Systematic genetic array analysis links the Saccharomyces cerevisiae SAGA/SLIK and NuA4 component Tra1 to multiple cellular processes

Abstract

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.

Figure 1

Hierarchal cluster analysis of tra1_SRR3413 SSL interactions. Agglomerative hierarchical clustering based on the average linkage of uncentered correlations using CLUSTER 3.0 software [50] was performed on the profile obtained with the tra1_SRR3413 strain and the combined data sets of Tong et al. [11], Measday et al. [12], Reguly et al. [13], Pan et al. [14], Mitchell et al. [15] and including SSL interactions of additional SAGA/SLIK and NuA4 components as listed in the Saccharomyces Genome Database. Only the profiles clustering close to tra1_SRR3413 are shown.

Figure 2

Sorbitol partially suppresses slow growth at 37°C due to tra1_SRR3413. Yeast strains CY2222 (tra1_SRR3413) and BY7092 (TRA1) were grown overnight to saturation in YPD at 30°C. Cells were diluted to approximately 1000 cells per μl and 10 μl of 10-fold serial dilutions spotted on synthetic complete media containing 2% glucose without (SC) or with 1.0 M sorbitol. Cells were grown for 4 days at 37°C.

Figure 3

Growth of tra1_SRR3413 double mutant strains in sorbitol. Strains containing the indicated single disruptions (mdm34, swc3, cog5 and mon2) and combinations with tra1_SRR3413 (tra1) were generated by sporulation of diploids obtained from SGA analysis. These strains plus CY2222 (tra1_SRR3413) and BY7092 (TRA1) were grown overnight to saturation in YPD at 30°C. Cells were diluted to approximately 2000 cells per μl and 5 μl of 10-fold serial dilutions spotted on synthetic complete media containing 2% glucose without (SC) or with 1.0 M sorbitol. Cells were grown for 3 days (upper grouping) or 2 days (lower grouping) at 33.5°C.

Figure 4

Growth of the tra1_SRR3413 strain under conditions of cellular stress. Yeast strains BY7092 (TRA1) and CY2222 (tra1_SRR3413) were grown overnight to saturation in YPD at 30°C. 5 μl of 10-fold serial dilutions were spotted onto synthetic complete media containing 2% glucose (SC) or with the addition of 2 nM rapamycin, 2 μg/ml staurosporine (stauro), 7.5 μg/ml calcofluor white (CW), or 2 μg/ml staurosporine plus 7.5 μg/ml calcofluor white (CW stauro). Cells were grown for 3 days on SC and 5 days on selective plates, at 30°C.

Figure 5

Phenotypic comparison of tra1_SRR3413 with deletions of SAGA/SLIK and NuA4 components. Yeast strains BY3534 (ngg1Δ0), BY4741 (wild-type background for deletion strains), BY7285 (gcn5Δ0), BY4282 (adaΔ0), BY3281 (spt7Δ0), BY4240 (yaf9Δ0), BY2940 (eaf7Δ0), BY7092 (TRA1) and CY2222 (tra1_SRR3413) were grown overnight to saturation in YPD at 30°C. Cells were diluted to approximately 2000 cells per μl and 5 μl of 10-fold serial dilutions spotted on synthetic complete media containing 2% glucose (SC), with 2 μg/ml staurosporine (ST), 7.5 μg/ml calcofluor white (CW), or 2 μg/ml staurosporine plus 7.5 μg/ml calcofluor white (CW ST) or YPD with 2 nM rapamycin (RAP). SC and ST were grown for 2 days at 30°C, CW and CW ST for 3 days, and RAP for 4 days. For the rapamycin image, TRA1 and tra1_SRR were grown on a separate plate.

Figure 6

GFP-Tra1 is found predominately in the nucleus. Yeast strain BY4741, expressing either GFP, GFP-Ngg1 or GFP-Tra1 from a URA3-centromeric plasmid was grown in synthetic complete media, stained with DAPI and examined by fluorescence microscopy. BF, bright field.