RESUMO
Tra1 is a component of the Saccharomyces cerevisiae SAGA and NuA4 complexes and a member of the PIKK family, which contain a C-terminal phosphatidylinositol 3-kinase-like (PI3K) domain followed by a 35-residue FATC domain. Single residue changes of L3733A and F3744A, within the FATC domain, resulted in transcriptional changes and phenotypes that were similar but not identical to those caused by mutations in the PI3K domain or deletions of other SAGA or NuA4 components. The distinct nature of the FATC mutations was also apparent from the additive effect of tra1-L3733A with SAGA, NuA4, and tra1 PI3K domain mutations. Tra1-L3733A associates with SAGA and NuA4 components and with the Gal4 activation domain, to the same extent as wild-type Tra1; however, steady-state levels of Tra1-L3733A were reduced. We suggest that decreased stability of Tra1-L3733A accounts for the phenotypes since intragenic suppressors of tra1-L3733A restored Tra1 levels, and reducing wild-type Tra1 led to comparable growth defects. Also supporting a key role for the FATC domain in the structure/function of Tra1, addition of a C-terminal glycine residue resulted in decreased association with Spt7 and Esa1, and loss of cellular viability. These findings demonstrate the regulatory potential of mechanisms targeting the FATC domains of PIKK proteins.
Assuntos
Regulação Fúngica da Expressão Gênica , Histona Acetiltransferases/química , Histona Acetiltransferases/genética , Domínios e Motivos de Interação entre Proteínas , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Oxirredutases do Álcool/genética , Sequência de Aminoácidos , Sequência de Bases , Western Blotting , Análise Mutacional de DNA , DNA Fúngico/genética , DNA Fúngico/metabolismo , Proteínas de Ligação a DNA/genética , Expressão Gênica , Perfilação da Expressão Gênica , Histona Acetiltransferases/metabolismo , Mutação , Fosfatidilinositol 3-Quinase , Mutação Puntual , Reação em Cadeia da Polimerase , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Deleção de Sequência , Transativadores/química , Transativadores/genética , Transativadores/metabolismo , Transcrição Gênica , Ativação TranscricionalRESUMO
Tra1 is an essential component of the Saccharomyces cerevisiae SAGA and NuA4 complexes. Using targeted mutagenesis, we identified residues within its C-terminal phosphatidylinositol-3-kinase (PI3K) domain that are required for function. The phenotypes of tra1-P3408A, S3463A, and SRR3413-3415AAA included temperature sensitivity and reduced growth in media containing 6% ethanol or calcofluor white or depleted of phosphate. These alleles resulted in a twofold or greater change in expression of approximately 7% of yeast genes in rich media and reduced activation of PHO5 and ADH2 promoters. Tra1-SRR3413 associated with components of both the NuA4 and SAGA complexes and with the Gal4 transcriptional activation domain similar to wild-type protein. Tra1-SRR3413 was recruited to the PHO5 promoter in vivo but gave rise to decreased relative amounts of acetylated histone H3 and histone H4 at SAGA and NuA4 regulated promoters. Distinct from other components of these complexes, tra1-SRR3413 resulted in generation-dependent telomere shortening and synthetic slow growth in combination with deletions of a number of genes with roles in membrane-related processes. While the tra1 alleles have some phenotypic similarities with deletions of SAGA and NuA4 components, their distinct nature may arise from the simultaneous alteration of SAGA and NuA4 functions.
Assuntos
Fosfatidilinositol 3-Quinases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Alelos , Western Blotting , Imunoprecipitação da Cromatina , DNA Fúngico/genética , DNA Fúngico/metabolismo , Perfilação da Expressão Gênica , Histona Acetiltransferases , Mutação/genética , Fenótipo , Fosfatidilinositol 3-Quinases/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Telômero/fisiologia , beta-Galactosidase/metabolismoRESUMO
BACKGROUND: Tra1 is an essential 437-kDa component of the Saccharomyces cerevisiae SAGA/SLIK and NuA4 histone acetyltransferase complexes. It is a member of a group of key signaling molecules that share a carboxyl-terminal domain related to phosphatidylinositol-3-kinase but unlike many family members, it lacks kinase activity. To identify genetic interactions for TRA1 and provide insight into its function we have performed a systematic genetic array analysis (SGA) on tra1SRR3413, an allele that is defective in transcriptional regulation. RESULTS: The SGA analysis revealed 114 synthetic slow growth/lethal (SSL) interactions for tra1SRR3413. The interacting genes are involved in a range of cellular processes including gene expression, mitochondrial function, and membrane sorting/protein trafficking. In addition many of the genes have roles in the cellular response to stress. A hierarchal cluster analysis revealed that the pattern of SSL interactions for tra1SRR3413 most closely resembles deletions of a group of regulatory GTPases required for membrane sorting/protein trafficking. Consistent with a role for Tra1 in cellular stress, the tra1SRR3413 strain was sensitive to rapamycin. In addition, calcofluor white sensitivity of the strain was enhanced by the protein kinase inhibitor staurosporine, a phenotype shared with the Ada components of the SAGA/SLIK complex. Through analysis of a GFP-Tra1 fusion we show that Tra1 is principally localized to the nucleus. CONCLUSION: We have demonstrated a genetic association of Tra1 with nuclear, mitochondrial and membrane processes. The identity of the SSL genes also connects Tra1 with cellular stress, a result confirmed by the sensitivity of the tra1SRR3413 strain to a variety of stress conditions. Based upon the nuclear localization of GFP-Tra1 and the finding that deletion of the Ada components of the SAGA complex result in similar phenotypes as tra1SRR3413, we suggest that the effects of tra1SRR3413 are mediated, at least in part, through its role in the SAGA complex.
Assuntos
Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Alelos , Benzenossulfonatos/farmacologia , Núcleo Celular/genética , Regulação Fúngica da Expressão Gênica , Genes Fúngicos , Ligação Genética , Proteínas de Fluorescência Verde , Histona Acetiltransferases/genética , Membranas Intracelulares/metabolismo , Mitocôndrias/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Plasmídeos , Transporte Proteico , Proteínas Recombinantes de Fusão/genética , Saccharomyces cerevisiae/efeitos dos fármacos , Deleção de Sequência , Sorbitol/farmacologia , Estaurosporina/farmacologia , Transativadores/genéticaRESUMO
BACKGROUND: Spt7 is an integral component of the multi-subunit SAGA complex that is required for the expression of approximately 10% of yeast genes. Two forms of Spt7 have been identified, the second of which is truncated at its C-terminus and found in the SAGA-like (SLIK) complex. RESULTS: We have found that C-terminal processing of Spt7 to its SLIK form (Spt7SLIK) and to a distinct third form (Spt7Form3) occurs in the absence of the SAGA complex components Gcn5, Spt8, Ada1 and Spt20, the latter two of which are required for the integrity of the complex. In addition, N-terminally truncated derivatives of Spt7, including a derivative lacking the histone fold, are processed, indicating that the C-terminus of Spt7 is sufficient for processing and that processing does not require functional Spt7. Using galactose inducible Spt7 expression, we show that the three forms of Spt7 appear and disappear at approximately the same rate with full-length Spt7 not being chased into Spt7SLIK or Spt7Form3. Interestingly, reduced levels of Spt7SLIK and Spt7Form3 were observed in a strain lacking the SAGA component Ubp8, suggesting a regulatory role for Ubp8 in the truncation of Spt7. CONCLUSION: We conclude that truncation of Spt7 occurs early in the biosynthesis of distinct Spt7 containing complexes rather than being a dynamic process linked to the action of the SAGA complex in transcriptional regulation.
Assuntos
Processamento de Proteína Pós-Traducional , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Transativadores/deficiência , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo , Aminoácidos/genética , Aminoácidos/metabolismo , Epitopos/imunologia , Deleção de Genes , Peso Molecular , Ligação Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Transativadores/genética , Fatores de Transcrição/genéticaRESUMO
The SAGA (Spt-Ada-Gcn5 acetyltransferase) complex of Saccharomyces cerevisiae contains more than 20 components that acetylate and deubiquitylate nucleosomal histones. Its acetyltransferase, Gcn5, preferentially acetylates histones H3 and H2B and is regulated through interactions with Ada2 and Ngg1/Ada3. Sequence alignments of Ada2 homologs indicate a conserved approximately 120-amino-acid-residue central region. To examine the function of this region, we constructed ada2 alleles with mutations of clustered conserved residues. One of these alleles, ada2-RLR (R211S, L212A, and R215A), resulted in an approximately threefold reduction in transcriptional activation of the PHO5 gene and growth changes that parallel deletion of ada2. Microarray analyses further revealed that ada2-RLR alters expression of a subset of those genes affected by deletion of ada2. Indicative of Ada2-RLR affecting Gcn5 function, Ada2-RLR resulted in a decrease in Gcn5-mediated histone acetylation in vitro to a level approximately 40% that with wild-type Ada2. In addition, in vivo acetylation of K16 of histone H2B was almost totally eliminated at Ada2-regulated promoters in the ada2-RLR strain, while acetylation of K9 and K18 of histone H3 was reduced to approximately 40% of wild-type levels. We also show that the central region of Ada2 interacts with phospholipids. Since phosphatidylserine binding paralleled Ada2 function, we suggest that lipid binding may play a role in the function or regulation of the SAGA complex.
Assuntos
Regulação Fúngica da Expressão Gênica , Histona Acetiltransferases/metabolismo , Fosfolipídeos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/metabolismo , Acetilação , Fosfatase Ácida , Sequência de Aminoácidos , Substituição de Aminoácidos , Sequência Conservada , Perfilação da Expressão Gênica , Histonas/metabolismo , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Mutação de Sentido Incorreto , Análise de Sequência com Séries de Oligonucleotídeos , Ligação Proteica , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/biossíntese , Proteínas de Saccharomyces cerevisiae/genética , Alinhamento de Sequência , Fatores de Transcrição/genéticaRESUMO
In Saccharomyces cerevisiae histone H2B is ubiquitylated at lysine 123 in a process requiring the E2-ubiquitin conjugase, Rad6. We have analyzed gene expression in a strain containing a variant of histone H2B with lysine 123 converted to arginine to address the mechanisms by which ubiquitylation and deubiquitylation of histone H2B affect gene expression. The SAGA complex component, Ubp8, is one of two proteases that remove the ubiquitin moiety at lysine 123. We show that changes in gene expression observed upon deletion of ubp8 are suppressed by htb1 ( K123R ), which provides genetic evidence that Ubp8 alters gene expression through deubiquitylation of histone H2B. Microarray analyses of the htb1 ( K123R ) strain show that loss of histone ubiquitylation results in a twofold or greater change in expression of approximately 1.5% of the protein coding genes with approximately 75% of these increasing. For genes in which ubiquitylation represses expression, ubiquitylation principally acts through its effects on histone methylation. In contrast, decreased expression of the CWP1 gene was not paralleled by deletions of methyltransferase components and is thus likely independent of methylation. Finally, by comparing gene expression changes in the htb1 ( K123R ) strain with those in a strain deleted for rad6, we conclude that lysine 123 affects transcription primarily because of it being a site of ubiquitylation.