RESUMO
The thermotolerant yeast Ogataea parapolymorpha (formerly Hansenula polymorpha) is an industrially relevant production host that exhibits a fully respiratory sugar metabolism in aerobic batch cultures. NADH-derived electrons can enter its mitochondrial respiratory chain either via a proton-translocating complex I NADH-dehydrogenase or via three putative alternative NADH dehydrogenases. This respiratory entry point affects the amount of ATP produced per NADH/O2 consumed and therefore impacts the maximum yield of biomass and/or cellular products from a given amount of substrate. To investigate the physiological importance of complex I, a wild-type O. parapolymorpha strain and a congenic complex I-deficient mutant were grown on glucose in aerobic batch, chemostat, and retentostat cultures in bioreactors. In batch cultures, the two strains exhibited a fully respiratory metabolism and showed the same growth rates and biomass yields, indicating that, under these conditions, the contribution of NADH oxidation via complex I was negligible. Both strains also exhibited a respiratory metabolism in glucose-limited chemostat cultures, but the complex I-deficient mutant showed considerably reduced biomass yields on substrate and oxygen, consistent with a lower efficiency of respiratory energy coupling. In glucose-limited retentostat cultures at specific growth rates down to â¼0.001 h-1, both O. parapolymorpha strains showed high viability. Maintenance energy requirements at these extremely low growth rates were approximately 3-fold lower than estimated from faster-growing chemostat cultures, indicating a stringent-response-like behavior. Quantitative transcriptome and proteome analyses indicated condition-dependent expression patterns of complex I subunits and of alternative NADH dehydrogenases that were consistent with physiological observations.IMPORTANCE Since popular microbial cell factories have typically not been selected for efficient respiratory energy coupling, their ATP yields from sugar catabolism are often suboptimal. In aerobic industrial processes, suboptimal energy coupling results in reduced product yields on sugar, increased process costs for oxygen transfer, and volumetric productivity limitations due to limitations in gas transfer and cooling. This study provides insights into the contribution of mechanisms of respiratory energy coupling in the yeast cell factory Ogataea parapolymorpha under different growth conditions and provides a basis for rational improvement of energy coupling in yeast cell factories. Analysis of energy metabolism of O. parapolymorpha at extremely low specific growth rates indicated that this yeast reduces its energy requirements for cellular maintenance under extreme energy limitation. Exploration of the mechanisms for this increased energetic efficiency may contribute to an optimization of the performance of industrial processes with slow-growing eukaryotic cell factories.
Assuntos
Complexo I de Transporte de Elétrons/metabolismo , Metabolismo Energético , Glucose/metabolismo , Saccharomycetales/metabolismo , Técnicas de Cultura Celular por Lotes , Reatores Biológicos , Oxirredução , Pichia/enzimologia , Pichia/metabolismo , Saccharomycetales/enzimologiaRESUMO
Efficient production of fuels and chemicals by metabolically engineered micro-organisms requires availability of precursor molecules for product pathways. In eukaryotic cell factories, heterologous product pathways are usually expressed in the cytosol, which may limit availability of precursors that are generated in other cellular compartments. In Saccharomyces cerevisiae, synthesis of the precursor molecule succinyl-Coenzyme A is confined to the mitochondrial matrix. To enable cytosolic synthesis of succinyl-CoA, we expressed the structural genes for all three subunits of the Escherichia coli α-ketoglutarate dehydrogenase (αKGDH) complex in S. cerevisiae. The E. coli lipoic-acid scavenging enzyme was co-expressed to enable cytosolic lipoylation of the αKGDH complex, which is required for its enzymatic activity. Size-exclusion chromatography and mass spectrometry indicated that the heterologously expressed αKGDH complex contained all subunits and that its size was the same as in E. coli. Functional expression of the heterologous complex was evident from increased αKGDH activity in the cytosolic fraction of yeast cell homogenates. In vivo cytosolic activity of the αKGDH complex was tested by constructing a reporter strain in which the essential metabolite 5-aminolevulinic acid could only be synthetized from cytosolic, and not mitochondrial, succinyl-CoA. To this end HEM1, which encodes the succinyl-CoA-converting mitochondrial enzyme 5-aminolevulinic acid (ALA) synthase, was deleted and a bacterial ALA synthase was expressed in the cytosol. In the resulting strain, complementation of ALA auxotrophy depended on activation of the αKGDH complex by lipoic acid addition. Functional expression of a bacterial αKGDH complex in yeast represents a vital step towards efficient yeast-based production of compounds such as 1,4-butanediol and 4-aminobutyrate, whose product pathways use succinyl-CoA as a precursor.
Assuntos
Proteínas de Escherichia coli , Expressão Gênica , Complexo Cetoglutarato Desidrogenase , Saccharomyces cerevisiae , Escherichia coli/enzimologia , Escherichia coli/genética , Proteínas de Escherichia coli/biossíntese , Proteínas de Escherichia coli/genética , Complexo Cetoglutarato Desidrogenase/biossíntese , Complexo Cetoglutarato Desidrogenase/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genéticaRESUMO
A conditional gene expression system that is fast-acting, is tunable and achieves single-gene specificity was recently developed for yeast. A gene placed directly downstream of a modified GAL1 promoter containing six Zif268 binding sequences (with single nucleotide spacing) was shown to be selectively inducible in the presence of ß-estradiol, so long as cells express the artificial transcription factor, Z3EV (a fusion of the Zif268 DNA binding domain, the ligand binding domain of the human estrogen receptor and viral protein 16). We show the strength of Z3EV-responsive promoters can be modified using straightforward design principles. By moving Zif268 binding sites toward the transcription start site, expression output can be nearly doubled. Despite the reported requirement of estrogen receptor dimerization for hormone-dependent activation, a single binding site suffices for target gene activation. Target gene expression levels correlate with promoter binding site copy number and we engineer a set of inducible promoter chassis with different input-output characteristics. Finally, the coupling between inducer identity and gene activation is flexible: the ligand specificity of Z3EV can be re-programmed to respond to a non-hormone small molecule with only five amino acid substitutions in the human estrogen receptor domain, which may prove useful for industrial applications.
Assuntos
Regulação Fúngica da Expressão Gênica , Engenharia Genética , Saccharomyces cerevisiae/genética , Regiões 5' não Traduzidas , Sítios de Ligação , Estradiol/farmacologia , Galactoquinase/genética , Ligantes , Regiões Promotoras Genéticas , Receptores de Estrogênio/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Biologia Sintética/métodos , Fatores de Transcrição/metabolismoRESUMO
Malaria, caused by Plasmodium sp, results in almost one million deaths and over 200 million new infections annually. The World Health Organization has recommended that artemisinin-based combination therapies be used for treatment of malaria. Artemisinin is a sesquiterpene lactone isolated from the plant Artemisia annua. However, the supply and price of artemisinin fluctuate greatly, and an alternative production method would be valuable to increase availability. We describe progress toward the goal of developing a supply of semisynthetic artemisinin based on production of the artemisinin precursor amorpha-4,11-diene by fermentation from engineered Saccharomyces cerevisiae, and its chemical conversion to dihydroartemisinic acid, which can be subsequently converted to artemisinin. Previous efforts to produce artemisinin precursors used S. cerevisiae S288C overexpressing selected genes of the mevalonate pathway [Ro et al. (2006) Nature 440:940-943]. We have now overexpressed every enzyme of the mevalonate pathway to ERG20 in S. cerevisiae CEN.PK2, and compared production to CEN.PK2 engineered identically to the previously engineered S288C strain. Overexpressing every enzyme of the mevalonate pathway doubled artemisinic acid production, however, amorpha-4,11-diene production was 10-fold higher than artemisinic acid. We therefore focused on amorpha-4,11-diene production. Development of fermentation processes for the reengineered CEN.PK2 amorpha-4,11-diene strain led to production of > 40 g/L product. A chemical process was developed to convert amorpha-4,11-diene to dihydroartemisinic acid, which could subsequently be converted to artemisinin. The strains and procedures described represent a complete process for production of semisynthetic artemisinin.
Assuntos
Antimaláricos/metabolismo , Artemisininas/metabolismo , Saccharomyces cerevisiae/metabolismo , Sesquiterpenos/metabolismo , Antimaláricos/química , Artemisininas/química , Técnicas de Cultura Celular por Lotes , Códon/genética , Etanol/metabolismo , Fermentação , Galactose/metabolismo , Genes Fúngicos/genética , Genótipo , Glucose/metabolismo , Sesquiterpenos Policíclicos , Saccharomyces cerevisiae/genética , Sesquiterpenos/químicaRESUMO
Cytosolic acetyl-coenzyme A is a precursor for many biotechnologically relevant compounds produced by Saccharomyces cerevisiae. In this yeast, cytosolic acetyl-CoA synthesis and growth strictly depend on expression of either the Acs1 or Acs2 isoenzyme of acetyl-CoA synthetase (ACS). Since hydrolysis of ATP to AMP and pyrophosphate in the ACS reaction constrains maximum yields of acetyl-CoA-derived products, this study explores replacement of ACS by two ATP-independent pathways for acetyl-CoA synthesis. After evaluating expression of different bacterial genes encoding acetylating acetaldehyde dehydrogenase (A-ALD) and pyruvate-formate lyase (PFL), acs1Δ acs2Δ S. cerevisiae strains were constructed in which A-ALD or PFL successfully replaced ACS. In A-ALD-dependent strains, aerobic growth rates of up to 0.27 h(-1) were observed, while anaerobic growth rates of PFL-dependent S. cerevisiae (0.20 h(-1)) were stoichiometrically coupled to formate production. In glucose-limited chemostat cultures, intracellular metabolite analysis did not reveal major differences between A-ALD-dependent and reference strains. However, biomass yields on glucose of A-ALD- and PFL-dependent strains were lower than those of the reference strain. Transcriptome analysis suggested that reduced biomass yields were caused by acetaldehyde and formate in A-ALD- and PFL-dependent strains, respectively. Transcript profiles also indicated that a previously proposed role of Acs2 in histone acetylation is probably linked to cytosolic acetyl-CoA levels rather than to direct involvement of Acs2 in histone acetylation. While demonstrating that yeast ACS can be fully replaced, this study demonstrates that further modifications are needed to achieve optimal in vivo performance of the alternative reactions for supply of cytosolic acetyl-CoA as a product precursor.
Assuntos
Acetato-CoA Ligase , Acetilcoenzima A , Citosol/enzimologia , Proteínas de Saccharomyces cerevisiae , Acetato-CoA Ligase/biossíntese , Acetato-CoA Ligase/genética , Acetilcoenzima A/biossíntese , Acetilcoenzima A/genética , Acetiltransferases/biossíntese , Acetiltransferases/genética , Aldeído Oxirredutases/biossíntese , Aldeído Oxirredutases/genética , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/genética , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/biossíntese , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
Although the proteins comprising many signaling systems are known, less is known about their numbers per cell. Existing measurements often vary by more than 10-fold. Here, we devised improved quantification methods to measure protein abundances in the Saccharomyces cerevisiae pheromone response pathway, an archetypical signaling system. These methods limited variation between independent measurements of protein abundance to a factor of two. We used these measurements together with quantitative models to identify and investigate behaviors of the pheromone response system sensitive to precise abundances. The difference between the maximum and basal signaling output (dynamic range) of the pheromone response MAPK cascade was strongly sensitive to the abundance of Ste5, the MAPK scaffold protein, and absolute system output depended on the amount of Fus3, the MAPK. Additional analysis and experiment suggest that scaffold abundance sets a tradeoff between maximum system output and system dynamic range, a prediction supported by recent experiments.
Assuntos
Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Transdução de Sinais , Biologia de Sistemas , Fluorescência , Immunoblotting , Sistema de Sinalização das MAP Quinases , Modelos Biológicos , Feromônios/metabolismoRESUMO
During meiosis, two rounds of chromosome segregation occur after a single round of DNA replication, producing haploid progeny from diploid progenitors. Three innovations in chromosome behaviour during meiosis I accomplish this unique division. First, crossovers between maternal and paternal sister chromatids (detected cytologically as chiasmata) bind replicated maternal and paternal chromosomes together. Second, sister kinetochores attach to microtubules from the same pole (mono-polar orientation), causing maternal and paternal centromere pairs (and not sister chromatids) to be separated. Third, sister chromatid cohesion near centromeres is preserved at anaphase I when cohesion along chromosome arms is destroyed. The finding that destruction of mitotic cohesion is regulated by Polo-like kinases prompted us to investigate the meiotic role of the yeast Polo-like kinase Cdc5. We show here that cells lacking Cdc5 synapse homologues and initiate recombination normally, but fail to efficiently resolve recombination intermediates as crossovers. They also fail to properly localize the Lrs4 (ref. 3) and Mam1 (ref. 4) monopolin proteins, resulting in bipolar orientation of sister kinetochores. Cdc5 is thus required both for the formation of chiasmata and for cosegregation of sister centromeres at meiosis I.
Assuntos
Proteínas de Ciclo Celular , Centrômero/genética , Segregação de Cromossomos/genética , Células Eucarióticas/metabolismo , Meiose/genética , Proteínas Quinases/genética , Proteínas de Saccharomyces cerevisiae , Leveduras/enzimologia , Leveduras/genética , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Células Cultivadas , Cromossomos/genética , Replicação do DNA/genética , Imunofluorescência , Regulação Fúngica da Expressão Gênica/genética , Cinetocoros/metabolismo , Proteínas Quinases/deficiência , Proteínas Serina-Treonina Quinases , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismoRESUMO
Future manufacturing will focus on new, improved products as well as on new and enhanced production methods. Recent biotechnological and scientific advances, such as CRISPR/Cas and various omic technologies, pave the way to exciting novel biotechnological research, development, and commercialization of new sustainable products. Rigorous mathematical descriptions of microbial cells and consortia thereof will enable deeper biological understanding and lead to powerful in silico cellular models. Biological engineering, namely model-based design together with synthetic biology, will accelerate the construction of robust and high-performing microorganisms. Using these organisms, and ambitions towards zero-concepts with respect to emissions and excess resources in bioprocess engineering, industrial biotechnology is expected to become highly integrated into sustainable generations of technology systems.
Assuntos
Biotecnologia/métodos , Pesquisa/tendências , Biocombustíveis , Engenharia Genética/métodos , Química Verde , Microbiologia Industrial/métodos , Engenharia Metabólica/métodos , Consórcios Microbianos/fisiologia , ReciclagemRESUMO
CAK1 encodes an essential protein kinase in Saccharomyces cerevisiae that is required for activation of the Cdc28p Cdk. CAK1 also has several CDC28-independent functions that are unique to meiosis. The earliest of these functions is to induce S phase, which is regulated differently in meiosis than in mitosis. In mitosis, Cdc28p controls its own S-phase-promoting activity by signaling the destruction of its inhibitor, Sic1p. In meiosis, Sic1p destruction is signaled by the meiosis-specific Ime2p protein kinase. Our data show that Cak1p is required to activate Ime2p through a mechanism that requires threonine 242 and tyrosine 244 in Ime2p's activation loop. This activation promotes autophosphorylation and accumulation of multiply phosphorylated forms of Ime2p during meiotic development. Consistent with Cak1p's role in activating Ime2p, cells lacking Cak1p are deficient in degrading Sic1p. Deletion of SIC1 or overexpression of IME2 can partially suppress the S-phase defect in cak1 mutant cells, suggesting that Ime2p is a key target of Cak1p regulation. These data show that Cak1p is required for the destruction of Sic1p in meiosis, as in mitosis, but in meiosis, it functions through a sporulation-specific kinase.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Quinases Ciclina-Dependentes , Meiose/fisiologia , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/enzimologia , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Proteínas Inibidoras de Quinase Dependente de Ciclina , Ativação Enzimática , Deleção de Genes , Expressão Gênica , Genes Fúngicos , Peptídeos e Proteínas de Sinalização Intracelular , Meiose/genética , Mitose , Modelos Biológicos , Mutação , Fosforilação , Proteínas Quinases/química , Proteínas Quinases/genética , Proteínas Serina-Treonina Quinases/genética , Fase S , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Transdução de Sinais , Quinase Ativadora de Quinase Dependente de CiclinaRESUMO
A key transition in meiosis is the exit from prophase and entry into the nuclear divisions, which in the yeast Saccharomyces cerevisiae depends upon induction of the middle sporulation genes. Ndt80 is the primary transcriptional activator of the middle sporulation genes and binds to a DNA sequence element termed the middle sporulation element (MSE). Sum1 is a transcriptional repressor that binds to MSEs and represses middle sporulation genes during mitosis and early sporulation. We demonstrate that Sum1 and Ndt80 have overlapping yet distinct sequence requirements for binding to and acting at variant MSEs. Whole-genome expression analysis identified a subset of middle sporulation genes that was derepressed in a sum1 mutant. A comparison of the MSEs in the Sum1-repressible promoters and MSEs from other middle sporulation genes revealed that there are distinct classes of MSEs. We show that Sum1 and Ndt80 compete for binding to MSEs and that small changes in the sequence of an MSE can yield large differences in which protein is bound. Our results provide a mechanism for differentially regulating the expression of middle sporulation genes through the competition between the Sum1 repressor and the Ndt80 activator.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Regulação Fúngica da Expressão Gênica , Proteínas Nucleares/metabolismo , Sequências Reguladoras de Ácido Nucleico , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Fatores de Transcrição/metabolismo , Sequência de Aminoácidos , Ligação Competitiva/fisiologia , Proteínas de Ligação a DNA/genética , Meiose/genética , Proteínas Quinases Ativadas por Mitógeno/genética , Dados de Sequência Molecular , Mutação , Proteínas Nucleares/genética , Proteínas Repressoras , Proteínas de Saccharomyces cerevisiae/genética , Fatores de Transcrição/genéticaRESUMO
The energetic (ATP) cost of biochemical pathways critically determines the maximum yield of metabolites of vital or commercial relevance. Cytosolic acetyl coenzyme A (acetyl-CoA) is a key precursor for biosynthesis in eukaryotes and for many industrially relevant product pathways that have been introduced into Saccharomyces cerevisiae, such as isoprenoids or lipids. In this yeast, synthesis of cytosolic acetyl-CoA via acetyl-CoA synthetase (ACS) involves hydrolysis of ATP to AMP and pyrophosphate. Here, we demonstrate that expression and assembly in the yeast cytosol of an ATP-independent pyruvate dehydrogenase complex (PDH) from Enterococcus faecalis can fully replace the ACS-dependent pathway for cytosolic acetyl-CoA synthesis. In vivo activity of E. faecalis PDH required simultaneous expression of E. faecalis genes encoding its E1α, E1ß, E2, and E3 subunits, as well as genes involved in lipoylation of E2, and addition of lipoate to growth media. A strain lacking ACS that expressed these E. faecalis genes grew at near-wild-type rates on glucose synthetic medium supplemented with lipoate, under aerobic and anaerobic conditions. A physiological comparison of the engineered strain and an isogenic Acs(+) reference strain showed small differences in biomass yields and metabolic fluxes. Cellular fractionation and gel filtration studies revealed that the E. faecalis PDH subunits were assembled in the yeast cytosol, with a subunit ratio and enzyme activity similar to values reported for PDH purified from E. faecalis. This study indicates that cytosolic expression and assembly of PDH in eukaryotic industrial microorganisms is a promising option for minimizing the energy costs of precursor supply in acetyl-CoA-dependent product pathways. Importance: Genetically engineered microorganisms are intensively investigated and applied for production of biofuels and chemicals from renewable sugars. To make such processes economically and environmentally sustainable, the energy (ATP) costs for product formation from sugar must be minimized. Here, we focus on an important ATP-requiring process in baker's yeast (Saccharomyces cerevisiae): synthesis of cytosolic acetyl coenzyme A, a key precursor for many industrially important products, ranging from biofuels to fragrances. We demonstrate that pyruvate dehydrogenase from the bacterium Enterococcus faecalis, a huge enzyme complex with a size similar to that of a ribosome, can be functionally expressed and assembled in the cytosol of baker's yeast. Moreover, we show that this ATP-independent mechanism for cytosolic acetyl-CoA synthesis can entirely replace the ATP-costly native yeast pathway. This work provides metabolic engineers with a new option to optimize the performance of baker's yeast as a "cell factory" for sustainable production of fuels and chemicals.
Assuntos
Acetilcoenzima A/metabolismo , Proteínas de Bactérias/metabolismo , Enterococcus faecalis/enzimologia , Engenharia Metabólica , Complexo Piruvato Desidrogenase/genética , Complexo Piruvato Desidrogenase/metabolismo , Saccharomyces cerevisiae/metabolismo , Aerobiose , Anaerobiose , Proteínas de Bactérias/genética , Biomassa , Meios de Cultura/química , Citosol/enzimologia , Citosol/metabolismo , Enterococcus faecalis/genética , Perfilação da Expressão Gênica , Análise do Fluxo Metabólico , Dados de Sequência Molecular , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Análise de Sequência de DNARESUMO
Microscope-based cytometry provides a powerful means to study cells in high throughput. Here we present a set of refined methods for making sensitive measurements of large numbers of individual Saccharomyces cerevisiae cells over time. The set consists of relatively simple 'wet' methods, microscope procedures, open-source software tools and statistical routines. This combination is very sensitive, allowing detection and measurement of fewer than 350 fluorescent protein molecules per living yeast cell. These methods enabled new protocols, including 'snapshot' protocols to calculate rates of maturation and degradation of molecular species, including a GFP derivative and a native mRNA, in unperturbed, exponentially growing yeast cells. Owing to their sensitivity, accuracy and ability to track changes in individual cells over time, these microscope methods may complement flow-cytometric measurements for studies of the quantitative physiology of cellular systems.
Assuntos
Citometria por Imagem/métodos , Microscopia de Fluorescência/métodos , Proteínas/análise , Proteínas de Saccharomyces cerevisiae/análise , Saccharomyces cerevisiae/química , Citometria de Fluxo , Fluorescência , Proteínas de Fluorescência Verde/análise , Células HL-60 , Humanos , Proteínas/metabolismo , Estabilidade de RNA , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/metabolismo , Sensibilidade e Especificidade , Fatores de TempoRESUMO
Meiosis is thought to require the protein kinase Ime2 early for DNA replication and the cyclin-dependent kinase Cdc28 late for chromosome segregation. To elucidate the roles of these kinases, we inhibited their activities early and late using conditional mutants that are sensitive to chemical inhibitors. Our studies reveal that both Cdc28 and Ime2 have critical roles in meiotic S phase and M phase. Early inhibition of analog-sensitive cdc28-as1 blocked DNA replication, revealing a previously undetected role for Cdc28. Yet Cdc28 was dispensable for one of its functions in the mitotic cell cycle, degradation of Sic1. Late addition of inhibitor to ime2-as1 revealed unexpected roles of Ime2 in the initiation and execution of chromosome segregation. The requirement of Ime2 for M phase is partially explained by its stimulation of the key meiotic transcription factor Ndt80, which is needed in turn for high Cdc28 activity. In accordance with a late role for Ime2, we observed an increase in its activity during M phase that depended on Cdc28 and Ndt80. We speculate that several unique features of the meiotic cell division reflect a division of labor and regulatory coordination between Ime2 and Cdc28.
Assuntos
Proteína Quinase CDC28 de Saccharomyces cerevisiae/metabolismo , Proteínas de Ciclo Celular/metabolismo , Meiose/fisiologia , Proteínas Quinases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Proteína Quinase CDC28 de Saccharomyces cerevisiae/genética , Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas de Ciclo Celular/genética , Núcleo Celular/genética , Proteínas Inibidoras de Quinase Dependente de Ciclina , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Inibidores Enzimáticos/farmacologia , Fase G1/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular , Mitose/fisiologia , Mutação , Inibidores de Proteínas Quinases , Proteínas Quinases/genética , Proteínas Serina-Treonina Quinases , Fase S/fisiologia , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/antagonistas & inibidores , Proteínas de Saccharomyces cerevisiae/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transcrição GênicaRESUMO
The process by which the Saccharomyces cerevisiae strand transfer protein, Rad51, seeks out homologous sequences in vivo can be modeled by an in vitro reaction between a single-stranded DNA circle and a double-stranded linear DNA. In addition to the substrates and products, electrophoresis of reaction mixtures resolves two groups of low mobility bands. Here we show that the low mobility bands formed during strand transfer by Rad51 (or Escherichia coli RecA) represent joint molecules (JM) between the two substrates. One group, which we name JM1, is an obligatory reaction intermediate in which the complementary strand from the duplex substrate has been partially transferred to the single-stranded circle. Our assignment is based on pulse-chase and restriction enzyme digestion experiments and verified by electron microscopy. The slower moving group of bands, designated JM2, is formed by an unexpected reaction between JM1 and a second double-stranded linear substrate. Strand transfer of the second duplex initiates noncanonically from the end where the complementary strand is recessed. Thus JM2 is formed by two strand transfer reactions with the same single-stranded circular substrate but with opposite polarities. Finally, we show that the multiple sharp bands in JM1 and JM2 are the result of substrate sequences that pause strand transfer.