RESUMO
ATP synthase is the last enzyme of the OXPHOS system synthesizing ATP. Mutations in either mitochondrial or nuclear genes encoding subunits of this enzyme (17 polypeptides) cause neurodegenerative diseases. The ATP synthase subunits 8 (ATP8, alias A6L) and a (ATP6) are encoded by the MT-ATP8 and MT-ATP6 mitochondrial genes, respectively. 17 diseases associated mutations were identified in five nuclear genes coding for subunits of this enzyme. 58 mutations were described in the MT-ATP6 and MT-ATP8 genes, among them 36 were deposited in MITOMAP database. For most of them neither their pathogenic character nor the mechanisms are known. This review summarizes what is known about the molecular basis of the ATP synthase deficiencies. We review the mutations in the ATP synthase genes as well as biochemical data obtained from studies of patient's cells and cybrid or yeast models. We include yeast research about drugs selection and their mechanism of action. Moreover we position the mutations into a recently published structural model of the Fo complex and discuss their structural/functional consequences.
Assuntos
ATPases Mitocondriais Próton-Translocadoras/genética , Mutação , Trifosfato de Adenosina/biossíntese , Humanos , Mitocôndrias/enzimologia , Mitocôndrias/genética , ATPases Mitocondriais Próton-Translocadoras/efeitos dos fármacos , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Modelos Biológicos , Subunidades Proteicas/genética , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/efeitos dos fármacos , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
The author describes her life in science, starting from 1945 in post II world war Warsaw to an enthusiastic and devoted member of the international yeast community, recalling her collaboration with laboratories from US through Australia to France and Germany where she met great scientists and great friends.
Assuntos
Liberdade , Amigos , Ciência , Família , História do Século XX , História do Século XXI , Humanos , Política , ReligiãoRESUMO
The yeast protein Ccz1p is necessary for vacuolar protein trafficking and biogenesis. In a complex with Mon1p, it mediates fusion of transport intermediates with the vacuole membrane by activating the small GTPase Ypt7p. Additionally, genetic data suggest a role of Ccz1p in earlier transport steps, in the Golgi. In a search for further proteins interacting with Ccz1p, we identified the endosomal soluble N-ethylmaleimide-sensitive factor attachment protein receptor Pep12p as an interaction partner of Ccz1p. Combining the ccz1Delta mutation with deletions of PEP12 or other genes encoding components of the endosomal fusion machinery, VPS21, VPS9 or VPS45, results in synthetic growth phenotypes. The genes MON1 and YPT7 also interact genetically with PEP12. These results suggest that the Ccz1p-Mon1p-Ypt7p complex is involved in fusion of transport vesicles to multiple target membranes in yeast cells.
Assuntos
Fatores de Troca do Nucleotídeo Guanina/metabolismo , Mapeamento de Interação de Proteínas , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/fisiologia , Proteínas de Transporte Vesicular/metabolismo , Vesículas Citoplasmáticas/metabolismo , Deleção de Genes , Fatores de Troca do Nucleotídeo Guanina/genética , Ligação Proteica , Proteínas Qa-SNARE/genética , Proteínas Qa-SNARE/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Transporte Vesicular/genética , Proteínas rab de Ligação ao GTP/metabolismoRESUMO
Dolichol formation is examined in three Saccharomyces cerevisiae strains with mutations in the ERG20 gene encoding farnesyl diphosphate synthase (mevalonic acid pathway) and/or the ERG9 gene encoding squalene synthase (sterol synthesis pathway) differing in the amount and chain length of the polyisoprenoids synthesized. Our results suggest that the activities of two yeast cis-prenyltransferases Rer2p and Srt1p and polyprenol reductase are not co-regulated and that reductase may be the rate-limiting enzyme in dolichol synthesis if the amount of polyisoprenoids synthesized exceeds a certain level. We demonstrate that reductase preferentially acts on typical polyprenols with 13-18 isoprene residues but can reduce much longer polyprenols with even 32 isoprene residues.
Assuntos
Alquil e Aril Transferases/metabolismo , Dimetilaliltranstransferase/metabolismo , Oxirredutases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Transferases/metabolismo , Alquil e Aril Transferases/química , Alquil e Aril Transferases/genética , Cromatografia Líquida de Alta Pressão , Dimetilaliltranstransferase/química , Dimetilaliltranstransferase/genética , Dolicóis/biossíntese , Regulação Fúngica da Expressão Gênica , Oxirredutases/química , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Transferases/química , Transferases/genéticaRESUMO
Heme biosynthesis pathway is conserved in yeast and humans and hem12 yeast mutants mimic porphyria cutanea tarda (PCT), a hereditary human disease caused by mutations in the UROD gene. Even though mutations in other genes also affect UROD activity and predispose to sporadic PCT, the regulation of UROD is unknown. Here, we used yeast as a model to study regulation of Hem12 by ubiquitination and involvement of Rsp5 ubiquitin ligase in this process. We found that Hem12 is monoubiquitinated in vivo by Rsp5. Hem12 contains three conserved lysine residues located on the protein surface that can potentially be ubiquitinated and lysine K8 is close to the 36-LPEY-39 (PY) motif which binds WW domains of the Rsp5 ligase. The hem12-K8A mutation results in a defect in cell growth on a glycerol medium at 38°C but it does not affect the level of Hem12. The hem12-L36A,P37A mutations which destroy the PY motif result in a more profound growth defect on both, glycerol and glucose-containing media. However, after several passages on the glucose medium, the hem12-L36A,P37A cells adapt to the growth medium owing to higher expression of hem12-L36A,P37A gene and higher stability of the mutant Hem12-L36A,P37A protein. The Hem12 protein is downregulated upon heat stress in a Rsp5-independent way. Thus, Rsp5-dependent Hem12 monoubiquitination is important for its functioning, but not required for its degradation. Since Rsp5 has homologs among the Nedd4 family of ubiquitin ligases in humans, a similar regulation by ubiquitination might be also important for functioning of the human UROD.
Assuntos
Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Regulação Fúngica da Expressão Gênica , Mutação , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Complexos Ubiquitina-Proteína Ligase/metabolismo , Uroporfirinogênio Descarboxilase/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Regulação para Baixo , Glucose/química , Glicerol/química , Heme/metabolismo , Humanos , Lisina/química , Conformação Molecular , Dados de Sequência Molecular , Porfiria Cutânea Tardia/genética , Proteínas de Saccharomyces cerevisiae/fisiologia , Homologia de Sequência de Aminoácidos , Ubiquitina/química , Ubiquitinação , Uroporfirinogênio Descarboxilase/genética , Uroporfirinogênio Descarboxilase/fisiologiaRESUMO
Mitochondrial diseases are severe and largely untreatable. Owing to the many essential processes carried out by mitochondria and the complex cellular systems that support these processes, these diseases are diverse, pleiotropic, and challenging to study. Much of our current understanding of mitochondrial function and dysfunction comes from studies in the baker's yeast Saccharomyces cerevisiae. Because of its good fermenting capacity, S. cerevisiae can survive mutations that inactivate oxidative phosphorylation, has the ability to tolerate the complete loss of mitochondrial DNA (a property referred to as 'petite-positivity'), and is amenable to mitochondrial and nuclear genome manipulation. These attributes make it an excellent model system for studying and resolving the molecular basis of numerous mitochondrial diseases. Here, we review the invaluable insights this model organism has yielded about diseases caused by mitochondrial dysfunction, which ranges from primary defects in oxidative phosphorylation to metabolic disorders, as well as dysfunctions in maintaining the genome or in the dynamics of mitochondria. Owing to the high level of functional conservation between yeast and human mitochondrial genes, several yeast species have been instrumental in revealing the molecular mechanisms of pathogenic human mitochondrial gene mutations. Importantly, such insights have pointed to potential therapeutic targets, as have genetic and chemical screens using yeast.
Assuntos
Doenças Mitocondriais/metabolismo , Doenças Mitocondriais/terapia , Saccharomyces cerevisiae/metabolismo , Animais , DNA Fúngico/metabolismo , Humanos , Mitocôndrias/metabolismo , Modelos Biológicos , Pesquisa Translacional BiomédicaRESUMO
In Saccharomyces cerevisiae the nicotinic acid moiety of NAD+ can be synthesized from tryptophan using the kynurenine pathway or incorporated directly using nicotinate phosphoribosyl transferase (NPT1). We have identified the genes that encode the enzymes of the kynurenine pathway and for BNA5 (YLR231c) and BNA6 (YFR047c) confirmed that they encode kynureninase and quinolinate phosphoribosyl transferase respectively. We show that deletion of genes encoding kynurenine pathway enzymes are co-lethal with the Deltanpt1, demonstrating that no other pathway for the synthesis of nicotinic acid exists in S. cerevisiae. Also, we show that under anaerobic conditions S. cerevisiae is a nicotinic acid auxotroph.
Assuntos
NAD/metabolismo , Niacina/biossíntese , Saccharomyces cerevisiae/metabolismo , Aerobiose/fisiologia , Anaerobiose/fisiologia , Deleção de Genes , Genes Fúngicos , Hidrolases/genética , Hidrolases/metabolismo , Niacina/metabolismo , Pentosiltransferases/genética , Pentosiltransferases/metabolismo , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Proteínas Cotransportadoras de Sódio-Fosfato , Proteínas Cotransportadoras de Sódio-Fosfato Tipo III , Simportadores/fisiologia , Triptofano/metabolismoRESUMO
The yeast Saccharomyces cerevisiae strain W303 synthesizes in the early logarithmic phase of growth dolichols of 14-18 isoprene residues. The analysis of the polyisoprenoids present in the stationary phase revealed an additional family which proved to be also dolichols but of 19-24 isoprene residues, constituting 39% of the total dolichols. The transfer of early logarithmic phase cells to a starvation medium lacking glucose or nitrogen resulted in the synthesis of the longer chain dolichols. The additional family of dolichols represented 13.8% and 10.3% of total dolichols in the glucose and nitrogen deficient media, respectively. The level of dolichols in yeast cells increased with the age of the cultures. Since both families of dolichols are present in stationary phase cells we postulate that the longer chain dolichols may be responsible for the physico-chemical changes in cellular membranes allowing yeast cells to adapt to nutrient deficient conditions to maintain long-term viability.
Assuntos
Dolicóis/biossíntese , Glucose/deficiência , Nitrogênio/deficiência , Saccharomyces cerevisiae/metabolismo , Cromatografia Líquida de Alta Pressão , Meios de Cultura , Dolicóis/química , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Fatores de TempoRESUMO
Sporulation of the budding yeast Saccharomyces cerevisiae equivalent to gametogenesis in higher organisms, is a complex differentiation program induced by starvation of cells for nitrogen and carbon. Such environmental conditions activate coordinated, sequential changes in gene expression leading to production of haploid, stress-resistant spores. Sporulation comprises two rounds of meiosis coupled with spore morphogenesis and is tightly controlled to ensure viable progeny. This review concerns the regulation of differentiation process by nutritional and transcriptional signals.
Assuntos
Regulação Fúngica da Expressão Gênica/fisiologia , Saccharomyces cerevisiae/metabolismo , Esporos Fúngicos/metabolismo , Regulação Fúngica da Expressão Gênica/genética , Meiose/genética , Meiose/fisiologia , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Esporos Fúngicos/genéticaRESUMO
The CCZ1 gene is a member of the class B VPS (vacuolar protein sorting) genes and it is engaged in the last stage of delivery of multiple kinds of cargo to the yeast vacuole. In the process of fusion of the multivesicular body (MVB) with the vacuole, Ccz1p forms a complex with Ypt7p. Both genes are non-essential for vegetative growth, but their deletions cause a complete block in spore formation. The results of this study indicate that ccz1Δ cells initiate the meiotic program, properly proceed through premeiotic DNA replication and through the pairing of homologous chromosomes, but fail to progress through the first meiotic divisions and arrest in prophase I with a single nucleus. The mutant cells are defective in spindle formation as well as in duplication and/or separation of the SPBs. ypt7Δ cells, on the other hand, cannot execute DNA synthesis. We also show that expression of a mutated variant of the YPT7 gene suppresses the sporulation and autophagy defects of ccz1Δ cells to a quantitatively similar level, suggesting that restoration of autophagy in the ccz1Δ strain is sufficient to enable its sporulation.
Assuntos
Regulação Fúngica da Expressão Gênica , Fatores de Troca do Nucleotídeo Guanina/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/fisiologia , Proteínas de Transporte Vesicular/genética , Proteínas rab de Ligação ao GTP/genética , Sequência de Aminoácidos , Autofagia/genética , Pareamento Cromossômico , Meiose/genética , Mutagênese/fisiologia , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Esporos Fúngicos/fisiologia , Vacúolos/metabolismoRESUMO
The proteins Ccz1 and Mon1 are known to function together with the Rab-GTPase Ypt7 in membrane fusion reactions at the yeast vacuole. In a genome-wide analysis they have also been found to interact genetically with the nuclear-migration protein Num1. In this study we analyze these synthetic effects and we show that the mutants ccz1Delta num1Delta, mon1Delta num1Delta and ypt7Delta num1Delta exhibit severe defects in cell cycle progression. A large fraction of the mutant cells enter a new cell division cycle without having completed mitotic exit, leading to the accumulation of multinuclear, anuclear and multibudded cells. The double deletion strains display also increased sensitivity to calcium ions. The cell-cycle defects are only weakly observed if deletions of other vacuolar protein sorting genes are combined with num1Delta or if other nuclear-migration genes are deleted together with CCZ1, whereas the calcium sensitivity is characteristic for a large subset of the tested double mutants. Further, the cell-cycle defects of the ccz1Delta num1Delta strain can be partially rescued by overproduction of either the calcium pump Pmc1 or the nuclear-migration factors Kar9 and Bim1. Together, these results indicate that deregulation of the cell cycle in these mutants results from two separate mechanisms, one of which is related to calcium homeostasis.
Assuntos
Proteínas do Citoesqueleto/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/genética , Cálcio/metabolismo , Ciclo Celular/genética , Proteínas do Citoesqueleto/química , Proteínas do Citoesqueleto/metabolismo , Fatores de Troca do Nucleotídeo Guanina/química , Fatores de Troca do Nucleotídeo Guanina/genética , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Microscopia de Fluorescência , Mutação , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Vacúolos/metabolismo , Proteínas de Transporte Vesicular/química , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismo , Proteínas rab de Ligação ao GTP/química , Proteínas rab de Ligação ao GTP/genética , Proteínas rab de Ligação ao GTP/metabolismoRESUMO
Lysosomes are conserved organelles that are present in all eukaryotic cells. They are part of a complicated network of intracellular trafficking routes - the lysosomal transport system. Lysosomes are necessary for the maintenance of cellular homeostasis and for many specialized functions, including the activity of many components of the mammalian immune system. Dysfunctions of the lysosomal system are associated with numerous diseases, such as storage disorders, neuro- and myopathies, cancer and some types of albinism and immunological deficiencies. High conservation of the processes of lysosomal biogenesis and transport enables the use of yeast as a model for studying the mechanisms that underlie these diseases. In this review, we discuss several examples of such models in an attempt to present an overview of the most important experimental methods available in yeast research.
RESUMO
NARP (neuropathy, ataxia, and retinitis pigmentosa) and MILS (maternally inherited Leigh syndrome) are mitochondrial disorders associated with point mutations of the mitochondrial DNA (mtDNA) in the gene encoding the Atp6p subunit of the ATP synthase. The most common and studied of these mutations is T8993G converting the highly conserved leucine 156 into arginine. We have introduced this mutation at the corresponding position (183) of yeast Saccharomyces cerevisiae mitochondrially encoded Atp6p. The "yeast NARP mutant" grew very slowly on respiratory substrates, possibly because mitochondrial ATP synthesis was only 10% of the wild type level. The mutated ATP synthase was found to be correctly assembled and present at nearly normal levels (80% of the wild type). Contrary to what has been reported for human NARP cells, the reverse functioning of the ATP synthase, i.e. ATP hydrolysis in the F(1) coupled to F(0)-mediated proton translocation out of the mitochondrial matrix, was significantly compromised in the yeast NARP mutant. Interestingly, the oxygen consumption rate in the yeast NARP mutant was decreased by about 80% compared with the wild type, due to a selective lowering in cytochrome c oxidase (complex IV) content. This finding suggests a possible regulatory mechanism between ATP synthase activity and complex IV expression in yeast mitochondria. The availability of a yeast NARP model could ease the search for rescuing mechanisms against this mitochondrial disease.
Assuntos
Ataxia/enzimologia , Doença de Leigh/enzimologia , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Modelos Biológicos , Retinose Pigmentar/enzimologia , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Trifosfato de Adenosina/biossíntese , Trifosfato de Adenosina/genética , Substituição de Aminoácidos , Ataxia/genética , Complexo IV da Cadeia de Transporte de Elétrons/genética , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Hidrólise , Mitocôndrias/enzimologia , Mitocôndrias/genética , ATPases Mitocondriais Próton-Translocadoras/genética , Mutação de Sentido Incorreto , Consumo de Oxigênio/genética , Prótons , Retinose Pigmentar/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
Atp6p is an essential subunit of the ATP synthase proton translocating domain, which is encoded by the mitochondrial DNA (mtDNA) in yeast. We have replaced the coding sequence of Atp6p gene with the non-respiratory genetic marker ARG8m. Due to the presence of ARG8m, accumulation of rho-/rho0 petites issued from large deletions in mtDNA could be restricted to 20-30% by growing the atp6 mutant in media lacking arginine. This moderate mtDNA instability created favorable conditions to investigate the consequences of a specific lack in Atp6p. Interestingly, in addition to the expected loss of ATP synthase activity, the cytochrome c oxidase respiratory enzyme steady-state level was found to be extremely low (<5%) in the atp6 mutant. We show that the cytochrome c oxidase-poor accumulation was caused by a failure in the synthesis of one of its mtDNA-encoded subunits, Cox1p, indicating that, in yeast mitochondria, Cox1p synthesis is a key target for cytochrome c oxidase abundance regulation in relation to the ATP synthase activity. We provide direct evidence showing that in the absence of Atp6p the remaining subunits of the ATP synthase can still assemble. Mitochondrial cristae were detected in the atp6 mutant, showing that neither Atp6p nor the ATP synthase activity is critical for their formation. However, the atp6 mutant exhibited unusual mitochondrial structure and distribution anomalies, presumably caused by a strong delay in inner membrane fusion.
Assuntos
Genes Mitocondriais/genética , Mitocôndrias/enzimologia , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/enzimologia , Trifosfato de Adenosina/metabolismo , Forma Celular , Citocromos c/metabolismo , DNA Mitocondrial/genética , Deleção de Genes , Regulação Fúngica da Expressão Gênica , Genótipo , Hidrólise , Microscopia Eletrônica , Mitocôndrias/genética , ATPases Mitocondriais Próton-Translocadoras/genética , Oxirredutases/metabolismo , Oxigênio/metabolismo , Biossíntese de Proteínas/genética , Saccharomyces cerevisiae/genética , Transaminases/genética , Transaminases/metabolismo , Transcrição Gênica/genéticaRESUMO
The uncharacterized Saccharomyces cerevisiae proteins Fcf1 and Fcf2, encoded by the ORFs YDR339c and YLR051c, respectively, were identified in a tandem affinity purification experiment of the known 40S factor Faf1p. Most of the proteins associated with TAP-Faf1p are trans-acting factors involved in pre-rRNA processing and 40S subunit biogenesis, in agreement with the previously observed role of Faf1p in 18S rRNA synthesis. Fcf1p and Fcf2p are both essential and localize to the nucleolus. Depletion of Fcf1p and Fcf2p leads to a decrease in synthesis of the 18S rRNA, resulting in a deficit in 40S ribosomal subunits. Northern analysis indicates inefficient processing of pre-rRNA at the A(0), A(1), and A(2) cleavage sites.
Assuntos
Nucléolo Celular/metabolismo , Proteínas Nucleares/biossíntese , Precursores de RNA/biossíntese , RNA Fúngico/biossíntese , Proteínas de Saccharomyces cerevisiae/biossíntese , Saccharomyces cerevisiae/metabolismo , Sequência de Aminoácidos , Regulação Fúngica da Expressão Gênica , Dados de Sequência Molecular , Proteínas Nucleares/genética , Ligação Proteica , Processamento Pós-Transcricional do RNA , RNA Ribossômico 18S/biossíntese , Proteínas de Saccharomyces cerevisiae/genética , Homologia de Sequência de AminoácidosRESUMO
Hermansky-Pudlak syndrome (HPS) is a rare disorder caused by malfunctions of lysosomes and specialized lysosome-related organelles, resulting primarily in oculocutaneous albinism and bleeding diathesis. The majority of the HPS genes have been described as novel, but herein we report the identification of a conserved protein family which includes human HPS4, as well as distant homologs for other HPS genes. Our results suggest that the cellular machinery involved in the HPS syndrome is ancient.
Assuntos
Síndrome de Hermanski-Pudlak/fisiopatologia , Sequência de Aminoácidos , Animais , Fatores de Troca do Nucleotídeo Guanina , Síndrome de Hermanski-Pudlak/genética , Humanos , Camundongos , Dados de Sequência Molecular , Proteínas/química , Proteínas/genética , Homologia de Sequência de AminoácidosRESUMO
The CCZ1 (YBR131w) gene encodes a protein required for fusion of various transport intermediates with the vacuole. Ccz1p, in a complex with Mon1p, is a close partner of Ypt7p in the processes of fusion of endosomes to vacuoles and homotypic vacuole fusion. In this work, we exploited the Ca(2+)-sensitivity of the ccz1Delta mutant to identify genes specifically interacting with CCZ1, basing on functional multicopy suppression of calcium toxicity. The presented results indicate that Ccz1p functions in the cell either in association with Mon1p and Ypt7p in fusion at the vacuolar membrane, or--separately--with Arl1p at early steps of vacuolar transport. We also show that suppression of calcium toxicity by the calcium pumps Pmr1p and Pmc1p is restricted only to the subset of mutants defective in vacuole morphology. The mechanisms of Ca(2+)-pump-mediated suppression also differ from each other, since the action of Pmr1p, but not Pmc1p, appears to require Arl1p function.
Assuntos
Fatores de Troca do Nucleotídeo Guanina/fisiologia , Proteínas de Saccharomyces cerevisiae/fisiologia , Saccharomyces cerevisiae/metabolismo , Proteínas de Transporte Vesicular/fisiologia , Cálcio/metabolismo , Proliferação de Células , DNA/metabolismo , Endossomos/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Mutação , Fenótipo , Plasmídeos/metabolismo , Ligação Proteica , Proteínas de Saccharomyces cerevisiae/metabolismo , Temperatura , Vacúolos/química , Vacúolos/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Proteínas rab de Ligação ao GTP/metabolismoRESUMO
Pan1p is a yeast actin cytoskeleton-associated protein localized in actin patches. It activates the Arp2/3 complex, which is necessary for actin polymerization and endocytosis. We isolated the pan1-11 yeast mutant unable to grow on oleate as a sole carbon source and, therefore, exhibiting the Oleate- phenotype. In addition, mutant cells are temperature-sensitive and grow more slowly on glycerol or succinate-containing medium but similarly to the wild type on ethanol, pyruvate or acetate-containing media; this indicates proper functioning of the mitochondrial respiratory chain. However, growth on ethanol medium is compromised when oleic acid is present. Cells show growth arrest in the apical growth phase, and accumulation of cells with abnormally elongated buds is observed. The growth defects of pan1-11 are suppressed by overexpression of the END3 gene encoding a protein that binds Pan1p. The morphology of peroxisomes and induction of peroxisomal enzymes are normal in pan1-11, indicating that the defect in growth on oleate medium does not result from impairment in peroxisome function. The pan1-11 allele has a deletion of a fragment encoding amino acids 1109-1126 that are part of (QPTQPV)7 repeats. Surprisingly, the independently isolated pan1-9 mutant, which expresses a truncated form of Pan1p comprising aa 1-859, is able to grow on all media tested. Our results indicate that Pan1p, and possibly other components of the actin cytoskeleton, are necessary to properly regulate growth of dividing cells in response to the presence of some alternative carbon sources in the medium.
Assuntos
Proteínas Fúngicas/genética , Ácido Oleico/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/genética , Actinas/química , Actinas/metabolismo , Alelos , Parede Celular/genética , Parede Celular/metabolismo , Parede Celular/ultraestrutura , Meios de Cultura/metabolismo , Proteínas do Citoesqueleto/química , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Citoesqueleto/genética , Citoesqueleto/metabolismo , Endocitose/genética , Proteínas Fúngicas/análise , Proteínas Fúngicas/metabolismo , Proteínas dos Microfilamentos , Mutação , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/análise , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
We present the characteristics of the Csm1 (Spo86) protein of Saccharomyces cerevisiae that are important for meiotic division. The level of Csm1p does not change throughout the cell cycle, but this protein is absent in mature spores. Deletion of CSM1 causes incorrect spore formation and meiotic chromosome missegregation together with increased sensitivity of vegetative cells to benomyl and manganese. In a two-hybrid analysis with Csm1p as bait, we detected interactions with three members of the Mcm2-7 family of proteins involved in the initiation of DNA replication, and with Clf1p also implicated in replication. The Csm1p-Mcm3, Mcm5 and Mcm7p interactions were confirmed by co-immunoprecipitation. Three other interacting proteins, Mgs1p, Ulp2, and Plp2, participate in chromosome assembling and segregation, whereas the function of two others has not been established. Genetic experiments showed that the two-hybrid isolates MGS1, CLF1, MCM3, 5, 7 (CDC47), and YDL089w, when overexpressed, partially suppress the csm1Delta/csm1Delta sporulation defect. We propose that, besides its other functions, Csm1p may be involved in premeiotic DNA replication.
Assuntos
Proteínas de Ciclo Celular/genética , Segregação de Cromossomos/genética , Replicação do DNA/genética , Meiose/genética , Proteínas Nucleares/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas de Ciclo Celular/metabolismo , Núcleo Celular/metabolismo , DNA Helicases/genética , DNA Helicases/metabolismo , Endopeptidases/genética , Endopeptidases/metabolismo , Regulação Fúngica da Expressão Gênica/genética , Lipoproteínas/genética , Lipoproteínas/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas Nucleares/metabolismo , Reprodução/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Esporos Fúngicos/genética , Esporos Fúngicos/metabolismo , Técnicas do Sistema de Duplo-HíbridoRESUMO
The heme biosynthesis pathway in the yeast Saccharomyces cerevisiae is a highly regulated system, but the mechanisms accounting for this regulation remain unknown. In an attempt to identify rate-limiting steps in heme synthesis, which may constitute potential regulatory points, we constructed yeast strains overproducing two enzymes of the pathway: the porphobilinogen synthase (PBG-S) and deaminase (PBG-D). Biochemical analysis of the enzyme-overproducing strains revealed intracellular porphobilinogen and porphyrin accumulation. These results indicate that both enzymes play a rate-limiting role in yeast heme biosynthesis.