The N-terminal TPR region is the functional domain of SSN6, a nuclear phosphoprotein of Saccharomyces cerevisiae.
The SSN6 protein functions as a negative regulator of a variety of genes in Saccharomyces cerevisiae and is required for normal growth, mating, and sporulation. It is a member of a family defined by a repeated amino acid sequence, the TPR (tetratricopeptide repeat) motif. Here, we have used specific antibody to identify and characterize the SSN6 protein. Both SSN6 and a bifunctional SSN6-beta-galactosidase fusion protein were localized in the nucleus by immunofluorescence staining. The N-terminal one-third of the protein containing the TPR units was identified as the region that is important for SSN6 function. Analysis of four nonsense alleles, isolated as intragenic suppressors of an ssn6::URA3 insertion, revealed that polypeptides truncated after TPR unit 7 provide SSN6 function. Deletion analysis suggested that TPR units are required but that 4 of the 10 TPR units are sufficient. In addition, deletion studies indicated that three very long, homogeneous tracts of polyglutamine and poly(glutamine-alanine) are dispensable. Previous genetic evidence suggested the SSN6 protein as a possible target of the SNF1 protein kinase. Here, we show that the C terminus of SSN6 is phosphorylated in vivo and that the SNF1 kinase is not responsible for most of the phosphorylation. Finally, SSN6 has a modest effect on the maintenance of minichromosomes.
Department of Genetics and Development, Columbia University College of Physicians and Surgeons, New York, New York 10032.
J
J
Schultz
L
L
Marshall-Carlson
M
M
Carlson
D000483:Alleles; D002467:Cell Nucleus / Q000378:metabolism; D002872:Chromosome Deletion; D003001:Cloning, Molecular; D004268:DNA-Binding Proteins; D005455:Fluorescent Antibody Technique; D005656:Fungal Proteins / Q000097:blood / Q000235:genetics* / Q000302:isolation & purification; D005800:Genes, Fungal; D005838:Genotype; D006026:Glycoside Hydrolases / Q000378:metabolism; D009687:Nuclear Proteins / Q000235:genetics*; D010750:Phosphoproteins / Q000235:genetics*; D010957:Plasmids; D012091:Repetitive Sequences, Nucleic Acid; D012097:Repressor Proteins; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins; D043324:beta-Fructofuranosidase
C065455:CYC8 protein, S cerevisiae; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; D009687:Nuclear Proteins; D010750:Phosphoproteins; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; D006026:Glycoside Hydrolases; D043324:beta-Fructofuranosidase