The N-terminal TPR region is the functional domain of SSN6, a nuclear phosphoprotein of Saccharomyces cerevisiae.

Abstract

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-j0-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.The Saccharomyces cerevisiae SSN6 gene encodes a protein that functions as a negative regulator of gene expression with a broad range of action and that is required for normal growth. Mutations at the locus cause diverse pleiotropic phenotypes, suggesting that SSN6 affects the expression of many genes (6,52,63). The ssn6 mutants show slow growth at 30°C, temperature sensitivity for growth, extreme clumpiness, defects in utilization of glycerol, and high-level, glucose-insensitive expression of SUC2 and other glucoserepressible genes. MATa ssn6 strains exhibit mating defects because of failure to repress genes that are normally expressed only in MATa strains. Homozygous mutant diploids are defective in sporulation. In addition, ssn6 is allelic to cyc8, which causes overproduction of iso-2-cytochrome c (49). SSN6 affects SUC2 (invertase) gene expression at the transcriptional level, and overexpression of the SSN6 gene prevents full derepression of SUC2, which is consistent with a role for the SSN6 protein as a negative regulator of SUC2 (52). Taken together, this evidence indicates that SSN6 has a role in regulating expression of genes with a variety of functions and that SSN6 is important for normal cell growth, mating, and sporulation.Sequence analysis of the SSN6 gene predicted a 107-kilodalton (kDa) product with several unusual structural features (52, 64). Near its N terminus, the SSN6 protein includes 10 tandem repeats of a 34-amino-acid sequence, termed the TPR (tetratricopeptide repeat) motif, which was recently identified by Sikorski et al. (54). This repeated sequence defines a family of six genes, from several organisms, that encode structurally similar proteins. In additio...

A Complex Composed of Tup1 and Ssn6 Represses Transcription in Vitro

“…The only known function of TPRs is to mediate protein-protein interactions. The TPRs of Ssn6 are essential for repression in vivo and mediate in vitro binding to both Tup1 and ␣2 (35,37,38). A complex composed of several Tup1 proteins and Ssn6 would, therefore, contain numerous proteinprotein interaction domains.…”

“…Ssn6 contains a repeated motif termed the tetratricopeptide repeats, TPRs (35). This 34 amino acid repeat is found in proteins involved in a variety of cellular functions from cell cycle progression to mitochondrial protein import (for review, see Ref.…”

“…Glycerol Gradient Sedimentation-A 5-ml gradient was made by stepwise addition of 1 ml of 35,30,25,20, and 15% glycerol plus 20 mM HEPES, pH 7.6, 10 mM MgOAc, 1 mM EDTA, and 1 mM DTT. The gradient was allowed to diffuse for 3 h at 4°C.…”

A sequence resembling a peroxisomal targeting sequence directs the interaction between the tetratricopeptide repeats of Ssn6 and the homeodomain of α2

“…Ssn6 is a transcriptional repressor found in the nucleus of S. cerevisiae (9,10). It contains 10 TPR repeats (2,10), and together with its corepressor, Tup1, is responsible for the repression of more than 150 genes in yeast (9,11). The Ssn6-Tup1 complex is not capable of binding DNA on its own, but is brought to the promoters of the genes it represses by interactions with sequence-specific DNA binding proteins (9,12,13).…”

“…Ssn6 is a transcriptional repressor found in the nucleus of S. cerevisiae (9,10). It contains 10 TPR repeats (2,10), and together with its corepressor, Tup1, is responsible for the repression of more than 150 genes in yeast (9,11).…”