C-terminal processing of yeast Spt7 occurs in the absence of functional SAGA complex

<p>Abstract</p> <p>Background</p> <p>Spt7 is an integral component of the multi-subunit SAGA complex that is required for the expression of ~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.</p> <p>Results</p> <p>We have found that C-terminal processing of Spt7 to its SLIK form (Spt7_SLIK) and to a distinct third form (Spt7_Form3) 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 Spt7_SLIKor Spt7_Form3. Interestingly, reduced levels of Spt7_SLIKand Spt7_Form3were observed in a strain lacking the SAGA component Ubp8, suggesting a regulatory role for Ubp8 in the truncation of Spt7.</p> <p>Conclusion</p> <p>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.</p

Mutational analysis of the C-terminal FATC domain of Saccharomyces cerevisiae Tra1

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

C-terminal processing of yeast Spt7 occurs in the absence of functional SAGA complex-3

<p><b>Copyright information:</b></p><p>Taken from "C-terminal processing of yeast Spt7 occurs in the absence of functional SAGA complex"</p><p>http://www.biomedcentral.com/1471-2091/8/16</p><p>BMC Biochemistry 2007;8():16-16.</p><p>Published online 8 Aug 2007</p><p>PMCID:PMC1976419.</p><p></p>:5 into YP media containing 2% glucose (lane 2) or 2%galactose (lane 3) and grown for 30 minutes. Extracts were prepared by glass bead disruption and 100 μg separated by SDS-PAGE (5%). Flag-Spt7 was detected by Western blotting with anti-Flag antibody. Lane one contains 100 μg of protein extract prepared after growth of BY4741 in galactose-containing media. . CY1811 containing was grown in raffinose. Expression of Spt7 was induced by the addition of galactose then inhibited by the addition of YP media containing 2% glucose. Cells were grown for 90 minutes which was empirically determined as time 0. Additional equal volume samples were taken at time 0, 30, 60, 120 and 240 minutes (lanes 2–7). Protein extracts were prepared and equal volumes separated by SDS-Page (5%). Flag-Spt7 was detected by Western blotting (top panel) or stained with Coomassie Brilliant Blue (bottom panel)