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.
Hoke
Stephen M T
SM
Department of Biochemistry, University of Western Ontario, London, Canada.
Irina Mutiu
A
A
Genereaux
Julie
J
Kvas
Stephanie
S
Buck
Michael
M
Yu
Michael
M
Gloor
Gregory B
GB
Brandl
Christopher J
CJ
D000429:Alcohol Oxidoreductases / Q000235:genetics; D000595:Amino Acid Sequence; D001483:Base Sequence; D015153:Blotting, Western; D004252:DNA Mutational Analysis; D004271:DNA, Fungal / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics; D015870:Gene Expression; D020869:Gene Expression Profiling; D015966:Gene Expression Regulation, Fungal; D051548:Histone Acetyltransferases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D009154:Mutation; D058539:Phosphatidylinositol 3-Kinase; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D054730:Protein Interaction Domains and Motifs; D012441:Saccharomyces cerevisiae / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D017384:Sequence Deletion; D015534:Trans-Activators / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014158:Transcription, Genetic; D015533:Transcriptional Activation
D004271:DNA, Fungal; D004268:DNA-Binding Proteins; C507785:SAGA complex, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; C491364:TRA1 protein, S cerevisiae; D015534:Trans-Activators; D000429:Alcohol Oxidoreductases; C096794:C-terminal binding protein; D051548:Histone Acetyltransferases; C504740:NuA4 protein, S cerevisiae; D058539:Phosphatidylinositol 3-Kinase