PMCCPMCCPMCC

Search tips
Search criteria 

Advanced

 
Logo of molcellbPermissionsJournals.ASM.orgJournalMCB ArticleJournal InfoAuthorsReviewers
 
Mol Cell Biol. 1986 July; 6(7): 2500–2510.
PMCID: PMC367804

The PEP4 gene encodes an aspartyl protease implicated in the posttranslational regulation of Saccharomyces cerevisiae vacuolar hydrolases.

Abstract

pep4 mutants of Saccharomyces cerevisiae accumulate inactive precursors of vacuolar hydrolases. The PEP4 gene was isolated from a genomic DNA library by complementation of the pep4-3 mutation. Deletion analysis localized the complementing activity to a 1.5-kilobase pair EcoRI-XhoI restriction enzyme fragment. This fragment was used to identify an 1,800-nucleotide mRNA capable of directing the synthesis of a 44,000-dalton polypeptide. Southern blot analysis of yeast genomic DNA showed that the PEP4 gene is unique; however, several related sequences exist in yeasts. Tetrad analysis and mitotic recombination experiments localized the PEP4 gene proximal to GAL4 on chromosome XVI. Analysis of the DNA sequence indicated that PEP4 encodes a polypeptide with extensive homology to the aspartyl protease family. A comparison of the PEP4 predicted amino acid sequence with the yeast protease A protein sequence revealed that the two genes are, in fact, identical (see also Ammerer et al., Mol. Cell. Biol. 6:2490-2499, 1986). Based on our observations, we propose a model whereby inactive precursor molecules produced from the PEP4 gene self-activate within the yeast vacuole and subsequently activate other vacuolar hydrolases.

Full text

Full text is available as a scanned copy of the original print version. Get a printable copy (PDF file) of the complete article (2.1M), or click on a page image below to browse page by page. Links to PubMed are also available for Selected References.

Images in this article

Click on the image to see a larger version.

Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Baudys M, Kostka V. Covalent structure of chicken pepsinogen. Eur J Biochem. 1983 Oct 17;136(1):89–99. [PubMed]
  • Birnboim HC, Doly J. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 1979 Nov 24;7(6):1513–1523. [PMC free article] [PubMed]
  • Blundell T, Sibanda BL, Pearl L. Three-dimensional structure, specificity and catalytic mechanism of renin. Nature. 1983 Jul 21;304(5923):273–275. [PubMed]
  • Botstein D, Falco SC, Stewart SE, Brennan M, Scherer S, Stinchcomb DT, Struhl K, Davis RW. Sterile host yeasts (SHY): a eukaryotic system of biological containment for recombinant DNA experiments. Gene. 1979 Dec;8(1):17–24. [PubMed]
  • Burnette WN. "Western blotting": electrophoretic transfer of proteins from sodium dodecyl sulfate--polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A. Anal Biochem. 1981 Apr;112(2):195–203. [PubMed]
  • Carlson M, Botstein D. Two differentially regulated mRNAs with different 5' ends encode secreted with intracellular forms of yeast invertase. Cell. 1982 Jan;28(1):145–154. [PubMed]
  • Chen HR, Dayhoff MO, Barker WC, Hunt LT, Yeh LS, George DG, Orcutt BC. Nucleic acid sequence database IV. DNA. 1982;1(4):365–374. [PubMed]
  • Chou PY, Fasman GD. Prediction of protein conformation. Biochemistry. 1974 Jan 15;13(2):222–245. [PubMed]
  • Davis RW, Thomas M, Cameron J, St John TP, Scherer S, Padgett RA. Rapid DNA isolations for enzymatic and hybridization analysis. Methods Enzymol. 1980;65(1):404–411. [PubMed]
  • Devenish RJ, Newlon CS. Isolation and characterization of yeast ring chromosome III by a method applicable to other circular DNAs. Gene. 1982 Jun;18(3):277–288. [PubMed]
  • Distel B, Al EJ, Tabak HF, Jones EW. Synthesis and maturation of the yeast vacuolar enzymes carboxypeptidase Y and aminopeptidase I. Biochim Biophys Acta. 1983 Oct 13;741(1):128–135. [PubMed]
  • Emr SD, Schekman R, Flessel MC, Thorner J. An MF alpha 1-SUC2 (alpha-factor-invertase) gene fusion for study of protein localization and gene expression in yeast. Proc Natl Acad Sci U S A. 1983 Dec;80(23):7080–7084. [PubMed]
  • Falco SC, Li Y, Broach JR, Botstein D. Genetic properties of chromosomally integrated 2 mu plasmid DNA in yeast. Cell. 1982 Jun;29(2):573–584. [PubMed]
  • Faust PL, Kornfeld S, Chirgwin JM. Cloning and sequence analysis of cDNA for human cathepsin D. Proc Natl Acad Sci U S A. 1985 Aug;82(15):4910–4914. [PubMed]
  • Harboe MK, Foltmann B. Bovine pepsin: the sequence of the first 65 amino acid residues (completing the sequence of the first 110 residues of bovine pepsinogen). FEBS Lett. 1975 Dec 1;60(1):133–136. [PubMed]
  • Harris TJ, Lowe PA, Lyons A, Thomas PG, Eaton MA, Millican TA, Patel TP, Bose CC, Carey NH, Doel MT. Molecular cloning and nucleotide sequence of cDNA coding for calf preprochymosin. Nucleic Acids Res. 1982 Apr 10;10(7):2177–2187. [PMC free article] [PubMed]
  • Hasilik A, Tanner W. Carbohydrate moiety of carboxypeptidase Y and perturbation of its biosynthesis. Eur J Biochem. 1978 Nov 15;91(2):567–575. [PubMed]
  • Hawthorne DC, Mortimer RK. Chromosome Mapping in Saccharomyces: Centromere-Linked Genes. Genetics. 1960 Aug;45(8):1085–1110. [PubMed]
  • Hemmings BA, Zubenko GS, Hasilik A, Jones EW. Mutant defective in processing of an enzyme located in the lysosome-like vacuole of Saccharomyces cerevisiae. Proc Natl Acad Sci U S A. 1981 Jan;78(1):435–439. [PubMed]
  • Hereford LM, Rosbash M. Number and distribution of polyadenylated RNA sequences in yeast. Cell. 1977 Mar;10(3):453–462. [PubMed]
  • Hsiao CL, Carbon J. High-frequency transformation of yeast by plasmids containing the cloned yeast ARG4 gene. Proc Natl Acad Sci U S A. 1979 Aug;76(8):3829–3833. [PubMed]
  • Ito H, Fukuda Y, Murata K, Kimura A. Transformation of intact yeast cells treated with alkali cations. J Bacteriol. 1983 Jan;153(1):163–168. [PMC free article] [PubMed]
  • James MN, Hsu IN, Delbaere LT. Mechanism of acid protease catalysis based on the crystal structure of penicillopepsin. Nature. 1977 Jun 30;267(5614):808–813. [PubMed]
  • James MN, Sielecki AR. Stereochemical analysis of peptide bond hydrolysis catalyzed by the aspartic proteinase penicillopepsin. Biochemistry. 1985 Jul 2;24(14):3701–3713. [PubMed]
  • James MN, Sielecki AR. Molecular structure of an aspartic proteinase zymogen, porcine pepsinogen, at 1.8 A resolution. Nature. 1986 Jan 2;319(6048):33–38. [PubMed]
  • Johnston SA, Hopper JE. Isolation of the yeast regulatory gene GAL4 and analysis of its dosage effects on the galactose/melibiose regulon. Proc Natl Acad Sci U S A. 1982 Nov;79(22):6971–6975. [PubMed]
  • Jones EW. Proteinase mutants of Saccharomyces cerevisiae. Genetics. 1977 Jan;85(1):23–33. [PubMed]
  • Jones EW. The synthesis and function of proteases in Saccharomyces: genetic approaches. Annu Rev Genet. 1984;18:233–270. [PubMed]
  • Jones EW, Zubenko GS, Parker RR. PEP4 gene function is required for expression of several vacuolar hydrolases in Saccharomyces cerevisiae. Genetics. 1982 Dec;102(4):665–677. [PubMed]
  • Kaneko Y, Tamai Y, Toh-e A, Oshima Y. Transcriptional and post-transcriptional control of PHO8 expression by PHO regulatory genes in Saccharomyces cerevisiae. Mol Cell Biol. 1985 Jan;5(1):248–252. [PMC free article] [PubMed]
  • Kaneko Y, Toh-e A, Oshima Y. Identification of the genetic locus for the structural gene and a new regulatory gene for the synthesis of repressible alkaline phosphatase in Saccharomyces cerevisiae. Mol Cell Biol. 1982 Feb;2(2):127–137. [PMC free article] [PubMed]
  • Kuo CL, Campbell JL. Cloning of Saccharomyces cerevisiae DNA replication genes: isolation of the CDC8 gene and two genes that compensate for the cdc8-1 mutation. Mol Cell Biol. 1983 Oct;3(10):1730–1737. [PMC free article] [PubMed]
  • Kyte J, Doolittle RF. A simple method for displaying the hydropathic character of a protein. J Mol Biol. 1982 May 5;157(1):105–132. [PubMed]
  • Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. [PubMed]
  • Larkin JC, Woolford JL., Jr Molecular cloning and analysis of the CRY1 gene: a yeast ribosomal protein gene. Nucleic Acids Res. 1983 Jan 25;11(2):403–420. [PMC free article] [PubMed]
  • Last RL, Stavenhagen JB, Woolford JL., Jr Isolation and characterization of the RNA2, RNA3, and RNA11 genes of Saccharomyces cerevisiae. Mol Cell Biol. 1984 Nov;4(11):2396–2405. [PMC free article] [PubMed]
  • Laughon A, Gesteland RF. Isolation and preliminary characterization of the GAL4 gene, a positive regulator of transcription in yeast. Proc Natl Acad Sci U S A. 1982 Nov;79(22):6827–6831. [PubMed]
  • Mechler B, Müller M, Müller H, Meussdoerffer F, Wolf DH. In vivo biosynthesis of the vacuolar proteinases A and B in the yeast Saccharomyces cerevisiae. J Biol Chem. 1982 Oct 10;257(19):11203–11206. [PubMed]
  • Mechler B, Müller M, Müller H, Wolf DH. In vivo biosynthesis of vacuolar proteinases in proteinase mutants of Saccharomyces cerevisiae. Biochem Biophys Res Commun. 1982 Aug;107(3):770–778. [PubMed]
  • Mechler B, Wolf DH. Analysis of proteinase A function in yeast. Eur J Biochem. 1981 Dec;121(1):47–52. [PubMed]
  • Messenguy F, Colin D, ten Have JP. Regulation of compartmentation of amino acid pools in Saccharomyces cerevisiae and its effects on metabolic control. Eur J Biochem. 1980 Jul;108(2):439–447. [PubMed]
  • Miyazaki H, Fukamizu A, Hirose S, Hayashi T, Hori H, Ohkubo H, Nakanishi S, Murakami K. Structure of the human renin gene. Proc Natl Acad Sci U S A. 1984 Oct;81(19):5999–6003. [PubMed]
  • Müller M, Müller H. Synthesis and processing of in vitro and in vivo precursors of the vacuolar yeast enzyme carboxypeptidase Y. J Biol Chem. 1981 Dec 10;256(23):11962–11965. [PubMed]
  • Navon G, Shulman RG, Yamane T, Eccleshall TR, Lam KB, Baronofsky JJ, Marmur J. Phosphorus-31 nuclear magnetic resonance studies of wild-type and glycolytic pathway mutants of Saccharomyces cerevisiae. Biochemistry. 1979 Oct 16;18(21):4487–4499. [PubMed]
  • Orr-Weaver TL, Szostak JW, Rothstein RJ. Yeast transformation: a model system for the study of recombination. Proc Natl Acad Sci U S A. 1981 Oct;78(10):6354–6358. [PubMed]
  • Panthier JJ, Foote S, Chambraud B, Strosberg AD, Corvol P, Rougeon F. Complete amino acid sequence and maturation of the mouse submaxillary gland renin precursor. Nature. 1982 Jul 1;298(5869):90–92. [PubMed]
  • Petes TD, Broach JR, Wensink PC, Hereford LM, Fink GR, Botstein D. Isolation and analysis of recombinant DNA molecules containing yeast DNA. Gene. 1978 Sep;4(1):37–49. [PubMed]
  • Ricciardi RP, Miller JS, Roberts BE. Purification and mapping of specific mRNAs by hybridization-selection and cell-free translation. Proc Natl Acad Sci U S A. 1979 Oct;76(10):4927–4931. [PubMed]
  • Salhany JM, Yamane T, Shulman RG, Ogawa S. High resolution 31P nuclear magnetic resonance studies of intact yeast cells. Proc Natl Acad Sci U S A. 1975 Dec;72(12):4966–4970. [PubMed]
  • Sanger F, Nicklen S, Coulson AR. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. [PubMed]
  • Sepulveda P, Marciniszyn J, Jr, Liu D, Tang J. Primary structure of porcine pepsin. III. Amino acid sequence of a cyanogen bromide fragment, CB2A, and the complete structure of porcine pepsin. J Biol Chem. 1975 Jul 10;250(13):5082–5088. [PubMed]
  • Shewale JG, Tang J. Amino acid sequence of porcine spleen cathepsin D. Proc Natl Acad Sci U S A. 1984 Jun;81(12):3703–3707. [PubMed]
  • Shortle D, Haber JE, Botstein D. Lethal disruption of the yeast actin gene by integrative DNA transformation. Science. 1982 Jul 23;217(4557):371–373. [PubMed]
  • Sogawa K, Fujii-Kuriyama Y, Mizukami Y, Ichihara Y, Takahashi K. Primary structure of human pepsinogen gene. J Biol Chem. 1983 Apr 25;258(8):5306–5311. [PubMed]
  • Southern EM. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol. 1975 Nov 5;98(3):503–517. [PubMed]
  • Stevens T, Esmon B, Schekman R. Early stages in the yeast secretory pathway are required for transport of carboxypeptidase Y to the vacuole. Cell. 1982 Sep;30(2):439–448. [PubMed]
  • Trumbly RJ, Bradley G. Isolation and characterization of aminopeptidase mutants of Saccharomyces cerevisiae. J Bacteriol. 1983 Oct;156(1):36–48. [PMC free article] [PubMed]
  • von Heijne G. Signal sequences. The limits of variation. J Mol Biol. 1985 Jul 5;184(1):99–105. [PubMed]
  • Wiemken A, Dürr M. Characterization of amino acid pools in the vacuolar compartment of Saccharomyces cerevisiae. Arch Microbiol. 1974;101(1):45–57. [PubMed]
  • Yanisch-Perron C, Vieira J, Messing J. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene. 1985;33(1):103–119. [PubMed]
  • Zubenko GS, Mitchell AP, Jones EW. Septum formation, cell division, and sporulation in mutants of yeast deficient in proteinase B. Proc Natl Acad Sci U S A. 1979 May;76(5):2395–2399. [PubMed]
  • Zubenko GS, Park FJ, Jones EW. Genetic properties of mutations at the PEP4 locus in Saccharomyces cerevisiae. Genetics. 1982 Dec;102(4):679–690. [PubMed]
  • Zubenko GS, Park FJ, Jones EW. Mutations in PEP4 locus of Saccharomyces cerevisiae block final step in maturation of two vacuolar hydrolases. Proc Natl Acad Sci U S A. 1983 Jan;80(2):510–514. [PubMed]

Articles from Molecular and Cellular Biology are provided here courtesy of American Society for Microbiology (ASM)