of mitochondrial cytochrome b (COB) mRNA. A CCG triplet within the 5′UTR of COB mRNA is essential for Cbp1-dependent stability. Like cbp1 mutations, mutation of any nucleotide in CCG results in degradation of COB transcripts. In this study, CBP1-linked pseudorevertants of the temperature-sensitive CCU strain were isolated. The suppressors are missense mutations within a central cluster or a carboxyl cluster in the linear sequence of Cbp1. Strains with mutations in the carboxyl half of the central cluster or the carboxyl cluster respire better than those with mutations in the amino half of the central cluster. COB mRNA levels in the suppressor strains were increased compared with that in the CCU strain and were positively correlated with respiratory capability. This correlation supports a model in which the primary role of Cbp1 is to protect COB mRNAs and deliver them to the mitochondrial translational apparatus.
• Islas-Osuna, M. A., Ellis, T. P., Marnell, L. L., Mittelmeier, T. M., & Dieckmann, C. L. (2002). Cbp1 is required for translation of the mitochondrial cytochrome b mRNA of Saccharomyces cerevisiae. Journal of Biological Chemistry, 277(41), 37987-37990.
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  PMID: 12149267;Abstract: Expression of the yeast mitochondrial cytochrome b gene (COB) is controlled by at least 15 nuclear-encoded proteins. One of these proteins, Cbp1, is required for COB mRNA stability. Δcbp1 null strains fail to accumulate mature COB mRNA and cannot respire. Since Δcbp1 null strains lack mature COB transcripts, the hypothesis that Cbp1 also plays a role in translation of these mRNAs could not be tested previously. 5′-End trimming of precursor COB RNA and other mitochondrial transcripts is dependent on Pet127. pet127 mutants accumulate high levels of precursor COB mRNA and have no mature mRNA. pet127 mutants respire well; this phenotype implies that COB precursor RNA is translated efficiently. With the expectation that a Δcbp1Δpet127 strain might accumulate substantial levels of COB RNA, the double null strain was constructed and analyzed to test the hypothesis that Cbp1 is required for translation of COB RNA. The Δcbp1Δpet127 strain does accumulate levels of COB precursor mRNA that are ∼60% of the level of COB mRNA in the wild-type strain. However, cytochrome b protein is not synthesized, and thus the Δcbp1Δpet127 strain does not respire. These results suggest that Cbp1 is required for translation of COB RNAs.
• G., D., Lamb, M. R., & Dieckmann, C. L. (2001). Characterization of the EYE2 gene required for eyespot assembly in Chlamydomonas reinhardtii. Genetics, 158(3), 1037-1049.
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  PMID: 11454753;PMCID: PMC1461727;Abstract: The unicellular biflagellate green alga Chlamydomonas reinhardtii can perceive light and respond by altering its swimming behavior. The eyespot is a specialized structure for sensing light, which is assembled de novo at every cell division from components located in two different cellular compartments. Photoreceptors and associated signal transduction components are localized in a discrete patch of the plasma membrane. This patch is tightly packed against an underlying sandwich of chloroplast membranes and carotenoid-filled lipid granules, which aids the cell in distinguishing light direction. In a prior screen for mutant strains with eyespot defects, the EYE2 locus was defined by the single eye2-1 allele. The mutant strain has no eyespot by light microscopy and has no organized carotenoid granule layers as judged by electron microscopy. Here we demonstrate that the eye2-1 mutant is capable of responding to light, although the strain is far less sensitive than wild type to low light intensities and orients imprecisely. Therefore, pigment granule layer assembly in the chloroplast is not required for photoreceptor localization in the plasma membrane. A plasmid-insertion mutagenesis screen yielded the eye2-2 allele, which allowed the isolation and characterization of the EYE2 gene. The EYE2 protein is a member of the thioredoxin superfamily. Site-directed mutagenesis of the active site cysteines demonstrated that EYE2 function in eyespot assembly is redox independent, similar to the auxiliary functions of other thioredoxin family members in protein folding and complex assembly.
• Dieckmann, C., Chen, W., Islas-Osuna, M. A., & Dieckmann, C. L. (1999). Suppressor analysis of mutations in the 5'-untranslated region of COB mRNA identifies components of general pathways for mitochondrial mRNA processing and decay in Saccharomyces cerevisiae. Genetics, 151(4).
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  The cytochrome b gene in Saccharomyces cerevisiae, COB, is encoded by the mitochondrial genome. Nuclear-encoded Cbp1 protein is required specifically for COB mRNA stabilization. Cbp1 interacts with a CCG element in a 64-nucleotide sequence in the 5'-untranslated region of COB mRNA. Mutation of any nucleotide in the CCG causes the same phenotype as cbp1 mutations, i.e., destabilization of both COB precursor and mature message. In this study, eleven nuclear suppressors of single-nucleotide mutations in CCG were isolated and characterized. One dominant suppressor is in CBP1, while the other 10 semidominant suppressors define five distinct linkage groups. One group of four mutations is in PET127, which is required for 5' end processing of several mitochondrial mRNAs. Another mutation is linked to DSS1, which is a subunit of mitochondrial 3' --> 5' exoribonuclease. A mutation linked to the SOC1 gene, previously defined by recessive mutations that suppress cbp1 ts alleles and stabilize many mitochondrial mRNAs, was also isolated. We hypothesize that the products of the two uncharacterized genes also affect mitochondrial RNA turnover.
• Lamb, M. R., Dutcher, S. K., Worley, C. K., & Dieckmann, C. L. (1999). Eyespot-assembly mutants in Chlamydomonas reinhardtii. Genetics, 153(2), 721-729.
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  PMID: 10511552;PMCID: PMC1460774;Abstract: Chlamydomonas reinhardtii is a single-celled green alga that phototaxes toward light by means of a light-sensitive organelle, the eyespot. The eyespot is composed of photoreceptor and Ca++-channel signal transduction components in the plasma membrane of the cell and reflective carotenoid pigment layers in an underlying region of the large chloroplast. To identify components important for the positioning and assembly of a functional eyespot, a large collection of nonphototactic mutants was screened for those with aberrant pigment spots. Four loci were identified, eye2 and eye3 mutants have no pigmented eyespots. min1 mutants have smaller than wild-type eyespots, mlt1(ptx4) mutants have multiple eyespots. The MINI, MLT1(PTX4), and EYE2 loci are closely linked to each other; EYE3 is unlinked to the other three loci. The eye2 and eye3 mutants are epistatic to min1 and mlt1 mutations; all double mutants are eyeless, min1 mlt1 double mutants have a synthetic phenotype; they are eyeless or have very small, misplaced eyespots. Ultrastructural studies revealed that the min1 mutants are defective in the physical connection between the plasma membrane and the chloroplast envelope membranes in the region of the pigment granules. Characterization of these four loci will provide a beginning for the understanding of eyespot assembly and localization in the cell.
• Sparks, K. A., & Dieckmann, C. L. (1998). Regulation of poly(A) site choice of several yeast mRNAs. Nucleic Acids Research, 26(20), 4676-4687.
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  PMID: 9753737;PMCID: PMC147891;Abstract: Several yeast genes produce multiple transcripts with different 3'-ends. Of these, four genes are known to produce truncated transcripts that end within the coding sequence of longer transcripts: CBP1, AEP2/ATP13, RNA 14 and SIR1. It has been shown that the level of the truncated CBP1 transcript increases during the switch to respiratory growth while that of the full-length transcript decreases. To determine whether this phenomenon is unique to CBP1, northern analysis was used to determine whether the levels of other truncated transcripts are regulated similarly by carbon source. The levels of the shortest transcripts of AEP2/ATP13 and RNA 14 increased during respiration while the shortest SIR1 transcript remained constant. However, two longer SIR1 transcripts were regulated reciprocally by carbon source. Mapping the 3'-ends of each transcript by sequencing partial cDNA clones revealed multiple 3'-ends for each transcript. Examination of the sequences surrounding the 3'-ends of the induced transcripts failed to identify a consensus sequence but did reveal weak putative 3'end formation signals in all of the transcripts. Similarly, no consensus sequence was found when the sequences surrounding the 3'-ends of the longest transcripts were compared, but again weak putative 3'-end formation signals were identified. These data are suggestive of carbon source regulation of alternative poly(A) site choice in yeast.
• Chen, W., & Dieckmann, C. L. (1997). Genetic evidence for interaction between Cbp1 and specific nucleotides in the 5' untranslated region of mitochondrial cytochrome B mRNA in Saccharomyces cerevisiae. Molecular and Cellular Biology, 17(11), 6203-6211.
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  PMID: 9343381;PMCID: PMC232471;Abstract: The cytochrome b (COB) gene is encoded by the mitochondrial genome; however, its expression requires the participation of several nuclearly encoded protein factors. The yeast Cbp1 protein, which is encoded by the nuclear CBP1 gene, is required for the stabilization of COB mRNA. A previous deletion analysis identified an 11-nucleotide-long sequence within the 5' untranslated region of COB mRNA that is important for Cbp1-dependent COB mRNA stability. In the present study, site-directed mutagenesis experiments were carried out to define further the features of this cis element. The CCG sequence within this region was shown to be necessary for stability. A change in residue 533 of Cbp1 from aspartate to tyrosine suppresses the effects of a single-base change in the CCG element. This is strong genetic evidence that the nuclearly encoded Cbp1 protein recognizes and binds directly to the sequence containing CCG and thus protects COB mRNA from degradation.
• Dieckmann, C., Sparks, K. A., Mayer, S. A., & Dieckmann, C. L. (1997). Premature 3'-end formation of CBP1 mRNA results in the downregulation of cytochrome b mRNA during the induction of respiration in Saccharomyces cerevisiae. Molecular and cellular biology, 17(8).
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  The yeast mitochondrial genome encodes only seven major components of the respiratory chain and ATP synthase; more than 200 other mitochondrial proteins are encoded by nuclear genes. Thus, assembly of functional mitochondria requires coordinate expression of nuclear and mitochondrial genes. One example of coordinate regulation is the stabilization of mitochondrial COB (cytochrome b) mRNA by Cbp1, the product of the nuclear gene CBP1 (cytochrome b processing). CBP1 produces two types of transcripts with different 3' ends: full-length 2.2-kb transcripts and 1.2-kb transcripts truncated within the coding sequence of Cbp1. Upon induction of respiration, the steady-state level of the long transcripts decreases while that of the short transcripts increases reciprocally, an unexpected result since the product of the long transcripts is required for COB mRNA stability and thus for respiration. Here we have tested the hypothesis that the short transcripts, or proteins translated from the short transcripts, are also required for respiration. A protein translated from the short transcripts was not detected by Western analysis, although polysome gradient fractions were shown to contain both long and short CBP1 transcripts. A mutant strain in which production of the short transcripts was abolished showed wild-type growth properties, indicating that the short transcripts are not required for respiration. Due to mutation of the carbon source-responsive element, the long transcript level in the mutant strain did not decrease during induction of respiration. The mutant strain had increased levels of COB RNA, suggestive that production of short CBP1 transcripts is a mechanism for downregulation of the levels of long CBP1 transcripts, Cbp1, and COB mRNA during the induction of respiration.
• Sparks, K. A., Mayer, S. A., & Dieckmann, C. L. (1997). Premature 3'-end formation of CBP1 mRNA results in the downregulation of cytochrome b mRNA during the induction of respiration in Saccharomyces cerevisiae. Molecular and Cellular Biology, 17(8), 4199-4207.
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  PMID: 9234677;PMCID: PMC232273;Abstract: The yeast mitochondrial genome encodes only seven major components of the respiratory chain and ATP synthase; more than 200 other mitochondrial proteins are encoded by nuclear genes. Thus, assembly of functional mitochondria requires coordinate expression of nuclear and mitochondrial genes. One example of coordinate regulation is the stabilization of mitochondrial COB (cytochrome b) mRNA by Cbp1, the product of the nuclear gene CBP1 (cytochrome b processing). CBP1 produces two types of transcripts with different 3' ends: full-length 2.2-kb transcripts and 1.2-kb transcripts truncated within the coding sequence of Cbp1. Upon induction of respiration, the steady-state level of the long transcripts decreases while that of the short transcripts increases reciprocally, an unexpected result since the product of the long transcripts is required for COB mRNA stability and thus for respiration. Here we have tested the hypothesis that the short transcripts, or proteins translated from the short transcripts, are also required for respiration. A protein translated from the short transcripts was not detected by Western analysis, although polysome gradient fractions were shown to contain both long and short CBP1 transcripts. A mutant strain in which production of the short transcripts was abolished showed wild-type growth properties, indicating that the short transcripts are not required for respiration. Due to mutation of the carbon source-responsive element, the long transcript level in the mutant strain did not decrease during induction of respiration. The mutant strain had increased levels of COB RNA, suggestive that production of short CBP1 transcripts is a mechanism for downregulation of the levels of long CBP1 transcripts, Cbp1, and COB mRNA during the induction of respiration.
• Liu, Y., Gu, K. L., & Dieckmann, C. L. (1996). Independent regulation of full-length and 5′-truncated PAS5 mRNAs in Saccharomyces cerevisiae. Yeast, 12(2), 135-143.
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  PMID: 8686377;Abstract: Peroxisome assembly in Saccharomyces cerevisiae requires the products of several genes. In this report, the PAS5 gene has been characterized. The gene is on the left arm of chromosome X and encodes a polypeptide with similarity to the mammalian peroxisome assembly factor-1 (PAF-1). Two different length transcripts are produced from the yeast PAS5 gene. The longer mRNAs encompass an open reading frame, while the shorter transcripts initiate 46-110 base pairs downstream of the first in-frame AUG. The longer transcripts are induced four-fold on medium containing fatty acids as the sole carbon source, while the shorter transcripts are induced up to ten-fold on medium containing glycerol as a carbon source. The full-length coding sequence encodes a protein with a calculated molecular weight of 30-7 kDa. A protein of 25 kDa could be translated from the shorter transcripts and would lack a very acidic domain found in the ammo-terminal extension of the longer protein. The common portion of the proteins is very basic; the calculated pi of the longer polypeptide is 9-02 and that of the shorter protein is 10-06. The PAS5 GenBank accession number is M86538.
• Mittelmeier, T. M., & Dieckmann, C. L. (1995). In vivo analysis of sequences required for translation of cytochrome b transcripts in yeast mitochondria. Molecular and Cellular Biology, 15(2), 780-789.
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  PMID: 7823946;PMCID: PMC231951;Abstract: Respiratory chain proteins encoded by the yeast mitochondrial genome are synthesized within the organelle. Mitochondrial mRNAs lack a 5' cap structure and contain long AU-rich 5' untranslated regions (UTRs) with many potential translational start sites and no apparent Shine-Dalgarno-like complementarity to the 15S mitochondrial rRNA. However, translation initiation requires specific interactions between the 5' UTRs of the mRNAs, mRNA-specific activators, and the ribosomes. In an initial step toward identifying potential binding sites for the mRNA-specific translational activators and the ribosomes, we have analyzed the effects of deletions in the 5' UTR of the mitochondrial COB gene on translation of COB transcripts in vivo. The deletions define two regions of the COB 5' UTR that are important for translation and indicate that sequence just 5' of the AUG is involved in selection of the correct start codon. Taken together, the data implicate specific regions of the 5' UTR of COB mRNA as possible targets for the mitochondrial translational machinery.
• Chen, W., & Dieckmann, C. L. (1994). Cbp1p is required for message stability following 5′-processing of COB mRNA. Journal of Biological Chemistry, 269(24), 16574-16578.
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  PMID: 8206974;Abstract: Cbplp is a nuclear encoded protein required for the stabilization of mitochondrial COB mRNA, which codes for apocytochrome b in the yeast Saccharomyce8 cerevisiae. The COB gene is cotranscribed with the upstream tRNAGlu gene. Release of tRNAGlu from the initial transcript generates a precursor mRNA with a 5′-end at position -1098. The 5′-end of mature COB message is generated by cleavage of the pre-mRNA at nucleotide -955 or -954. Previous work indicated that Cbp1p acts through cis-elements near these cleavage sites. Here we have tested whether Cbp1p stabilizes COB mRNA solely by stimulating the processing of COB precursor RNA at nucleotide -955/-954. Yeast strain TG955 was constructed such that the -955 COB mRNA 5′-processing site coincides with the upstream tRNA 3′-endonuclease site at position -1098, allowing the 5′-end of COB mRNA to be formed by the tRNA 3′-processing enzyme. Respiratory growth and stability of COB mRNA in TG955 are Cbp1p-dependent. Therefore, we conclude that Cbp1p is important for the stabilization of COB mRNA after 5′-processing.
• Dieckmann, C. L., & Staples, R. R. (1994). Regulation of mitochondrial gene expression in Saccharomyces cerevisiae. International Review of Cytology, 152, 145-181.
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  PMID: 8206703;
• Dieckmann, C., Staples, R. R., & Dieckmann, C. L. (1994). Suppressor analyses of temperature-sensitive cbp1 strains of Saccharomyces cerevisiae: the product of the nuclear gene SOC1 affects mitochondrial cytochrome b mRNA post-transcriptionally. Genetics, 138(3).
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  The induction of mitochondrial function is dependent upon both nuclearly encoded and mitochondrially encoded gene products. To understand nuclear-mitochondrial interactions, we must first understand gene-specific interactions. The accumulation of mitochondrial cytochrome b (COB) RNA is dependent upon Cbp1p, encoded by the nuclear gene CBP1. Thus, respiration is dependent upon Cbp1p. In this study, suppressors of temperature-sensitive cbp1 (cbp1ts) strains were selected for restoration of respiratory capability at the restrictive temperature Ts+). One nuclearly encoded suppressor, extragenic to CBP1, is recessive with respect to the wild-type suppressor allele and is unlinked to other known genetic loci whose gene products are necessary for expression of COB mRNA. The suppressor, called soc1 for Suppressor of cbp1, suppresses several other cbp1ts alleles but does not operate via a bypass mechanism. Molecular analyses indicate that soc1 allows the steady-state level of COB mRNA to increase at high temperature but has little or no effect on the levels of COB pre-mRNA. These data have led us to propose that the product of the nuclear gene SOC1 is required for normal turnover of COB mRNA.
• Staples, R. R., & Dieckmann, C. L. (1994). Suppressor analyses of temperature-sensitive cbp1 strains of Saccharomyces cerevisiae: The product of the nuclear gene SOC1 affects mitochondrial cytochrome b mRNA post-transcriptionally. Genetics, 138(3), 565-575.
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  PMID: 7851755;PMCID: PMC1206208;Abstract: The induction of mitochondrial function is dependent upon both nuclearly encoded and mitochondrially encoded gene products. To understand nuclear- mitochondrial interactions, we must first understand gene-specific interactions. The accumulation of mitochondrial cytochrome b (COB) RNA is dependent upon Cbp1p, encoded by the nuclear gene CBP1. Thus, respiration is dependent upon Cbp1p. In this study, suppressors of temperature-sensitive cbp1 (cbp1(ts)) strains were selected for restoration of respiratory capability at the restrictive temperature (Ts+). One nuclearly encoded suppressor, extragenic to CBP1, is recessive with respect to the wild-type suppressor allele and is unlinked to other known genetic loci whose gene products are necessary for expression of COB mRNA. The suppressor, called soc1 for Suppressor of cbp1, suppresses several other cbp1(ts) alleles but does not operate via a bypass mechanism. Molecular analyses indicate that soc1 allows the steady-state level of COB mRNA to increase at high temperature but has little or no effect on the levels of COB pre-mRNA. These data have led us to propose that the product of the nuclear gene SOC1 is required for normal turnover of COB mRNA.
• Dieckmann, C., Mittelmeier, T. M., & Dieckmann, C. L. (1993). In vivo analysis of sequences necessary for CBP1-dependent accumulation of cytochrome b transcripts in yeast mitochondria. Molecular and cellular biology, 13(7).
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  In Saccharomyces cerevisiae, cytochrome b, an essential component of the respiratory chain, is encoded by the mitochondrial gene cob. The cob transcription unit includes the tRNA(Glu) gene from positions -1170 to -1099 relative to the cob ATG at +1. The initial tRNA(Glu)-cob transcript undergoes several processing events, including removal of tRNA(Glu) and production of the mature 5' end of cob mRNA at nucleotide -954. The nuclear gene product CBP1 is specifically required for the accumulation of cob mRNA. In cbp1 mutant strains, cob transcripts are not detectable by Northern (RNA) blot analysis, but the steady-state level of tRNA(Glu) is similar to that of wild type. The results of a previous study led to the conclusion that a 400-nucleotide region just downstream of tRNA(Glu) is sufficient for CBP1 function. In the present study, the microprojectile bombardment method of mitochondrial transformation was used to introduce deletions within this region of cob. The analysis of cob transcripts in strains carrying the mitochondrial deletion genomes indicates that a 63-nucleotide sequence that encompasses the cleavage site at -954 is sufficient both for CBP1 function and for correct positioning of the cleavage. Furthermore, the data indicate that CBP1 prevents the degradation of unprocessed cob transcripts produced by endonucleolytic cleavage at the 3' end of tRNA(Glu).
• Dieckmann, C., Staples, R. R., & Dieckmann, C. L. (1993). Generation of temperature-sensitive cbp1 strains of Saccharomyces cerevisiae by PCR mutagenesis and in vivo recombination: characteristics of the mutant strains imply that CBP1 is involved in stabilization and processing of cytochrome b pre-mRNA. Genetics, 135(4).
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  Mitochondrial biogenesis is dependent on both nuclearly and mitochondrially encoded proteins. Study of the nuclearly encoded mitochondrial gene products and their effect on mitochondrial genome expression is essential to understanding mitochondrial function. Mutations in the nuclear gene CBP1 of Saccharomyces cerevisiae result in degradation of mitochondrially encoded cytochrome b (cob) RNA; thus, the cells are unable to respire. Putative roles for the CBP1 protein include processing of precursor RNA to yield the mature 5' end of cob mRNA and/or physical protection of the mRNA from degradation by nucleases. To examine the activity of CBP1, we generated temperature-sensitive cbp1 mutant strains by polymerase chain reaction (PCR) mutagenesis and in vivo recombination. These temperature-sensitive cbp1 strains lack cob mRNA only at the nonpermissive temperature. Quantitative primer extension analyses of RNA from these strains and from a cbp1 deletion strain demonstrated that CBP1 is required for the stability of precursor RNAs in addition to production of the stable mature mRNA. Thus, CBP1 is not involved solely in the protection of mature cob mRNA from nucleases. Moreover, we found that mature mRNAs are undetectable while precursor RNAs are reduced only slightly at the nonpermissive temperature. Collectively, these data lead us to favor a hypothesis whereby CBP1 protects cob precursor RNAs and promotes the processing event that generates the mature 5' end of the mRNA.
• Mittelmeier, T. M., & Dieckmann, C. L. (1993). In vivo analysis of sequences necessary for CBP1-dependent accumulation of cytochrome b transcripts in yeast mitochondria. Molecular and Cellular Biology, 13(7), 4203-4213.
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  PMID: 8321224;PMCID: PMC359970;Abstract: In Saccharomyces cerevisiae, cytochrome b, an essential component of the respiratory chain, is encoded by the mitochondrial gene cob. The cob transcription unit includes the tRNAGlu gene from positions -1170 to -1099 relative to the cob ATG at +1. The initial tRNAGlu-cob transcript undergoes several processing events, including removal of tRNAGlu and production of the mature 5′ end of cob mRNA at nucleotide -954. The nuclear gene product CBP1 is specifically required for the accumulation of cob mRNA. In cbp1 mutant strains, cob transcripts are not detectable by Northern (RNA) blot analysis, but the steady-state level of tRNAGlu is similar to that of wild type. The results of a previous study led to the conclusion that a 400-nucleotide region just downstream of tRNAGlu is sufficient for CBP1 function. In the present study, the microprojectile bombardment method of mitochondrial transformation was used to introduce deletions within this region of cob. The analysis of cob transcripts in strains carrying the mitochondrial deletion genomes indicates that a 63-nucleotide sequence that encompasses the cleavage site at -954 is sufficient both for CBP1 function and for correct positioning of the cleavage. Furthermore, the data indicate that CBP1 prevents the degradation of unprocessed cob transcripts produced by endonucleolytic cleavage at the 3′ end of tRNAGlu.
• Staples, R. R., & Dieckmann, C. L. (1993). Generation of temperature-sensitive cbp1 strains of Saccharomyces cerevisiae by PCR mutagenesis and in vivo recombination: Characteristics of the mutant strains imply that CBP1 is involved in stabilization and processing of cytochrome b pre-mRNA. Genetics, 135(4), 981-991.
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  PMID: 8307338;PMCID: PMC1205759;Abstract: Mitochondrial biogenesis is dependent on both nuclearly and mitochondrially encoded proteins. Study of the nuclearly encoded mitochondrial gene products and their effect on mitochondrial genome expression is essential to understanding mitochondrial function. Mutations in the nuclear gene CBP1 of Saccharomyces cerevisiae result in degradation of mitochondrially encoded cytochrome b (cob) RNA; thus, the cells are unable to respire. Putative roles for the CBP1 protein include processing of precursor RNA to yield the mature 5' end of cob mRNA and/or physical protection of the mRNA from degradation by nucleases. To examine the activity of CBP1, we generated temperature-sensitive cbp1 mutant strains by polymerase chain reaction (PCR) mutagenesis and in vivo recombination. These temperature- sensitive cbp1 strains lack cob mRNA only at the nonpermissive temperature. Quantitative primer extension analyses of RNA from these strains and from a cbp1 deletion strain demonstrated that CBP1 is required for the stability of precursor RNAs in addition to production of the stable mature mRNA. Thus, CBP1 is not involved solely in the protection of mature cob mRNA from nucleases. Moreover, we found that mature mRNAs are undetectable while precursor RNAs are reduced only slightly at the nonpermissive temperature. Collectively, these data lead us to favor a hypothesis whereby CBP1 protects cob precursor RNAs and promotes the processing event that generates the mature 5' end of the mRNA.
• Mayer, S. A., & Dieckmann, C. L. (1991). Yeast CBP1 mRNA 3′ End Formation Is Regulated during the Induction of Mitochondrial Function. Molecular and Cellular Biology, 11(2), 813-821.
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  PMID: 1990285;PMCID: PMC359733;Abstract: Alternative mRNA processing is one mechanism for generating two or more polypeptides from a single gene. While many mammalian genes contain multiple mRNA 3′ cleavage and polyadenylation signals that change the coding sequence of the mature mRNA when used at different developmental stages or in different tissues, only one yeast gene has been identified with this capacity. The Saccharomyces cerevisiae nuclear gene CBP1 encodes a mitochondrial protein that is required for cytochrome b mRNA stability. This 66-kDa protein is encoded by a 2.2-kb mRNA transcribed from CBP1. Previously we showed that a second 1.2-kb transcript is initiated at the CBP1 promoter but has a 3′ end near the middle of the coding sequence. Furthermore, it was shown that the ratio of the steady-state level of 2.2-kb CBP1 message to 1.2-kb message decreases 10-fold during the induction of mitochondrial function, while the combined levels of both messages remain constant. Having proposed that regulation of 3′ end formation dictates the amount of each CBP1 transcript, we now show that a 146-bp fragment from the middle of CBP1 is sufficient to direct carbon source-regulated production of two transcripts when inserted into the yeast URA3 gene. This fragment contains seven polyadenylation sites for the wild-type 1.2-kb mRNA, as mapped by sequence analysis of CBP1 cDNA clones. Deletion mutations upstream of the polyadenylation sites abolished formation of the 1.2-kb transcript, whereas deletion of three of the sites only led to a reduction in abundance of the 1.2-kb mRNA. Our results indicate that regulation of the abundance of both CBP1 transcripts is controlled by elements in a short segment of the gene that directs 3′ end formation of the 1.2-kb transcript, a unique case in yeast cells.
• Dieckmann, C., Mittelmeier, T. M., & Dieckmann, C. L. (1990). CBP1 function is required for stability of a hybrid cob-oli1 transcript in yeast mitochondria. Current genetics, 18(5).
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  The nuclear gene product CBP1 stabilizes cytochrome b transcripts in yeast mitochondria. In cbp1 mutant strains, cytochrome b gene (cob) transcripts are not detectable by Northern blot analysis. The results of previous studies led to the hypothesis that CBP1 interacts with the 5'-untranslated sequence of the cob mRNA, or pre-mRNA, to stabilize the message. To determine what portion of the cob leader is sufficient for interaction with CBP1, we have investigated the stability of transcripts from a novel hybrid gene, cob-oli1, in which the 5'-terminal third of the cob leader sequence was fused to the coding sequence of the gene for ATP synthase subunit 9, oli1. The hybrid cob-oli1 transcript was stable in a strain wild-type at the CBP1 locus, but was undetectable in the cbp1 mutant background. That the cob-oli1 transcript was translated to produce ATP synthase subunit 9 in CBP1 strains containing the cob-oli1 gene was verified by 35S-methionine labeling of mitochondrial proteins. We conclude that the 5'-terminal portion of the cob message is sufficient for CBP1 function and discuss the hypothesis that CBP1 interacts directly with this region of the transcript to promote cob mRNA stability.
• Mittelmeier, T. M., & Dieckmann, C. L. (1990). CBP1 function is required for stability of a hybrid cob-oli1 transcript in yeast mitochondria. Current Genetics, 18(5), 421-428.
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  PMID: 2150347;Abstract: The nuclear gene product CBP1 stabilizes cytochrome b transcripts in yeast mitochondria. In cbp1 mutant strains, cytochrome b gene (cob) transcripts are not detectable by Northern blot analysis. The results of previous studies led to the hypothesis that CBP1 interacts with the 5'-untranslated sequence of the cob mRNA, or pre-mRNA, to stabilize the message. To determine what portion of the cob leader is sufficient for interaction with CBP1, we have investigated the stability of transcripts from a novel hybrid gene, cob-oli1, in which the 5'-terminal third of the cob leader sequence was fused to the coding sequence of the gene for ATP synthase subunit 9, oli1. The hybrid cob-oli1 transcript was stable in a strain wild-type at the CBP1 locus, but was undetectable in the cbp1 mutant background. That the cob-oli1 transcript was translated to produce ATP synthase subunit 9 in CBP1 strains containing the cob-oli1 gene was verified by 35S-methionine labeling of mitochondrial proteins. We conclude that the 5'-terminal portion of the cob message is sufficient for CBP1 function and discuss the hypothesis that CBP1 interacts directly with this region of the transcript to promote cob mRNA stability.
• Tzagoloff, A., & Dieckmann, C. L. (1990). PET genes of Saccharomyces cerevisiae. Microbiological Reviews, 54(3), 211-225.
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  PMID: 2215420;PMCID: PMC372773;
• Weber, E. R., & Dieckmann, C. L. (1990). Identification of the CBP1 polypeptide in mitochondrial extracts from Saccharomyces cerevisiae. Journal of Biological Chemistry, 265(3), 1594-1600.
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  PMID: 2104848;Abstract: CBP1 is a nuclearly encoded yeast protein required for stability of mitochondrial cytochrome 6 pre-mRNA. Previous studies have shown that CBP1 stabilizes the cytochrome b transcripts via interaction with the 5′-end. For the present study, both rabbit polyclonal and mouse monoclonal antibodies against CBP1 were prepared using a trpE-CBP1 fusion polypeptide as a source of antigen. CBP1 was undetectable in a crude mitochondrial fraction from a wild-type strain by Western blot assay, but a 66-kDa immunoreactive protein was detected in a more purified fraction. The 66-kDa protein was absent in the equivalent fraction from a strain with a deletion in CBP1. Assignment of Mr = 66,000 to the mature CBP1 polypeptide was verified by Western analysis of mitochondria from a strain which over-expresses CBP1. Mitochondrial localization was verified by transcribing CBP1 in vitro with T3 polymerase, translating the artificial mRNA in a rabbit reticulocyte system and importing 35S-CBP1 precursor polypeptides into isolated mitochondria. The mature protein product was 66 kDa, whereas the precursor protein migrated as if it were 68 kDa rather than 76 kDa as predicted from the sequence. Analysis of polypeptides truncated at the carboxyl terminus showed that CBP1 polypeptides migrate anomalously fast in the Laemmli system due to a property of the carboxyl two-thirds of the primary sequence, several sections of which are extremely basic.
• Liu, Y., & Dieckmann, C. L. (1989). Overproduction of yeast viruslike particles by strains deficient in a mitochondrial nuclease. Molecular and Cellular Biology, 9(8), 3323-3331.
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  PMID: 2552292;PMCID: PMC362377;
• Mayer, S. A., & Dieckmann, C. L. (1989). The yeast CBP1 gene produces two differentially regulated transcripts by alternative 3'-end formation. Molecular and Cellular Biology, 9(10), 4161-4169.
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  PMID: 2573826;PMCID: PMC362494;
• Mayer, S. A., & Dieckmann, C. L. (1989). The yeast CBPI gene produces two differentially regulated transcripts by alternative 3′-end formation. Trends in Genetics, 5(C), 392-.
• Dieckmann, C. L., & Gandy, B. (1987). Preferential recombination between GC clusters in yeast mitochondrial DNA.. EMBO Journal, 6(13), 4197-4203.
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  PMID: 3327690;PMCID: PMC553904;Abstract: Yeast mitochondrial DNA molecules have long, AT-rich intergenic spacers punctuated by short GC clusters. GC-rich elements have previously been characterized by others as preferred sites for intramolecular recombination leading to the formation of subgenomic petite molecules. In the present study we show that GC clusters are favored sites for intermolecular recombination between a petite and the wild-type grande genome. The petite studied retains 6.5 kb of mitochondrial DNA reiterated tandemly to form molecules consisting of repeated units. Genetic selection for integration of tandem 6.5 kb repeats of the petite into the grande genome yielded a novel recombination event. One of two crossovers in a double exchange event occurred as expected in the 6.5 kb of matching sequence between the genomes, whereas the second exchange involved a 44 bp GC cluster in the petite and another 44 bp GC cluster in the grande genome 700 bp proximal to the region of homology. Creation of a mitochondrial DNA molecule with a repetitive region led to secondary recombination events that generated a family of molecules with zero to several petite units. The finding that 44 bp GC clusters are preferred as sites for intermolecular exchange adds to the data on petite excision implicating these elements as recombinational hotspots in the yeast mitochondrial genome.
• Dieckmann, C. L., & Mittelmeier, T. M. (1987). Nuclearly-encoded CBP1 interacts with the 5′ end of mitochondrial cytochrome b pre-mRNA. Current Genetics, 12(6), 391-397.
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  PMID: 3329053;Abstract: CBP1 is a nuclearly-encoded protein that is imported into mitochondria and confers stability on the mRNA for cytochrome b. Previous work has shown that CBP1 interacts with the cytochrome b transcript upstream of the coding sequence; a region encompassing some 1,100 nucleotides. The work presented here narrows the site of action of CBP1 to the distal third of this upstream sequence through analysis of mRNA produced from a novel recombinant gene containing segments of the gene for cytochrome b, cob, and the ATP synthase subunit 9 gene, olil. In a wild-type CBP1 strain, the cob-olil-cob gene produces stable, mature mRNA that is translated and contributes a portion of the cytochrome b necessary for optimal growth on non-fermentable medium. © 1987 Springer-Verlag.
• Brody, S., Dieckmann, C., & Mikolajczyk, S. (1985). Circadian rhythms in Neurospora crassa: the effects of point mutations on the proteolipid portion of the mitochondrial ATP synthetase. MGG Molecular & General Genetics, 200(1), 155-161.
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  PMID: 2863735;Abstract: Five oligomycin-resistant (olir) mutant strains of Neurospora crassa were analyzed for their growth rate and for the periodicity of their circadian rhythm. The most resistant strains had periods of 18-19 h while the least resistant strain had a normal period of 21.0 h. There was a rough correlation between the in vivo degree of oligomycinresistance and the amount of change in the period. Several of the olir mutations have been previously described by Sebald et al. (1977) in terms of known amino acid changes in the primary structure of the proteolipid, or DCCD-binding protein, found in the F0 membrane portion of the mitochondrial ATP synthetase. Amino acid changes in the structure of this protein are reported here for two other olir mutations. The proteolipid isolation procedures were slightly modified to include a delipidation step, and an HPLC procedure was developed to separate the hydrophobic peptides of this protein. Analysis of heterocaryons carrying both the olir and olis markers indicated that the olir and olis mutations were codominant to each other in terms of period and growth rate. The changes in the primary structure of this DCCD-binding protein reported here are the first known examples of changes in the primary structure of a protein which alter the period of a circadian rhythm. © 1985 Springer-Verlag.
• Dieckmann, C. L., & Tzagoloff, A. (1985). Assembly of the mitochondrial membrane system. CBP6, a yeast nuclear gene necessary for synthesis of cytochrome b. Journal of Biological Chemistry, 260(3), 1513-1520.
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  PMID: 2981859;Abstract: A new gene function involved in the expression of mitochondrial cytochrome b is described. E158 is a respiratory deficient strain of Saccharomyces cerevisiae with a recessive mutation in nuclear DNA. The block in mitochondrial respiration is a consequence of the mutant's inability to synthesize cytochrome b. This has been confirmed by analysis of the mitochondrial translation products in E158. Since the mutant has wild-type concentrations of mature cytochrome b mRNA, the absence of the cytochrome b protein cannot be due to a transcriptional or RNA-processing defect. The wild-type gene (CBP6) responsible for the observed phenotype has been cloned by transformation of E158 with a genomic library of yeast nuclear DNA. The cloned gene has been sequenced and shown to code for a basic protein with a molecular weight of 18,657. Both deletion and disruption of the CBP6 coding sequence in chromosomal DNA of wild-type yeast lead to respiratory deficiency with a concomitant loss of cytochrome b. Part of the CBP6 gene has been fused to the trpE gene on a high expression Escherichia coli vector. The hybrid protein encoded by the trpE/CBP6 fusion has been purified from E. coli and used as an antigen for antibody production. Antibodies to the hybrid protein cross-react with an 18-kDa protein present in yeast mitochondria.
• Dieckmann, C. L., Homison, G., & Tzagoloff, A. (1984). Assembly of the mitochondrial membrane system. Nucleotide sequence of a yeast nuclear gene (CBP1) involved in 5' end processing of cytochrome b pre-mRNA. Journal of Biological Chemistry, 259(8), 4732-4738.
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  PMID: 6325407;Abstract: A nuclear gene (CBP1) of Saccharomyces cerevisiae involved in 5' end processing of the cytochrome b pre-mRNA has been cloned and sequenced. The gene was originally selected by transformation of cbp1 mutants with a recombinant plasmid bank of random fragments of yeast nuclear DNA ligated to a plasmid vector with autonomous replicative function in yeast. The recombinant plasmid pG60/T10 with a nuclear DNA insert of 6.7 kilobase pairs (kb) was used to construct a new plasmid pool with the CBP1 gene on smaller fragments of nuclear DNA. A number of subclones have been isolated with plasmids carrying inserts of 2.4 to 6.1 kb. The plasmid designated pG60/T31 confers respiratory competency to cbp1 mutants and restores their ability to synthesize mature cytochrome b mRNA. The pG60/T31 plasmid has a nuclear DNA insert of 2.4 kb. The sequence of the cloned fragment reveals only one open reading frame capable of coding for a protein. The reading frame is 1962 nucleotides long and codes for a basic polypeptide with a molecular weight of 76,140. A transcript of a size commensurate with the length of the gene has been detected in wild type yeast.
• Dieckmann, C. L., Koerner, T. J., & Tzagoloff, A. (1984). Assembly of the mitochondrial membrane system. CBP1, a yeast nuclear gene involved in 5' end processing of cytochrome b pre-mRNA. Journal of Biological Chemistry, 259(8), 4722-4731.
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  PMID: 6370993;Abstract: Noncomplementing mutations in a nuclear gene (CBP1) of Saccharomyces cerevisiae D273-10B specifically affect the synthesis of cytochrome b, a mitochondrially encoded carrier of the respiratory chain. The nuclear mutants have been shown to have lowered levels of cytochrome b-specific transcripts. This phenotype is attributed to the inability of the mutant strains to process the 5' end of the cytochrome b pre-mRNA. Impairment of the processing function encoded by the CBP1 gene introduces an instability in the transcripts and promotes nucleolytic degradation. Mutations in CBP1 can be suppressed by a ρ- genome in which the 5' untranslated leader of the oli1 gene (subunit 9 of the ATPase) is fused near the 5' side of the cytochrome b coding sequence. The rearranged genome allows the cytochrome b gene to be transcribed from the oli1 promoter and results in novel cytochrome b transcripts with the 5' leader sequence of the oli1 mRNA. The presence of the oli1 leader sequence confers stability to the RNA and circumvents the CBP1 processing function.
• Dieckmann, C. L., & Tzagoloff, A. (1983). [35] Transformation of nuclear respiratory deficient mutants of yeast. Methods in Enzymology, 97(C), 355-361.
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  PMID: 6361477;
• Dieckmann, C. L., & Tzagoloff, A. (1983). [36] Analysis of yeast mitochondrial genes. Methods in Enzymology, 97(C), 361-373.
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  PMID: 6318032;
• Nobrega, F. G., Dieckmann, C. L., & Tzagoloff, A. (1983). A rapid method for detecting specific RNA transcripts by hybridization to DNA probes in solution. Analytical Biochemistry, 131(1), 141-145.
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  PMID: 6193726;Abstract: A method is described for detecting specific transcripts in crude mixtures of RNA. The method employs hybridization of single-stranded or double-stranded radioactive DNA probes in solution, followed by electrophoretic separation of the hybrid and probe on agarose and visualization by radioautography. The procedure offers the advantages of decreased preparation time and increased sensitivity over currently used methods. © 1983.
• Dieckmann, C. L., Pape, L. K., & Tzagoloff, A. (1982). Identification and cloning of a yeast nuclear gene (CBP1) involved in expression of mitochondrial cytochrome b. Proceedings of the National Academy of Sciences of the United States of America, 79(6 I), 1805-1809.
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  PMID: 7043464;PMCID: PMC346069;Abstract: Nuclear pet mutants of Saccharomyces cerevisiae deficient in mitochondrial respiration have been studied genetically and biochemically. Seven noncomplementing mutations leading to a deficiency of mitochondrial cytochrome b have been assigned to a single complementation group (group 60). Examination of mitochondrial RNA by blot hybridization on diazobenzyloxy-methyl-paper has revealed that group 60 mutants produce a large number of nobel apocytochrome b transcripts not detected in wild-type yeast. The product of the gene affected in the mutants, therefore, appears to be required either for correct transcription or for processing of apocytochrome b premessenger RNA. The gene has been designated CBP1. A representative mutant from complementation group (N5-26) has been transformed to respiratory competency with a recombinant plasmid pool consisting of random fragments of wild-type yeast nuclear DNA inserted into a vector capable of replicating in yeast and Escherichia coli. The complementation of the N5-26 mutation has been shown for a number of independent transformants to be due to the presence of plasmid DNA. The plasmid pG60/T10 was further characterized to have a nuclear DNA insert of 6.7 kilobase pairs. This plasmid complements the mutations of all group 60 mutants, thus confirming that it contains the CBP1 gene.
• Tzagoloff, A., Thalenfeld, B. E., Pape, L. K., Mcgraw, P., Homison, G., Hill, J. E., Dieckmann, C. L., & Bonitz, S. G. (1982). Structure of the Apocytochrome- b Gene and Processing of Apocytochrome- b Transcripts in Saccharomyces cerevisiae. Cold Spring Harbor Monograph Archive, 12, 213-223. doi:10.1101/087969145.12.213
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  Cytochrome b is a catalytic subunit of the coenzyme QH 2 –cytochrome c reductase complex of the mitochondrial respiratory chain. Although there are two distinct cytochromes b ( b 562 and b 566 ) associated with this complex, biochemical and genetic evidence indicates the existence of only one polypeptide with two separate heme-binding sites (Tzagoloff and Nobrega 1980). The spectral properties and redox potentials that distinguish cytochromes b 562 and b 566 (Chance et al. 1970) can therefore be most easily rationalized in terms of two different environments of the heme groups in the protein. Cytochrome b is the only subunit of the coenzyme QH 2 –cytochrome c reductase complex synthesized on mitochondrial ribosomes (Weiss et al. 1973; Katan et al. 1976). Earlier studies of mit − mutants of yeast confirmed that the protein is also encoded in mtDNA (Tzagoloff et al. 1975; Slonimski et al. 1978; Haid et al. 1979). The complete sequences of the yeast gene (Nobrega and Tzagoloff 1980), bovine gene, and human gene (Anderson et al. 1981) have revealed that, although the proteins are highly homologous, the gene itself has undergone extensive evolutionary change. This is most dramatically illustrated by the presence of intervening sequences in the fungal genes and by their absence in the mammalian genes. In this paper we describe the gene structure and some aspects of the processing of the apocytochrome- b messenger in Saccharomyces cerevisiae strain D273-10B. Of the yeast strains studied (Dhawale et al. 1981), S. cerevisiae D273-10B has the least complex gene organization, thereby making the analysis of the apocytochrome- b ...
• Dieckmann, C., & Brody, S. (1980). Circadian rhythms in Neurospora crassa: oligomycin-resistant mutations affect periodicity. Science, 207(4433), 896-898.
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  PMID: 6444467;

Presentations

• Dieckmann, C. L. (2020, June 9). MLT1 shows the way to the eyespot. Seminar. Zoom: Ohio State University - Patrice Hamel host.
• Dieckmann, C. L. (2020, November 20). How investigating mitochondrial RNA processing led us into the realm of fatty acid/lipoic acid biosynthesis and the intersection with glycine catabolism.. Seminar. Zoom: Ohio State University - host Patrice Hamel.
• Dieckmann, C. L. (2018, October 5). Chlamydomonas Eyespot - Asymmetry and Assembly. Mitochondria and Beyond. New York: Columbia University.
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  Symposium Talk
• Dieckmann, C. L., Mittelmeier, T. M., & Thompson, M. D. (2018, June). Choosing the right path to the eyespot. 18th International Conference on the Cell and Molecular Biology of Chlamydomonas. Washington D. C.: Carnegie Institute and others.
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  Choosing the Right Path to the Eyespot (Talk)Telsa M. Mittelmeier1, Mark D. Thompson2, and Carol L. Dieckmann11MCB Department, U. Arizona, 2Biological Industries USA, Cromwell CT. (dieckman@u.arizona.edu) Eyespot placement is asymmetrical with respect to bilateral positioning of Chlamydomonas flagella. Recognition of the association between the eyespot and the daughter four-membered D4 rootlet was the first hint at the underlying importance of the stable cortical microtubule rootlet structures in asymmetric placement1. A simple screen for non-phototactic mutant strains after UV mutagenesis, followed by microscopic inspection for eyespot irregularities, defined four genes required for eyespot placement and assembly2. The MLT1 gene is required for the exclusive choice of the D4 rootlet for eyespot placement; a percentage of mlt1 cells have eyespots on the mother M4 rootlet as well3. Immunofluorescence IF microscopy revealed that MLT1 protein is exclusively associated with the D4, from the anterior minus end to the eyespot position, but not posterior to the eyespot4. Dissociation of MLT1 from the D4 occurs as the D4 matures into an M4 at cell division. In daughter cells, MLT1 reappears immediately on nascently assembling D4 rootlets before tubulin acetylation4. Channelrhodopsin photoreceptor ChR1 was visualized by IF deconvolution3 and super-resolution microscopy (Thompson, unpublished) on the outer surface of the D4, suggesting that photoreceptor is trafficked on this surface from the anterior end of the rootlet to the eyespot. How it gets “loaded” exclusively onto the D4 is a mystery. Centrin is a major component of the fibers that connect the mother and daughter basal bodies, and is also found on the apical surface of both two-membered M2 and D2 microtubule rootlets5. In a centrin mutant, MLT1 was found on both daughter rootlets, and ChR1 patches were observed at their ends4. These data suggest that rootlet identity is disturbed in the centrin knockdown, and MLT1 and ChR1 are deposited erroneously. Without MLT1, eyespots can form 1, but ChR delivery to the eyespot after cell division is delayed. Preceding ChR localization, the chloroplast envelope eyespot protein EYE2 forms a patch in the correct position at the end of the D4 rootlet6. To definitively rule out64the ChRs as masters of eyespot assembly, a strain lacking both ChR1 and ChR2 photoreceptors was sought by insertional mutagenesis of a cop3 (ChR1-) strain. A strain lacking both photoreceptor proteins cannot phototax, but has eyespots visible by light microscopy, exclusive deposition of MLT1 on the D4, and proper positioning of EYE2. Thus, the still unknown “master” directs the assembly of the eyespot at the distal end of the D4 microtubule rootlet. 1) Holmes JA and Dutcher SK, 1989. J Cell Sci. 94:273-85. 2) Lamb MR, Worley CK, Dutcher SK, Dieckmann CL, 1999. Genetics 153:721-9. 3) Mittelmeier TM, Boyd JS, Lamb MR, Dieckmann CL, 2011. J Cell Biol. 193:741-53. 4) Mittelmeier TM, Thompson MD, Lamb MR, Lin H, Dieckmann, CL, 2015. Cytoskeleton 72:113-23. 5) Geimer S and Melkonian M, 2005. Euk. Cell 4:1253-63 6) Mittelmeier TM, Thompson MD, Öztürk E, Dieckmann CL. 2013. Euk. Cell 12:1258-70.
• Dieckmann, C. L. (2014, April). Cellular asymmetry & organelle biogenesis in Chlamydomonas reinhardtii. Seminar. Columbia University Department of Biological Sciences.
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  This seminar presented our work on Chlamydomonas eyespot placement and assembly
• Dieckmann, C. L. (2014, October). LA, where and how?. Seminar. Los Angeles: UCLA Department of Chemistry and Biochemistry.
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  This seminar summarized our work to date on elucidation of the mitochondrial fatty acid/lipoic acid biosynthetic pathway.
• Dieckmann, C. L. (2014, September). Cellular asymmetry & organelle biogenesis in Chlamydomonas reinhardtii. Seminar. University of Mississippi Medical Center Department of Biochemistry.
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  This seminar summarized our work to date on the placement and assembly of the eyespot in the single-celled, green alga Chlamydomonas.
• Dieckmann, C. L. (2013, October). Cellular asymmetry & organelle biogenesis in Chlamydomonas reinhardtii. Seminar. Sandia National Laboratories, Bioenergy and Defense Technologies, Albuquerque NM.
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  This seminar was given to a group of algal and microtubule biologists at Sandia in Bioenergy and Defense Technologies. It was simulcast to another sister lab in California.
• Dieckmann, C. L. (2012, June). Order of Assembly of Eyespot Proteins after Cell Division. 15th International Conference on the Cell and Molecular Biology of Chlamydomonas. Potsdam, Germany: Many including NIGMS/NIH and DRG.
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  T. M. Mittelmeier, M. D. Thompson, C. L. Dieckmann (2012) Order of Assembly of Eyespot Proteins after Cell Division. Abstract No. 40 (Potsdam, Germany) Platform talk.
• Dieckmann, C. L. (2012, October). A Study of Cellular Asymmetry and Organelle Biogenesis In Chlamydomonas reinhardtii. Seminar. University of Massachusetts Amherst Department of Biochemistry and Molecular Biology.

Poster Presentations

• Dieckmann, C. L. (2014, December). Large, low-complexity MLT1 protein is associated with unique, eyespot-associated microtubules in the green alga Chlamydomonas reinhardtii. American Society for Cell Biology Meeting. Philadelphia PA: ASCB.
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  Mittelmeier, T. M., Thompson, M. D., Lamb, M.R., & Dieckmann, C. L (2014) Large, low-complexity MLT1 protein is associated with unique, eyespot-associated microtubules in the green alga Chlamydomonas reinhardtii. Mol.Biol.Cell_(suppl), Abstract No. P2071 (Philadelphia)
• Dieckmann, C. L. (2014, June). Key Protein Domain Functions in Assembly and Placement of the Eyespot. 16th International Conference on the Cell and Molecular Biology of Chlamydomonas. Pacific Grove CA: Genetics Society of America.
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  M. D. Thompson,T. M. Mittelmeier, Figueroa, E, C. L. Dieckmann (2014) Key Protein Domain Functions in Assembly and Placement of the Eyespot. Abstract No. 130 (Pacific Grove, CA)
• Dieckmann, C. L. (2013, December). Assembly of an Algal Eyespot. American Society for Cell Biology Meeting. New Orleans LA: ASCB.
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  Mittelmeier, T. M., Thompson, M. D., Oztürk, E., & Dieckmann, C. L (2013) Assembly of an Algal Eyespot. Mol.Biol.Cell_(suppl), Abstract No. 818 (New Orleans)
• Dieckmann, C. L. (2012, December). Eyespot Assembly in Chlamydomonas reinhardtii After Cell Division. American Society for Cell Biology Meeting. San Francisco CA: ASCB.
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  T. M. Mittelmeier, M. D. Thompson, C. L. Dieckmann (2012). Eyespot Assembly in Chlamydomonas reinhardtii After Cell Division. Mol.Biol.Cell_(suppl), Abstract No. 552 (San Francisco)

Case Studies

• Baker, P. R., Friederich, M. W., Swanson, M. A., Shaikh, T., Bhattacharya, K., Scharer, G. H., Aicher, J., Creadon-Swindell, G., Geiger, E., MacLean, K. N., Lee, W., Deshpande, C., Freckmann, M., Shih, L., Wasserstein, M., Rasmussen, M. B., Lund, A. M., Procopis, P., Cameron, J. M., , Robinson, B. H., et al. (2014. Variant non ketotic hyperglycinemia is caused by mutations in LIAS, BOLA3 and the novel gene GLRX5(pp 366-79).
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  Patients with nonketotic hyperglycinemia and deficient glycine cleavage enzyme activity, but without mutations in AMT, GLDC or GCSH, the genes encoding its constituent proteins, constitute a clinical group which we call 'variant nonketotic hyperglycinemia'. We hypothesize that in some patients the aetiology involves genetic mutations that result in a deficiency of the cofactor lipoate, and sequenced genes involved in lipoate synthesis and iron-sulphur cluster biogenesis. Of 11 individuals identified with variant nonketotic hyperglycinemia, we were able to determine the genetic aetiology in eight patients and delineate the clinical and biochemical phenotypes. Mutations were identified in the genes for lipoate synthase (LIAS), BolA type 3 (BOLA3), and a novel gene glutaredoxin 5 (GLRX5). Patients with GLRX5-associated variant nonketotic hyperglycinemia had normal development with childhood-onset spastic paraplegia, spinal lesion, and optic atrophy. Clinical features of BOLA3-associated variant nonketotic hyperglycinemia include severe neurodegeneration after a period of normal development. Additional features include leukodystrophy, cardiomyopathy and optic atrophy. Patients with lipoate synthase-deficient variant nonketotic hyperglycinemia varied in severity from mild static encephalopathy to Leigh disease and cortical involvement. All patients had high serum and borderline elevated cerebrospinal fluid glycine and cerebrospinal fluid:plasma glycine ratio, and deficient glycine cleavage enzyme activity. They had low pyruvate dehydrogenase enzyme activity but most did not have lactic acidosis. Patients were deficient in lipoylation of mitochondrial proteins. There were minimal and inconsistent changes in cellular iron handling, and respiratory chain activity was unaffected. Identified mutations were phylogenetically conserved, and transfection with native genes corrected the biochemical deficiency proving pathogenicity. Treatments of cells with lipoate and with mitochondrially-targeted lipoate were unsuccessful at correcting the deficiency. The recognition of variant nonketotic hyperglycinemia is important for physicians evaluating patients with abnormalities in glycine as this will affect the genetic causation and genetic counselling, and provide prognostic information on the expected phenotypic course.