Abstract
To clarify the transcriptional regulation by nutrient limitation of the gene encoding carboxypeptidase yscS in Saccharomyces cerevisiae (CPS1), we performed an analysis of its 5′ noncoding region. In deletion experiments a sequence located between positions −644 and −591 was found to be responsible for transcriptional repression of the CPS1 gene in yeast cells grown on rich nitrogen sources. Furthermore, a 162 bp fragment spanning positions −644 to −482 of the promoter of the CPS1 gene repressed gene expression when placed 3′ to the upstream activation sequence (UAS) of the heterologous gene CYC1. A fragment containing this putative upstream repression sequence (URS) was shown specifically to bind protein from a yeast extract as demonstrated by gel retardation experiments. Although a sequence mediating the control of gene expression by GCN4 was found within the URS element, the GCN4 gene product is not required for DNA-binding activity. In addition, at least three other upstream activation UASs responsible for the activation of CPS1 expression by glucose under nitrogen starvation conditions were found to be located between positions −673 and −644, −482 and −353, and −243 and −186, respectively. The putative mechanism of the nitrogen limitation-dependent regulation of CPS1 expression via these regulatory elements is discussed.
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Ammerer G, Hunter CP, Rothman JH, Saari GC, Valls LA, Stevens TH (1986) PEP4 gene of Saccharomyces cerevisiae encodes proteinase A, a vacuolar enzyme required for processing of vacuolar precursors. Mol. Cell. Biol. 6:2490–2499
Ares M, Mangin M, Weiner AM (1985) Orientation-dependent transcriptional activator upstream of a human U2 snRNA gene. Mol Cell Biol 5:1560–1570
Bankaitis VA, Johnson LM, Emr SD (1986) Isolation of yeast mutants defective in protein targeting to the vacuole. Proc Natl Acad Sci USA 83:9075–9079
Banta LM, Robinson JS, Klionsky DJ, Emr SD (1988) Organelle assembly in yeast: characterization of yeast mutants defective in vacuolar biogenesis and protein sorting. J. Cell. Biol. 107:1369–1383
Bergman Y, Rice D, Grosschedl R, Baltimore D (1984) Two regulatory elements for immunoglobulin κ light chain gene expression. Proc. Natl. Acad. Sci. USA 81:7041–7045
Birnboim HC, Doly J (1979) A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic. Acid Res. 7:1513–1523
Bordallo J, Bordallo C, Gascón S, Suárez-Rendueles P (1991) Molecular cloning and sequencing of genomic DNA encoding yeast vacuolar carboxypeptidase yscS. FEBS Lett. 283:27–32
Bordallo J, Suárez-Rendueles P (1993) Control of Saccharomyces cerevisiae Carboxypeptidase S (CPS1) gene expression under nutrient limitation. Yeast 9:339–349
Bullock WO, Fernandez JM, Short JM (1987) XL1 Blue: a high efficiency plasmid transforming rec E. coli strain with β-galactosidase selection. Biotechniques 5:376–379
Cobianchi F, Wilson SH (1987) Enzymes for modifying and labelling DNA and RNA. Methods Enzymol. 152:94–110
Courtois G, Baumhueter S, Crabtree GR (1988) Purified hepatocyte nuclear factor 1 interacts with a family of hepatocytespecific promoters. Proc Natl Acad Sci USA 85:7937–7941
Chang Y-H, Smith JA (1989) Molecular cloning and sequencing of genomic DNA encoding aminopeptidase I from Saccharomyces cerevisiae. J Biol Chem 264:6979–6983
Cueva R, Garcia-Alvarez N, Suárez-Rendueles P (1989) Yeast vacuolar aminopeptidase yscI. Isolation and regulation of the APE1 (LAP4) structural gene. FEBS Lett 259:125–129
Falkner FG, Zachau HG (1984) Correct transcription of an immunoglobulin k gene requires an upstream fragment containing conserved sequence elements. Nature 310:71–74
Germino J, Gray JG, Charbonneau H, Vanaman T, Bastia D (1983) Use of gene fusions and protein interaction in the isolation of a biologically active regulatory protein: the replication initiator of plasmid R6 K. Proc Natl Acad Sci USA 80:6848–6852
Gershoni JM, Palade GE (1983) Protein blotting: principles and applications. Anal Biochem 131:1–15
Guarente L (1983) Yeast promoters and lacZ fusions designed to study expression of cloned genes in yeast. Methods Enzymol 101:181–191
Guarente L (1992) Messenger RNA transcription and its control in Saccharomyces cerevisiae. In: Broach JR, Pringle JR, Jones EW (eds) The molecular and cellular biology of the yeast Saccharomyces. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York
Herman PK, Stack JH, Emr SD (1991) A genetic and structural analysis of the yeast Vps15 protein kinase: evidence for a direct role of Vps15 in vacuolar protein delivery. EMBO J. 10:4049–4060
Hinnebusch AG (1988) Mechanisms of gene regulation in the general control of amino acid biosynthesis in Saccharomyces cerevisiae. Microbiol Rev 52:248
Hinnebusch AG (1992) General and pathway-specific regulatory mechanisms controlling the synthesis of amino acid biosynthetic enzymes in Saccharomyces cerevisiae. In: Broach JR, Pringle JR, Jones EW (eds) The Molecular and Cellular Biology of the Yeast Saccharomyces. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York
Hinnebusch AG, Fink GR (1983) Positive regulation in the general amino acid control of Saccharomyces cerevisiae. Proc Natl Acad Sci USA 80:5374
Ito H, Fukuda Y, Murata K, Kimura A (1983) Transformation of intact yeast cells treated with alkali cations. J Bacteriol 153:163–168
Klebe RJ, Harris JV, Sharp ZD, Douglas MG (1983) A general method for polyethylene-glycol induced genetic transformation of bacteria and yeast. Gene 25:333–341
Klionsky DJ, Emr SD (1990) A new class of lysosomal/vacuolar protein sorting signal. J Biol Chem 265:5349–5352
Klionsky DJ, Herman PK, Emr SD (1990) The fungal vacuole: composition, function and biogenesis. Microbiol Rev 54:266–292
Klionsky DJ, Cueva R, Yaver D (1992) Aminopeptidase I of Saccharomyces cerevisiaeis localized to the vacuole independent of the secretory pathway. J Cell Biol 119:287–299
Kovari L, Sumrada R, Kovari I, Cooper TG (1990) Multiple positive and negative cis acting elements mediated induced argmase (CAR1) gene expression in Saccharomyces cerevisiae. Mol Cell Biol 10:5087–5097
Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193:265–275
Luche RM, Sumrada R, Cooper TG (1990) A cis-acting element present in multiple genes serves as a repressor protein binding site for the yeast CAR1 gene. Mol Cell Biol 10:3884–3895
Mechler B, Müller H, and Wolf DH (1987) Maturation of vacuolar (lysosomal) enzymes in yeast: proteinase yscA and proteinase yscB are catalysts of the processing and activation event of carboxypeptidase yscY. EMBO J 6:2157–2163
Mechler B, Hirsch HH, Müller H, Wolf DH (1988) Biogenesis of the yeast lysosome (vacuole): biosynthesis and maturation of proteinase yscB. EMBO J 7:1705–1710
Miller JH (1972) Experiments in molecular genetics, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, p. 403
Moehle CM, Tizard R, Lemmon SK, Smart J, Jones EW (1987) Protease B of the lysosome-like vacuole of the yeast Saccharomyces cerevisiae is homologous to the subtilisin family of serene proteases. Mol Cell Biol 7:4390–4399
Moehle CM, Dixon CK, Jones EW (1989) Processing pathway for protease B of Saccharomyces cerevisiae J Cell Biol 108:39–55
Myers AL, Tzagoloff A, Kinney DM, Lusty CJ (1986) Yeast shuttle and integrative vectors with multiple cloning sites for construction of lacZ fusions. Gene 45:299–310
Nishizawa M, Araki R, Teranishi Y (1989) Identification of an upstream activating sequence and an upstream repressible sequence of the pyruvate kinase gene of the yeast Saccharomyces cerevisiae. Mol Cell Biol 9:442–451
Parslow TG, Blair DL, Murphy WJ, Granner DK (1984) Structure of the 5'-ends of immunoglobulin genes: a novel conserved sequence. ProcNatl Acad Sci USA 81:2650–2654
Roberts CJ, Pohlig G, Rothman JH, Stevens TH (1989) Structure, biosynthesis and localization of dipeptidyl aminopeptidase B, an integral membrane glycoprotein of the yeast vacuole. J Cell Biol 108:1363–1373
Rüther U, Müller-Hill B (1983) Easy identification of cDNA clones. EMBO J 2:1791–1794
Spormann DO, Heim J, Wolf DH (1992) Biogenesis of the yeast vacuole. The precursor forms of the soluble hydrolase carboxypeptidase yscS are associated with the vacuolar membrane. J Biol Chem 267:8021–8029
Stevens TH, Rothman JH, Payne GS, Schekman R (1986) Gene dosage dependent secretion of yeast vacuolar carboxypeptidase Y. J Cell Biol 102:1551–1557
Struhl K (1987) Promoters, activator proteins and the mechanism of transcriptional initiation in yeast. Cell 49:295–297
Suárez-Rendueles P, Wolf DH (1987) Identification of the structural gene for dipeptidyl aminopeptidase yscY (DAP2) in Saccharomyces cerevisiae. J Bacteriol 169:4041–4048
Suárez-Rendueles P, Wolf DH (1988) Proteinase function in yeast: Biochemical and genetic approaches to a central mechanism of post-translational control in the eukaryote cell. FEMS Microbiol Rev 54:17–46
Sumrada R, Cooper TG (1987) Ubiquitous upstream repression sequences control activation of the inducible arginase gene in yeast. Proc Natl Acad Sci USA 84:3997–4001
Tabor S, Richardson CC (1987) DNA sequence analysis with a modified bacteriophage T7 DNA polymerise. Proc Natl Acad Sci USA 84:4767–4771
Teichert T, Mechler B, Müller H, Wolf DH (1989) Lysosomal (vacuolar) proteinases of yeast are essential catalysts for protein degradation, differentiation and cell survival. J Biol Chem 264:16037–16045
Towbin H, Staehelin T, Gordon J (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets procedure and some applications. Proc Natl Acad Sci USA 76:4350–4354
Tso JY, Sun XH, Wu R (1985) Structure of the unlinked Drosophila melanogaster. Glyceraldehyde 3-phosphate dehydrogenase genes. J Biol Chem 260:8220–8228
Valls LA, Hunter CP, Rothman JH, Stevens TH (1987) Protein sorting in yeast: the localization determinant of yeast vacuolar carboxypeptidase Y resides in the propeptide. Cell 48:887–897
West RW, Yocum RR, Ptashne M (1984) Saccharomyces cerevisiae GAL1-GAL10 divergent promoter region: location and function of the upstream activating sequences UASG. Mol Cell Biol 4:2467–2478
Westin G, Lund E, Murphy JT, Petterson U, Dahlberg JE (1984) Human U2 and Ul RNA genes use similar transcription signals. EMBO J 3:3295–3301
Wolf DH, Ehmann C (1978) Carboxypeptidase S from yeast: regulation of its activity during vegetative growth and differentiation. FEBS Lett. 91:59–62
Wolf DH, Ehmann C (1981) Carboxypeptidase Y and carboxypeptidase S-deficient mutants of Saccharomyces cerevisiae. J Bacteriol 147:418–426
Woolford CA, Daniels LB, Park FJ, Jones EW, van Arsdell JN, Innis MA. (1986) The PEP4 gene encodes an aspartyl protease implicated in the post-translational regulation of Saccharomyces cerevisiae vacuolar hydrolases. Mol Cell Biol 6:2500–2510
Yanisch-Perron C, Vieira J, Messing J (1985) Improved M13 phage cloning vectors and host strains: nucleotide sequences of M13mpl8 and pUC19 vectors. Gene 33:103–119
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Communicated by C.P. Hollenberg.
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Bordallo, J., Suárez-Rendueles, P. Cis and trans-acting regulatory elements required for regulation of the CPS1 gene in Saccharomyces cerevisiae . Molec. Gen. Genet. 246, 580–589 (1995). https://doi.org/10.1007/BF00298964
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DOI: https://doi.org/10.1007/BF00298964