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

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

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

Supplemental Material for Berg et al.,2018

Supplemental Information contains Table S1 with strain information, Table S2 with oligonucleotide sequences, Figure S1 with sequence of synthetic DNA fragments, Figure S2 with <i>S. cereivisae</i> PIKK alignments, Figure S3 with half life of WT Tra1, Figure S4 with Tra1pr-LacZ data, Figure S5 with synthetic growth interactions between <i>tra1_Q3 </i>and various SAGA and NuA4 deletions, Figure S6 with mapping of mutated residues on Tra1 structure, Figure S7 with fractionation data, Figure S8 with Spt7 localization data, Figure S9 with western blot of Spt7, Figure S10 with coverage map of Spt20 from mass spectrometry data, Figure S11 with tagged Spt20 spot plate and Figure S12 with half life of various <i>tra1</i>_Q3suppressors. Tra1Q3 Spt20 Mass Spectrometry Data file contains peaks identified from mass spectrometry of Spt20 isolated from TAP-Ada2 pull downs of SAGA

Saccharomyces cerevisiae Tti2 Regulates PIKK Proteins and Stress Response

The TTT complex is composed of the three essential proteins Tel2, Tti1, and Tti2. The complex is required to maintain steady state levels of phosphatidylinositol 3-kinase-related kinase (PIKK) proteins, including mTOR, ATM/Tel1, ATR/Mec1, and TRRAP/Tra1, all of which serve as regulators of critical cell signaling pathways. Due to their association with heat shock proteins, and with newly synthesized PIKK peptides, components of the TTT complex may act as cochaperones. Here, we analyze the consequences of depleting the cellular level of Tti2 in Saccharomyces cerevisiae. We show that yeast expressing low levels of Tti2 are viable under optimal growth conditions, but the cells are sensitive to a number of stress conditions that involve PIKK pathways. In agreement with this, depleting Tti2 levels decreased expression of Tra1, Mec1, and Tor1, affected their localization and inhibited the stress responses in which these molecules are involved. Tti2 expression was not increased during heat shock, implying that it does not play a general role in the heat shock response. However, steady state levels of Hsp42 increase when Tti2 is depleted, and tti2L187P has a synthetic interaction with exon 1 of the human Huntingtin gene containing a 103 residue polyQ sequence, suggesting a general role in protein quality control. We also find that overexpressing Hsp90 or its cochaperones is synthetic lethal when Tti2 is depleted, an effect possibly due to imbalanced stoichiometry of a complex required for PIKK assembly. These results indicate that Tti2 does not act as a general chaperone, but may have a specialized function in PIKK folding and/or complex assembly

TOM1p, a yeast hect-domain protein which mediates transcriptional regulation through the ADA/SAGA coactivator complexes

Analysis of the surgical criteria for reintervention in Abdominal Surgery led to the accentuation of a certain number of pictures of occlusion, general infectious syndromes, postoperative peritonitis, gastro-intestinal fistula and hemorrhagic syndrome. In all cases, the clinical examination can be misleading in particular in the case of peritonitis, and the history and non-surgical criteria must be strongly borne in mind