Protein Kinase and Invertase Expression in Saccharomyces cerevisiae

The SNFl protein kinase and the associated SNF4 protein are required for release of glucose repression in Saccharomyces cereuisiae. To identify functionally related proteins, we selected genes that in multicopy suppress the raffinose growth defect of snf4 mutants. Among the nine genes recovered were two genes from the CAMP-dependent protein kinase (cAPK) pathway, MSZl and PDE2. Increased dosage of these genes partially compensates for defects in utrient utilization and sporulation in snfl and snf4 null mutants, but does not restore invertase expression. These results suggest that SNFl and cAPK affect some of the same cellular responses to nutrients. To examine the role of the cAPK pathway in regulation of invertase, we assayed mutants in which the cAPK is not modulated by CAMP. Expression of invertase was regulated in response to glucose and was dependent on SNFl function. Thus, a CAMP-responsive cAPK is dispensable for regulation of invertase. T HE yeast Saccharomyces cerevisiae prefers to utilize glucose as a carbon source. When glucose is plentiful, the cell represses the expression of genes that are involved in utilization of alternate carbon sources, a phenomenon known as glucose repression. This regulatory response is important to the cell and apparently involves many regulatory genes; however, the signaling pathway is not yet understood [for review see ENTIAN (1986), GANCEDO and GANCEDO (1 986) and CARLSON ( 1 987)]. One of the key genes is SNFI , which encodes a protein-serine/threonine kinase that is required for release of gene expression from glucose repression (CELENZA and CARLSON 1986). SNFl is the same gene as CAT1 and CCRl (CIRIACY 1977; DENIS 1984; ENTIAN and ZIMMERMANN 1982; SCHULLER and ENTIAN 1987). Mutations in SNFl cause defects in growth on carbon sources that are less preferred than glucose, general unhealthiness, and defects in sporulation of homozygous diploids (CARLSON, OSMOND and BOTSTEIN 198 1). While the SNFl kinase is clearly essential for the regulatory response to glucose, its exact role remains unclear. The SNFl kinase activity detected in vitro is not affected by the availability of glucose in the culture medium (CELENZA and CARLSON 1989); however, the physiologically relevant targets are as yet unidentified and it remains possible that their phosphorylation is regulated. Previously, we showed that the SNF4 gene (also known as CAT3; ENTIAN and ZIMMERMANN 1982; SCHULLER and ENTIAN 1988) encodes a protein that is functionally related to the SNFl kinase. The SNF4 protein is physically associated with the SNFl protein (kwetics 1 3 0 71-80 Uanuary, 1992) kinase and is required for maximal activity of the kinase in vitro, but SNF4 does not appear to convey regulatory signals (CELENZA and CARLSON 1989; CELENZA, ENG and CARLSON 1989; FIELDS and SONG 1989). Mutations in snf4 cause the same array of phenotypes as snfl, but are slightly less severe (NEIGEBORN and CARLSON 1984; CELENZA, ENG and CARLIn an effort to further our understanding of the regulatory pathway for the glucose response, we sought to identify other genes that encode proteins that are functionally related to the SNFl protein kinase. We selected for genes that in multicopy suppress the raffinose growth defect of a snf4 mutant. Although SNFl kinase activity is greatly reduced in a snf4 mutant, genetic and biochemical evidence indicates that some residual SNFl kinase activity remains (CELENZA and CARLSON 1989). We selected for suppression of reduced kinase activity, rather than no kinase activity, because we anticipated that a broader range of suppressor genes would be recovered. There are various possible mechanisms by which increased dosage of a gene could compensate for the snf4 defect. For example, the suppressor genes could encode additional activators of the kinase, substrates of the kinase, activators of a parai!el or partially redundant kinase pathway, or repressors of an antagonistic pathway. We report here that among the nine genes recovered using this strategy were two genes from the CAMP-dependent protein kinase (cAPK) pathway, MSII and PDE2. MSZI (also called J U N l ) was previously isolated as a multicopy suppressor of the heat SON 1989). 72 E. J. A. Hubbard, X. Yang and M. Carlson shock sensitive phenotype of iral and RAS2Va"9 mutants (NIKAWA, SASS and WICLER 1987; RUCCIERI et al. 1989), and PDE2 encodes a high-affinity cAMP phosphodiesterase (SASS et al. 1986; WILSON and TATCHELL 1988). T h e role of cAMP and cAPK in glucose repression in S. cerevisiae is problematic. cAMP does not function as a direct effector by a mechanism analogous to that in Escherichia coli (MATSUMOTO et al. 1982, 1983; ERASO and GANCEDO 1984). However, a RAS-dependent transient elevation in cAMP levels upon addition of glucose to glucose-starved cells has been documented (MBONYI et al. 1990, and references therein). Mutations in CYRl , the gene encoding adenylate cyclase, reduce expression of invertase and a-D-glucosidase (MATSUMOTO, UNO and ISHIKAWA 1984; SCHULTZ and CARLSON 1987). In contrast, mutants defective in BCYl , which encodes the CAMP-responsive negative regulatory subunit of cAPK, express invertase, galactokinase, and a-D-glucosidase at wildtype levels or a fewfold higher, and expression is still subject to glucose repression (MATSUMOTO et al. 1983; J. SCHULTZ and M. CARLSON, unpublished results). T h e cAPK pathway is known to affect expression of the glucose repressible gene ADH2 via phosphorylation of the transcriptional activator ADR 1 (BEMIS and DENIS 1988; CHERRY et al. 1989; TAYLOR and YOUNG 1990; THUKRAL et al. 1989); however, the data do not establish that this phosphorylation regulates glucose repression of ADH2. We have examined the ability of increased MSIl and PDE2 gene dosage to suppress various defects in both snf4 and snfl null mutants. We found that these multicopy suppressor genes did not restore invertase expression in response to glucose deprivation, but rather seemed to compensate for defects in nutrient utilization and sporulation. We also examined the effects of the cAPK pathway on regulation of invertase expression by using mutant strains in which cAPK activity is no longer responsive to the levels of CAMP; these strains lack BCYl and carry attenuating mutations in the genes encoding the catalytic subunits of cAPK (CAMERON et al. 1988). In these strains, expression of invertase was regulated in response to glucose and still dependent on SNFl function. MATERIALS AND METHODS Strains and general genetic methods: Strains of S. cerevisiae used in this study and their sources are listed in Table 1. MCY strains have the S288C genetic background except where other derivation is noted. Standard methods were used for genetic analysis (SHERMAN, FINK and LAWRENCE 1978) and transformation (ITO et al. 1983). Media contained 2% of the carbon source unless otherwise noted. Anaerobic growth was scored after incubation in a GasPak Disposable Anaerobic System (BBL). Isolation of multicopy suppressor plasmids: Strain MCY 1853 (snf4-A2 ura3) was transformed with a genomic library on the multicopy vector YEp24 (CARLSON and BOTSTEIN 1982). Approximately 24,000 Ura+ colonies were replica-plated onto supplemented synthetic medium (SHERMAN, FINK and LAWRENCE 1978) containing raffinose and lacking uracil (SR-Ura). The 476 Rap transformants were retested by spotting cell suspensions onto SR-Ura, and 170 colonies again scored Rap. We selected for further study 90 colonies that grew nearly as well as or better than MCY1853 carrying SNFl on a multicopy plasmid (pCE9; CELENZA and CARLSON 1989). Plasmid DNAs were recovered by passage through E. coli (HOFFMAN and WINSTON 1987). Five plasmids carrying SNF4 and 33 carrying SNFl were identified by diagnostic restriction digests and Southern blot analysis. Nine different plasmids, which conferred significant suppression upon retransformation of MCY1853, accounted for 37 of the remaining 52 plasmids. Plasmid constructions: DNA was manipulated and analyzed using standard methods (MANIATIS, FRITSCH and SAMBROOK 1982). pJHl0 contains the SalI/EcoRI fragment of pB37 cloned into pUC19 (YANISCH-PERRON, VIEIRA and MESSING 1985). pJH39 carries the 1.4-kb NruI fragment of pB37 cloned into the SmaI site of YEp24 (BOTSTEIN et al. 1979). To construct pJH44, pB37 was digested with XhoI plus BglII, the ends were filled in with Klenow fragment, and the vector-containing fragment was gel-purified and ligated. pJH46 and pJH49 contain the ClaI/SmaI and SalI/ XhoI fragments of pB37 cloned into the BamHI/SmaI and Sal1 sites of YEp24, respectively. pXY 1 contains the Sal1 to XbaI fragment of pB88 ligated to the large SalIINheI fragment of YEp24. pXY2 was constructed by cloning the XbaI/ SmaI fragment of pB88 into the NheIISmaI site of YEp24. pXY7 and pXY8 were constructed by cloning the SphI/ScaI and BamHIINheI fragments of pB88 between the SphI/SmaI and BamHIINheI sites of YEp24, respectively. Sequence analysis and computer methods: Restriction fragments were cloned into M 13 mp 18 or M 1 3 mpl9 (NORRANDER, KEMPE and MESSING 1983) and sequenced by the method of SANGER, NICKLEN and COULSON (1977) using Sequenase (U.S. Biochemical) and a 17-mer sequencing primer. Sequences were analyzed using DNA Strider (Commissariat a I'Energie Atomique-France) and the GCG package (DEVEREUX, HAEBERLI and SMITHIES 1984). Searches of reported sequences were made in the GenBank (BILOFSKY et al. 1986) and EMBL (HAMM and CAMERON 1986) databases using the GCG program TFASTA (PEARSON and LIPMAN 1988). Construction of the msil-Al::URA3 allele: First, the plasmid pJH5 1 was constructed by ligating the KpnIISphI fragment of pB37 to the KpnIISphI fragment of pUCl9. pJH5l was then cut with ClaI and BglII, filled in with Klenow, isolated, and ligated to a 1. l-kb SmaI fragment containing URA3 to obtain pJH52. The KpnI/SphI fragment of pJH52 was used to transform (ROTHSTEIN 1983) the diploid MCY 1093 X MCY 1094 to uracil prototrophy. The presence of the msil-Al::URA3 allele on one homologue of the diploid was confirmed by Southern blot analysis using probes prepared from pJH5 1. Construction of the snfl-l5::LEU2 allele: pJH80 was constructed by inserting the BglII fragment containing the LEU2 gene into the BglII site of the SNFl gene in pCC107 u. CELENZA and M. CARLSON, unpublished results). pCC107 contains SNFl on a HincIIBamHI partial fragment cloned in pUC18. The BamHI/SphI fragment of JH80 was used to transform SP1, TF1 .5prFHR, TF1.5prC H f and MCY2372. This results in a disruption of SNFl at codon 175 (CELENZA and CARLSON 1986). Southern blot and Northern blot analysis: Standard methods were used for preparation and analysis of genomic Yeast cAPK Pathway and Invertase 73