Molecular analysis of SSN6, a gene functionally related to the SNF1 protein kinase of Saccharomyces cerevisiae.

Abstract

Mutations in the SSN6 gene suppress the invertase derepression defect caused by a lesion in the SNF1 protein kinase gene. We cloned the SSN6 gene of Saccharomyces cerevisiae and identified its 3.3-kilobase poly(A)-containing RNA. Disruption of the gene caused phenotypes similar to, but more severe than, those caused by missense mutations: high-level constitutivity for invertase, clumpiness, temperature-sensitive growth, aspecific mating defects, and failure of homozygous diploids to sporulate. In contrast, the presence of multiple copies of SSN6 interfered with derepression of invertase. An ssn6 mutation was also shown to cause glucose-insensitive expression of a GAL1O-lacZ fusion and maltase. The mating defects of MATa ssn6 strains were associated with production of two a-specific products, a-factor and barrier, and reduced levels of a-factor; no deficiency of MATa2 RNA was detected. We showed that ssn6 partially restored invertase expression in a cyrl-2 mutant, although ssn6 was clearly not epistatic to cyri-2. We also determined the nucleotide sequence of SSN6, which is predicted to encode a 107-kilodalton protein with stretches of polyglutamine and poly(glutamine-alanine). Possible functions of the SSN6 product are discussed.The SNFJ gene of Saccharomyces cerevisiae encodes a protein kinase that is required for derepression of many glucose-repressible genes, including the SUC2 (invertase) gene (5,8). Mutations in the SSN6 gene have been isolated as suppressors of the invertase derepression defect in a snfl mutant (6). The ssn6 mutations were found to cause highlevel constitutive (glucose-insensitive) invertase synthesis in both snfl and wild-type (SNFI) genetic backgrounds. These findings suggested that SSN6 is a negative regulator of SUC2 and may be a substrate of the SNFJ protein kinase. The pleiotropic phenotypes of ssn6 mutants indicated that SSN6 also affects expression of other genes; the mutants exhibited extreme clumpiness, a-specific mating defects, and a failure to sporulate as homozygous diploids (6). Genetic mapping has led to the finding that ssn6 is allelic to cyc8, a mutation that causes overproduction of iso-2-cytochrome c (34). Additional cyc8/ssn6 alleles have recently been isolated by Trumbly (45).We report here the cloning and molecular analysis of the SSN6 gene. We constructed null mutations to determine the phenotype of strains lacking SSN6 function, tested the effects of multiple copies of SSN6 on SUC2 expression, and determined the sequence of the gene. We showed that ssn6 affects expression of two other glucose-repressible genes and also explored the molecular basis of the mating defects of MATa ssn6 strains. MATERIALS AND METHODSStrains and genetic methods. The S. cerevisiae strains used in this study are listed in Table 1. The strains were constructed by standard genetic methods (39), and the genotypes of double mutants were confirmed by complementation analysis. Media and methods for scoring markers have been described (28). Yeast transformations were carried out as described prev...

SNF6 encodes a nuclear protein that is required for expression of many genes in Saccharomyces cerevisiae.

“…An ssn6 mutation causes pleiotropic phenotypes, including high-level constitutive expression of invertase and extreme clumpiness (8,38). The snj2 ssn6 and snpf ssn6 double mutants display a phenotype that is intermediate between those of the two parent mutants; they show moderate derepression of invertase and partial glucose repression and are only slightly clumpy (28).…”

“…Poly(A)-containing RNAs were isolated from glucose-repressed or derepressed cultures as described previously (37). 32P-labeled singlestranded probes were prepared from pEM1, pEM2, and pEM3 by the M13 chain extension method (36), omitting chain terminators, as described previously (38).…”

Rgt1p of Saccharomyces cerevisiae, a Key Regulator of Glucose-Induced Genes, Is both an Activator and a Repressor of Transcription

“…The Ssn6p-Tup1p complex is involved in repression of transcription of several diversely regulated genes, including genes regulated by glucose repression, mating type, oxygen, and DNA damage (3,20,38,42,48). It seems that specific DNA-binding proteins recruit the Ssn6p-Tup1p repressor complex to different promoters (20,22,40,44,45).…”

Glucose regulates protein interactions within the yeast SNF1 protein kinase complex.

“…Mutation of SSN6 (encoding a transcriptional repressor) also relieves glucose repression of many genes, but SSN6 functions downstream of SNF1 (Schultz and Carlson 1987;Keleher et al 1992). Appropriately, the SNF1-SNF4 interaction was still inhibited by glucose in an ssn6 mutant.…”