The Pseudokinase Domain of Saccharomyces cerevisiae Tra1 Is Required for Nuclear Localization and Incorporation into the SAGA and NuA4 Complexes

Abstract

Tra1 is an essential component of the SAGA/SLIK and NuA4 complexes in S. cerevisiae, recruiting these co-activator complexes to specific promoters. As a PIKK family member, Tra1 is characterized by a C-terminal phosphoinositide 3-kinase (PI3K) domain. Unlike other PIKK family members (e.g., Tor1, Tor2, Mec1, Tel1), Tra1 has no demonstrable kinase activity. We identified three conserved arginine residues in Tra1 that reside proximal or within the cleft between the N- and C-terminal subdomains of the PI3K domain. To establish a function for Tra1’s PI3K domain and specifically the cleft region, we characterized a tra1 allele where these three arginine residues are mutated to glutamine. The half-life of the Tra1Q3 protein is reduced but its steady state level is maintained at near wild-type levels by a transcriptional feedback mechanism. The tra1Q3 allele results in slow growth under stress and alters the expression of genes also regulated by other components of the SAGA complex. Tra1Q3 is less efficiently transported to the nucleus than the wild-type protein. Likely related to this, Tra1Q3 associates poorly with SAGA/SLIK and NuA4. The ratio of Spt7SLIK to Spt7SAGA increases in the tra1Q3 strain and truncated forms of Spt20 become apparent upon isolation of SAGA/SLIK. Intragenic suppressor mutations of tra1Q3 map to the cleft region further emphasizing its importance. We propose that the PI3K domain of Tra1 is directly or indirectly important for incorporating Tra1 into SAGA and NuA4 and thus the biosynthesis and/or stability of the intact complexes.

Sfp1 regulates the SAGA component Tra1 in response to proteotoxic stress inSaccharomyces cerevisiae

“…Supporting these data, polyQ expression sensitized cells to stresses that result in slow growth of strains carrying TRA1 mutations, i.e. calcofluor white, growth at 39°C, 5% ethanol, MMS and caffeine 59 (Figure 2C). Expression of expanded polyQ proteins was also associated with decreased activity from SAGA regulated promoters ( HIS4 and PHO5 ) (Figure 2D) and histone H3 acetylation (Figure 2E), implicating that polyQ affects SAGA function in yeast.…”

“…Increased transcription was also observed from promoters of other components of SAGA and NuA4, i.e. NGG1 , SPT7 and EAF1 , but not for the control gene PGK1 as was the case for tra1 59 (Figure 4A). We also observed that the polyQ-induced increase in TRA1 expression was abolished when cells expressing 72Q were treated with the HDAC inhibitor trichostatin A (TSA) (Figure 4B), suggesting that changes in chromatin remodeling the regulate TRA1 expression in the presence of polyQ expansion.…”

“…We previously observed that Tra1 bearing mutations in its PI3K domain ( tra1 Q3 ) decrease nuclear localization of the protein 59 . We therefore assessed whether expressing polyQ expansion affects Tra1 localization using confocal microscopy.…”

“…Consequently, polyQ possibly alters SAGA function independently of Tra1 sequestration into cytoplasmic polyQ inclusions. Cells also respond to the defective tra1 Q3 allele by increasing the transcription of the TRA1 gene 59 . We assayed expression from the TRA1 promoter to evaluate whether expanded polyQ results in a Tra1 defect that also feedbacks to increase TRA1 expression.…”

“…Plasmids encoding fluorescently tagged Htt ex1 and LacZ reporter constructs carrying TRA1, PHO5, and PGK1 58,59 promoters in YCplac87 60 were previously described ( see Supplemental Table 2 ). SPT7 promoter sequences relative to the translational start, -633 to +68, NGG1 promoter sequences -430 to +5 and EAF1 promoter sequences -890 to +31 were engineered by PCR as BamHI/HindIII fragments using oligonucleotides listed in Table 3 and cloned into YCplac87 60 to generate transcriptional reporters.…”