Mutations Restore Invertase Lethality in Yeast Derepression and Cause Temperature-sensitive

Mutations in the SNF2 gene of Saccharomyces cerevisiae prevent derepression of the SUC2 (invertase) gene, and other glucose-repressible genes, in response to glucose deprivation. We have isolated 25 partial phenotypic revertants of a s n . mutant that are able to derepress secreted invertase. These revertants all carried suppressor mutations at a single locus, designated SSN20 (suppressor of snf2). Alleles with dominant, partially dominant and recessive suppressor phenotypes were recovered, but all were only partial suppressors of snf2, reversing the defect in invertase synthesis but not other defects. All alleles also caused recessive, temperature-sensitive lethality and a recessive defect in galactose utilization, regardless of the SNF2 genotype. No significant effect on SUC2 expression was detected in a wild-type (SNF2) genetic background. The ssn20 mutations also suppressed the defects in invertase derepression caused by snf5 and snf6 mutations, and selection for invertase-producing revertants of snf5 mutants yielded only additional ssn20 alleles. These findings suggest that the roles of the SNF2, SNF5 and SNF6 genes in regulation of SUC2 are functionally related and that SSN20 plays a role in expression of a variety of yeast genes. XPRESSION of the SUC2 (invertase) gene of Saccharomyces cerarisiae is E regulated by glucose (carbon catabolite) repression. The SUC2 gene offers a convenient system for studying glucose repression because the gene is not also inducible by sucrose or other substrates of invertase. The regulation of secreted invertase synthesis has been shown to occur at the mRNA level; the 1.9-kb mRNA encoding secreted invertase is produced only when cells are deprived of glucose (derepressing conditions) (CARLSON and BOTSTEIN 1982). In addition to the regulated 1.9-kb mRNA, the SUC2 gene also produces constitutively a minor, 1.8-kb species of mRNA with a different 5’ end (CARLSON et al. 1983); this mRNA encodes an intracellular form of invertase that appears to play no role in sucrose utilization (SAROKIN and CARLSON 1984). An upstream regulatory region that is required for regulated expression of secreted invertase has been identified (SAROKIN and CARLSON 1984) and shown to confer regulated expression to a heterologous gene (SAROKIN and CARLSON 1985a). Genetics 112: 741-753 April, 1986. 742 L. NEIGEBORN, K. RUBIN AND M. CARLSON T o identify genes involved in regulation by glucose repression, we have previously isolated mutants defective in derepression of secreted invertase. Six genes required for normal derepression were identified, SNFl through SNF6 (sucrose nonfermenting) (CARLSON, OSMOND and BOTSTEIN 198 1 ; NEICEBORN and CARLSON 1984). The snf l , snf2, snf4 and snf5 mutations almost completely prevent secreted invertase synthesis and cause defects in utilization of other carbon sources that are subject to glucose repression. The snf3 and snf6 mutations allow some invertase derepression, but in the case of snf6 we have only one allele, which may be leaky. The effects of these SNF genes on SUC2 expression appear to be mediated by the upstream regulatory region as snf mutations were found to affect the expression of a heterologous gene under the control of the SUC2 upstream region (SAROKIN and CARLSON 1985a). We previously isolated suppressors of a snfl mutation that fell into eight complementation groups, called ssnl-ssn8 for suppressor of sllfl (CARLSON et al. 1984). The ssn6 mutations were found to cause high-level constitutive secreted invertase synthesis in a wild-type (SNF) background. The interactions between ssnb and all of the snf mutations were examined, and ssnb suppressed the defects in invertase derepression caused by snfl-snf6; however, the snfl ssn6 and snf4 ssnb double mutants displayed the high-level constitutivity of an ssn6 single mutant, whereas the snf2 ssnb and snf5 ssn6 strains resembled the wild type more closely than either single mutant parent (NEIGEBORN and CARLSON 1984). These findings suggested that SNF2 and SNF5 play different roles from SNFl and SNF4 in regulation by glucose repression and that SNF2 and SNF5 may act antagonistically to SSN6. T o explore further the regulatory role of SNF2, we have isolated here partial suppressors of snf2 that relieve the defect in invertase derepression. Both dominant and recessive suppressors were recovered, and all of them were found to be recessive temperature-sensitive lethal mutations defining a single complementation group (ssn20). These mutations also suppressed snf5, and direct selection for suppressors of snf5 yielded only more alleles of the same gene. MATERIALS AND METHODS Yeast strains: All strains used in this study were isogenic or congenic to strain S288C (MATa SUC2 gall?). The origins of all alleles except snf4-A1 and snf5-5: :URA? have been previously described (CARLSON, OSMOND and BOTSTEIN 198 1; CARLSON et al. 1984; NEICEBORN and CARLSON 1984). The snf4-A1 allele is a deletion of part of the SNF4 gene (F. ENG and M. CARLSON, unpublished results), and snf5-5: :UM3 is an insertion of the URA? gene into SNF5 (E. ABRAMS and M. CARLSON, unpublished results). The SUC7 gene was introduced into the S288C background from strain FLlOO (LACROUTE 1968) through a series of ten backcrosses; SUC7, like SUC2, is regulated by glucose repression, but produces tenfold lower invertase activity than SUC2 in this genetic background, which is insufficient to confer a raffinose-fermenting phenotype (SAROKIN and CARLSON 1985b). The presence of SUC7 in some of these strains, therefore, is not relevant. The strains used to isolate revertants and their genotypes are as follows: MCY637 (MATa snfl-50 his4-539 lys2-801 ura?-52 SUC2 SUC7); MCY 1947 (MAT0 snf55: :URA3 his4-539 ade2-101 SUC2); MCY 1949 (MATa snf5-5: :URA? his4-539 lys2-801 Genetic methods: Standard genetic procedures of crossing, sporulation and tetrad analysis were followed (SHERMAN, FINK and LAWRENCE 1978). Media ad methods for SUC2). SUPPRESSORS OF Snf2 IN YEAST 743 scoring ability to utilize carbon sources have been described (CARLSON, OSMOND and BOTSTEIN 198 1). Scoring for glucose, sucrose, raffinose and galactose utilization was carried out under anaerobic conditions in a Gas Pak disposable system (BBL) or by addition of antimycin A (Sigma) to the medium at a final concentration of 1 rg/ml. Except in the original isolation of mutants, all scoring was determined by spotting cell suspensions onto YEP plates containing the appropriate carbon source. Isolation of mutants: Single colonies (10' cells) were suspended in water and were spread onto rich medium (YEP) containing 2% raffinose as a carbon source. Cells were then exposed to 100 J/m' of ultraviolet radiation; 20% of the cells remained viable. Revertants were selected by incubating the plates anaerobically at 30" for 5 days. Revertants were recovered from seven snfll! single colonies and five snf5 single colonies; however, it is likely that all revertants were independent, because mutagenesis stimulated the frequency of reversion by more than 1000-fold. Putative mutants were purified by isolation of single colonies and were tested. Complementation analysis: To test pairs of mutations for complementation, heterozygous diploids were constructed and isolated by prototrophic selection. The ability of the diploid to utilize raffinose and/or grow at 37" was then determined. Construction of double mutants: Pairwise heterozygous diploids were constructed by selecting for prototrophy. Diploids were sporulated and four-spored asci were dissected. Complete tetrads were tested for genetic markers, as well as for carbon source utilization and ability to grow at 37". The snf genotypes of double mutants were confirmed by complementation analysis. For use in these crosses, a ssn20-6 SNF2 SUCP segregant lacking the SUC7 gene [that is, carrying the suc7 " allele (CARLSON and BOTSTEIN 1983)] was identified by blot hybridization analysis (SOUTHERN 1975); EcoRI restriction fragments characteristic of the SUC7 and suc7 " loci were detected (CARLSON, CELENZA and ENG 1985). Assay for invertase: Preparation of glucose-repressed and derepressed cells was as described by CELENZA and CARLSON (1 984). Repressed cells were grown to exponential phase (Klett=50) in YEP medium containing 2% glucose, and derepressed cells were prepared by shifting repressed cells to YEP medium containing 0.05% glucose for 2.5 hr. In the case of clumpy yeast cultures, cell density was determined by measuring dry weight as described by CARISON et al. (1984). Extracellular invertase activity was quantitatively assayed in whole cells using the method of COLDSTEIN and LAMPEN (1975), as described by CELENZA and CARLSON (1 984). Assay for &galactosidase: Cells carrying the GALIO-lac2 fusion plasmid PRY 123 (WEST, YOCUM and PTASHNE 1984) were grown in supplemented minimal medium (SD) with selection for the plasmid marker URA3. Galactose-induced cells were prepared by growth to exponential phase in medium containing 2% galactose and 3% glycerol, and repressed cells were prepared by growth in 2% glucose, 2% galactose and 3% glycerol. Cells were permeabilized by treatment with SDS and chloroform and were assayed for &galactosidase as described by MILLER (1 972).