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

Abstract

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.Electronic supplementary materialThe online version of this article (doi:10.1007/s00294-010-0313-3) contains supplementary material, which is available to authorized users.

Domains of Tra1 Important for Activator Recruitment and Transcription Coactivator Functions of SAGA and NuA4 Complexes

“…A single deletion at the junction of the PI3K C-terminal lobe and FATC domain gave rise to viable yeast, whereas a complete deletion of the FATC domain was inviable, possibly due to poor protein stability, since this deletion resulted in a truncation in the C terminus (see below). This is consistent with a previous study suggesting that the FATC domain is important for integrity of complexes containing members of the PIKK protein family (36,41). We also found that Tra1 regions spanning HEAT repeats 8 and 9, 10 to 25, 20 to 35, and 46 to 53 and TPR repeats 1 to 6 and 10 to 14 were essential for viability.…”

“…The C terminus of PIKK proteins contains a FATC domain (FAT C-terminal) composed of one short and one long helix that are joined by a disulfide bond (20). However, one or both of these cysteines are missing in Tra1/TRRAP (36). Finally, the HEAT domain folds into a number of ␣-helical HEAT repeats that extend from the N terminus to the FAT domain, representing more than half the length of the protein.…”

Characterization of residue‐dependent differences in the peripheral membrane association of the FATC domain of the kinase ‘target of rapamycin’ by NMR and CD spectroscopy

“…S1A). This domain has been shown to play a very important role for the regulation of TOR and the other phosphoinositide-3 kinase-related kinase (PIKK) family members [8,[12][13][14][15][16][17].…”

“…S1B). The superpositions of the 1 H- 15 N-HSQC spectra of wild type and several mutant y1fatc proteins in the free form and with DMPC/DihepPC bicelles are shown in Fig. 3 (A-B untagged wild type and Y2463D/I2464D/W2466A, C-F Gb1-tagged y1fatc-L2459A, -Y2463A, -Y2463E/W2466E, -H2462R/Y2463D/I2464D/G2465S/W2466S/F2469D) and SI Fig.…”

“…Table 1 shows an overview of all prepared mutated FATC proteins. All proteins were overexpressed in Escherichia coli BL21 (DE3) in LB or 15 N-M9 minimal medium. The culture was grown until an OD 600 of $0.7-0.9, following induction with 1 mM IPTG for 3 h at 37°C.…”