Sucz Gene Expression by Glucose Repression in Saccharomyces Cerevisiae

Mutants of Saccharomyces cereoisiae with defects in sucrose or raffinose fermentation were isolated. In addition to mutations in the SUCZ structural gene for invertase, we recovered 18 recessive mutations that affected the regulation of invertase synthesis by glucose repression. These mutations included five new snfl (Sucrose ponfermenting) alleles and also defined five new complementation groups, designated snj2, snf3, snf4, snf5 and snf6. The snj2, snf4 and snf5 mutants produced little or no secreted invertase under derepressing conditions and were pleiotropically defective in galactose and glycerol utilization, which are both regulated by glucose repression. The snf6 mutant produced low levels of secreted invertase under derepressing conditions, and no pleiotropy was detected. The snf3 mutants derepressed secreted invertase to 10-35% the wildtype level but grew less well on sucrose than expected from their invertase activity; in addition, snf3 mutants synthesized some invertase under glucoserepressing conditions.-We examined the interactions between the different snf mutations and ssn6, a mutation causing constitutive (glucose-insensitive) high-level invertase synthesis that was previously isolated as a suppressor of snfl. The ssn6 mutation completely suppressed the defects in derepression of invertase conferred by snfl, snf3, snf4 and snf6, and each double mutant showed the constitutivity for invertase typical of ssn6 single mutants. In contrast, snj2 ssn6 and snf5 ssn6 strains produced only moderate levels of invertase under derepressing conditions and very low levels under repressing conditions. These findings suggest roles for the SNFl through SNF6 and SSN6 genes in the regulation of SUC2 gene expression by glucose repression. LUCOSE (carbon catabolite) repression is a general regulatory system in G Saccharomyces cerevisiae that affects the expression of a multitude of genes. The SUC2 structural gene for invertase provides an attractive system for studying glucose repression because glucose repression appears to be the only regulatory mechanism affecting expression of SUC.2. S. cerevisiae is able to utilize sucrose as a carbon source by derepressing synthesis of invertase, which cleaves sucrose to yield glucose and fructose. The SUC2 gene is the most extensively studied member of the SUC gene family (SUCl-SUCS and SUC7; MORTIMER and HAWTHORNE 1969; CARLSON and BOTSTEIN 1983); an individual haploid strain may contain zero, one or several SUC Genetics 108: 845-858 December, 1984. 846 L. NEIGEBORN AND M. CARLSON genes in its genome. Each SUC gene encodes two forms of invertase: a glycosylated form that is secreted into the periplasmic space and an intracellular, nonglycosylated form (NEUMANN and LAMPEN 1967; GASCON and LAMPEN 1968; CARLSON and BOTSTEIN 1982; CARLSON et al. 1983). The secreted enzyme is responsible for the utilization of sucrose; the in vivo function of the intracellular species is unclear. Synthesis of the secreted enzyme is regulated by glucose repression, and the intracellular enzyme is synthesized constitutively at a low level. The secreted and cytoplasmic invertases are encoded by two differently regulated SUC2 mRNAs (CARLSON and BOTSTEIN 1982). The secreted invertase is encoded by a 1.9-kb mRNA, the stable level of which is regulated by glucose repression. The cytoplasmic enzyme is translated from a 1.8-kb mRNA which is synthesized constitutively at a low level. These two mRNAs differ at their 5’ ends; the 1.9-kb species includes a signal peptide-coding sequence and, therefore, encodes a secreted form of invertase (CARLSON et al. 1983; PERLMAN, HALVORSON and CANNON 1982). Mutations preventing the expression of the SUC2 gene have been previously isolated. The snfl (Sucrose-nonferment.ing) mutations abolish derepression of secreted invertase synthesis but do not affect synthesis of the cytoplasmic enzyme (CARLSON, OSMOND and BOTSTEIN 1981). The defect in invertase synthesis probably lies at the transcriptional level; no stable 1.9-kb mRNA is synthesized in snfl mutants (CARLSON and BOTSTEIN 1982). The SNFl gene is also required to derepress expression of other glucose-repressible genes, and snfl mutants are deficient in growth on other carbon sources, the utilization of which is regulated by glucose repression (CARLSON, OSMOND and BOTSTEIN 1981). The SNFl gene has been cloned and genetically mapped to a position distal to m a 3 on chromosome ZV (CELENZA and CARLSON 1984a). Null mutations constructed at the chromosomal SNFl locus conferred the expected S n f phenotype. The gene encodes a 2.4-kb polyadenylated mRNA that is present in both glucose-repressed and -derepressed cells (CELENZA and CARLSON 1984b). Mutations causing constitutive synthesis of secreted invertase have also been described. The ssnb mutations were isolated as suppressors of a snfl mutation which restored capacity for growth on sucrose but not galactose or glycerol (CARLSON et al. 1984). Expression of the SUC2 gene was found to be resistant to glucose repression in either snfl ssnb or SNFl ssn6 strains, and secreted invertase was synthesized at levels as high as that of a derepressed wild-type strain. The ssnb mutations confer pleiotropic defects and are allelic to cyc8, a mutation causing overproduction of iso-2-cytochrome c (ROTHSTEIN and SHERMutations in hxk2, the structural gene for hexokinase PI1 (or B) also result in glucose-insensitive synthesis of secreted invertase, maltase, galactokinase and other enzymes, and it has been suggested that this effect is due not merely to a decreased rate of glucose metabolism, but rather to a defect in a regulatory function performed by this hexokinase (ENTIAN 1980; ENTIAN and MECKE 1982; MICHELS, HAHNENBERGER and SYLVESTRE 1983). MATSUMOTO, YOSHIMAN 1980). REGULATION OF SUCZ GENE EXPRESSION 847 MATSU and OSHIMA (1983) have isolated mutations at the REG1 locus that affect the glucose repressibility of galactokinase synthesis and cause some constitutive production of secreted invertase. The hex2 and cat80 mutations described by ENTIAN and ZIMMERMANN (1980) also cause constitutive invertase synthesis; hex2 and reg1 both map near the tr$l gene (ENTIAN and ZIMMERWe report here the isolation of additional mutations that affect the regulation of SUC2 gene expression. These mutations define five new complementation groups. We have examined the interactions of these new mutations with each other and with the snfl and ssnb mutations. MANN 1982). MATERIALS AND METHODS Yeast strains: All strains used in this study were isogenic or congenic to strain S288C (MATa SUC2 ga12), except where noted otherwise. The origins of snfl, suc2, a d d , his4, lys2, GAL2 and MATa alleles have been described previously (CARLSON, OSMOND and BOTSTEIN 1981). The ura35 2 allele was serially backcrossed into the S288C background as described by CARLSON et al. (1984). SUC7 was introduced into the S288C background from strain FLlOO (LACROUTE 1968) through a series of ten backcrosses. The h d 2 2 mutation was introduced into our strains from strain F445 (hxkl -I hxk2-2) by three serial backcrosses; segregants of genotype HXKl hxk2 were identified by their ability to utilize fructose and to secrete invertase constitutively. The genotypes and sources of strains used in this study are listed in Table 1. Genetic methods: Standard genetic procedures of crossing, sporulation and tetrad analysis were followed (MORTIMER and HAWTHORNE 1969; SHERMAN, FINK and LAWRENCE 1978). Media and methods for scoring ability to utilize carbon sources have been described (CARLSON, OSMOND and BOTSTEIN 1981). As before, scoring for glucose, sucrose, raffinose and galactose utilization was carried out under anaerobic conditions in a GasPak disposable anaerobic system (BBL). 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: Yeast cells were mutagenized with 3% ethyl methanesulfonate as described by CARLSON, OSMOND and BOTSTEIN (1981). As before, cells were stored under conditions nonpermissive for growth prior to plating for single colonies. This precaution was taken to ensure the independence of mutants recovered in a single experiment. Surviving cells were plated for single colonies on YEP-glucose and replica plated to YEP-sucrose in the experiments with strains MCY259, MCY517 and MCY527 and YEP-raffinose medium in those with strains MCY520 and DBY782. Raffinose is a poorer substrate than sucrose for invertase, and ability to utilize raffinose proved to be a more sensitive indicator for reduced levels of secreted invertase. Putative mutants were purified and retested for ability to ferment sucrose and raffinose by spotting cell suspensions. Complementation analysis: T o test pairs of mutations for complementation, heterozygous diploids were constructed and isolated, in most cases, by prototrophic selection; when prototrophic selection could not be employed, diploids were identified following single-colony purification by testing ability to sporulate. The ability of the diploid to utilize various carbon sources was then analyzed. Zdentzfcation of nonsense mutations: Each of the snj2 through snf6 mutations was tested for coreversion with the his4-539 and/or lys2-801 amber alleles by first plating each mutant on medium selective for reversion to prototrophy and then testing revertants for growth on sucrose and raffinose. The snf4-319 and snf5-Z8 mutations reverted simultaneously with amber markers. Corevertants were crossed to SNF strains carrying amber alleles for tetrad analysis; the phenotypic segregations observed were consistent with the segregation of an amber suppressor able to suppress the snf mutation. The snj2-141 allele was observed to corevert frequently with the ade2-lOl ochre mutation. Such a corevertant was crossed to a SNF2 strain carrying the hid-86 and lys2-802 ochre alleles. Tetrad analysis of this diploid indicated that snj2-141 is an ochre mutation. Assays for invertase: Preparation of glucose-repressed and -derepressed cells was as described by 848 L. NEIGEBORN AND M. CARLSON