Skip to main content
PMC home page
Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1991 Dec;11(12):5801–5812. doi: 10.1128/mcb.11.12.5801

Isolation and characterization of PEP3, a gene required for vacuolar biogenesis in Saccharomyces cerevisiae.

R A Preston 1, M F Manolson 1, K Becherer 1, E Weidenhammer 1, D Kirkpatrick 1, R Wright 1, E W Jones 1
PMCID: PMC361722  PMID: 1944264

Abstract

The Saccharomyces cerevisiae PEP3 gene was cloned from a wild-type genomic library by complementation of the carboxypeptidase Y deficiency in a pep3-12 strain. Subclone complementation results localized the PEP3 gene to a 3.8-kb DNA fragment. The DNA sequence of the fragment was determined; a 2,754-bp open reading frame predicts that the PEP3 gene product is a hydrophilic, 107-kDa protein that has no significant similarity to any known protein. The PEP3 predicted protein has a zinc finger (CX2CX13CX2C) near its C terminus that has spacing and slight sequence similarity to the adenovirus E1a zinc finger. A radiolabeled PEP3 DNA probe hybridized to an RNA transcript of 3.1 kb in extracts of log-phase and diauxic lag-phase cells. Cells bearing pep3 deletion/disruption alleles were viable, had decreased levels of protease A, protease B, and carboxypeptidase Y antigens, had decreased repressible alkaline phosphatase activity, and contained very few normal vacuolelike organelles by fluorescence microscopy and electron microscopy but had an abundance of extremely small vesicles that stained with carboxyfluorescein diacetate, were severely inhibited for growth at 37 degrees C, and were incapable of sporulating (as homozygotes). Fractionation of cells expressing a bifunctional PEP3::SUC2 fusion protein indicated that the PEP3 gene product is present at low abundance in both log-phase and stationary cells and is a vacuolar peripheral membrane protein. Sequence identity established that PEP3 and VPS18 (J. S. Robinson, T. R. Graham, and S. D. Emr, Mol. Cell. Biol. 11:5813-5824, 1991) are the same gene.

Full text

PDF
5801

Images in this article

5805Image
on p.5805
5805Image
on p.5805
5806Image
on p.5806
5806Image
on p.5806
5807Image
on p.5807
5808Image
on p.5808

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Ammerer G., Hunter C. P., Rothman J. H., Saari G. C., Valls L. A., Stevens T. H. PEP4 gene of Saccharomyces cerevisiae encodes proteinase A, a vacuolar enzyme required for processing of vacuolar precursors. Mol Cell Biol. 1986 Jul;6(7):2490–2499. doi: 10.1128/mcb.6.7.2490. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Banta L. M., Robinson J. S., Klionsky D. J., Emr S. D. Organelle assembly in yeast: characterization of yeast mutants defective in vacuolar biogenesis and protein sorting. J Cell Biol. 1988 Oct;107(4):1369–1383. doi: 10.1083/jcb.107.4.1369. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bauer H., Sigarlakie E. Localization of alkaline phosphatase in Saccharomyces cerevisiae by means of ultrathin frozen sections. J Ultrastruct Res. 1975 Feb;50(2):208–215. doi: 10.1016/s0022-5320(75)80052-9. [DOI] [PubMed] [Google Scholar]
  4. Birnboim H. C., Doly J. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 1979 Nov 24;7(6):1513–1523. doi: 10.1093/nar/7.6.1513. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Boyd D., Beckwith J. The role of charged amino acids in the localization of secreted and membrane proteins. Cell. 1990 Sep 21;62(6):1031–1033. doi: 10.1016/0092-8674(90)90378-r. [DOI] [PubMed] [Google Scholar]
  6. Broach J. R., Strathern J. N., Hicks J. B. Transformation in yeast: development of a hybrid cloning vector and isolation of the CAN1 gene. Gene. 1979 Dec;8(1):121–133. doi: 10.1016/0378-1119(79)90012-x. [DOI] [PubMed] [Google Scholar]
  7. Bruschi C. V., Comer A. R., Howe G. A. Specificity of DNA uptake during whole cell transformation of S. cerevisiae. Yeast. 1987 Jun;3(2):131–137. doi: 10.1002/yea.320030209. [DOI] [PubMed] [Google Scholar]
  8. Carlson M., Osmond B. C., Botstein D. Mutants of yeast defective in sucrose utilization. Genetics. 1981 May;98(1):25–40. doi: 10.1093/genetics/98.1.25. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Chou P. Y., Fasman G. D. Prediction of protein conformation. Biochemistry. 1974 Jan 15;13(2):222–245. doi: 10.1021/bi00699a002. [DOI] [PubMed] [Google Scholar]
  10. Davis R. W., Thomas M., Cameron J., St John T. P., Scherer S., Padgett R. A. Rapid DNA isolations for enzymatic and hybridization analysis. Methods Enzymol. 1980;65(1):404–411. doi: 10.1016/s0076-6879(80)65051-4. [DOI] [PubMed] [Google Scholar]
  11. Dulić V., Riezman H. Characterization of the END1 gene required for vacuole biogenesis and gluconeogenic growth of budding yeast. EMBO J. 1989 May;8(5):1349–1359. doi: 10.1002/j.1460-2075.1989.tb03515.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Dulić V., Riezman H. Saccharomyces cerevisiae mutants lacking a functional vacuole are defective for aspects of the pheromone response. J Cell Sci. 1990 Nov;97(Pt 3):517–525. doi: 10.1242/jcs.97.3.517. [DOI] [PubMed] [Google Scholar]
  13. Garnier J., Osguthorpe D. J., Robson B. Analysis of the accuracy and implications of simple methods for predicting the secondary structure of globular proteins. J Mol Biol. 1978 Mar 25;120(1):97–120. doi: 10.1016/0022-2836(78)90297-8. [DOI] [PubMed] [Google Scholar]
  14. Goldstein A., Lampen J. O. Beta-D-fructofuranoside fructohydrolase from yeast. Methods Enzymol. 1975;42:504–511. doi: 10.1016/0076-6879(75)42159-0. [DOI] [PubMed] [Google Scholar]
  15. Hereford L. M., Rosbash M. Number and distribution of polyadenylated RNA sequences in yeast. Cell. 1977 Mar;10(3):453–462. doi: 10.1016/0092-8674(77)90032-0. [DOI] [PubMed] [Google Scholar]
  16. Holm C., Meeks-Wagner D. W., Fangman W. L., Botstein D. A rapid, efficient method for isolating DNA from yeast. Gene. 1986;42(2):169–173. doi: 10.1016/0378-1119(86)90293-3. [DOI] [PubMed] [Google Scholar]
  17. Johnson L. M., Bankaitis V. A., Emr S. D. Distinct sequence determinants direct intracellular sorting and modification of a yeast vacuolar protease. Cell. 1987 Mar 13;48(5):875–885. doi: 10.1016/0092-8674(87)90084-5. [DOI] [PubMed] [Google Scholar]
  18. Jones E. W. Proteinase mutants of Saccharomyces cerevisiae. Genetics. 1977 Jan;85(1):23–33. doi: 10.1093/genetics/85.1.23. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Jones E. W. The synthesis and function of proteases in Saccharomyces: genetic approaches. Annu Rev Genet. 1984;18:233–270. doi: 10.1146/annurev.ge.18.120184.001313. [DOI] [PubMed] [Google Scholar]
  20. Jones E. W. Three proteolytic systems in the yeast saccharomyces cerevisiae. J Biol Chem. 1991 May 5;266(13):7963–7966. [PubMed] [Google Scholar]
  21. Jones E. W., Zubenko G. S., Parker R. R. PEP4 gene function is required for expression of several vacuolar hydrolases in Saccharomyces cerevisiae. Genetics. 1982 Dec;102(4):665–677. doi: 10.1093/genetics/102.4.665. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. KIRBY K. S. ISOLATION AND CHARACTERIZATION OF RIBOSOMAL RIBONUCLEIC ACID. Biochem J. 1965 Jul;96:266–269. doi: 10.1042/bj0960266. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Kimelman D., Miller J. S., Porter D., Roberts B. E. E1a regions of the human adenoviruses and of the highly oncogenic simian adenovirus 7 are closely related. J Virol. 1985 Feb;53(2):399–409. doi: 10.1128/jvi.53.2.399-409.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Klein P., Kanehisa M., DeLisi C. The detection and classification of membrane-spanning proteins. Biochim Biophys Acta. 1985 May 28;815(3):468–476. doi: 10.1016/0005-2736(85)90375-x. [DOI] [PubMed] [Google Scholar]
  25. Klionsky D. J., Emr S. D. Membrane protein sorting: biosynthesis, transport and processing of yeast vacuolar alkaline phosphatase. EMBO J. 1989 Aug;8(8):2241–2250. doi: 10.1002/j.1460-2075.1989.tb08348.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Kuo C. L., Campbell J. L. 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. doi: 10.1128/mcb.3.10.1730. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Kuranda M. J., Robbins P. W. Cloning and heterologous expression of glycosidase genes from Saccharomyces cerevisiae. Proc Natl Acad Sci U S A. 1987 May;84(9):2585–2589. doi: 10.1073/pnas.84.9.2585. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Kyte J., Doolittle R. F. A simple method for displaying the hydropathic character of a protein. J Mol Biol. 1982 May 5;157(1):105–132. doi: 10.1016/0022-2836(82)90515-0. [DOI] [PubMed] [Google Scholar]
  29. Lipman D. J., Pearson W. R. Rapid and sensitive protein similarity searches. Science. 1985 Mar 22;227(4693):1435–1441. doi: 10.1126/science.2983426. [DOI] [PubMed] [Google Scholar]
  30. McCusker J. H., Haber J. E. Efficient sporulation of yeast in media buffered near pH6. J Bacteriol. 1977 Oct;132(1):180–185. doi: 10.1128/jb.132.1.180-185.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Moehle C. M., Dixon C. K., Jones E. W. Processing pathway for protease B of Saccharomyces cerevisiae. J Cell Biol. 1989 Feb;108(2):309–325. doi: 10.1083/jcb.108.2.309. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Mortimer R. K., Schild D. Genetic map of Saccharomyces cerevisiae, edition 9. Microbiol Rev. 1985 Sep;49(3):181–213. doi: 10.1128/mr.49.3.181-213.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Petes T. D., Broach J. R., Wensink P. C., Hereford L. M., Fink G. R., Botstein D. Isolation and analysis of recombinant DNA molecules containing yeast DNA. Gene. 1978 Sep;4(1):37–49. doi: 10.1016/0378-1119(78)90013-6. [DOI] [PubMed] [Google Scholar]
  34. Pringle J. R., Preston R. A., Adams A. E., Stearns T., Drubin D. G., Haarer B. K., Jones E. W. Fluorescence microscopy methods for yeast. Methods Cell Biol. 1989;31:357–435. doi: 10.1016/s0091-679x(08)61620-9. [DOI] [PubMed] [Google Scholar]
  35. Robinson J. S., Graham T. R., Emr S. D. A putative zinc finger protein, Saccharomyces cerevisiae Vps18p, affects late Golgi functions required for vacuolar protein sorting and efficient alpha-factor prohormone maturation. Mol Cell Biol. 1991 Dec;11(12):5813–5824. doi: 10.1128/mcb.11.12.5813. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Robinson J. S., Klionsky D. J., Banta L. M., Emr S. D. Protein sorting in Saccharomyces cerevisiae: isolation of mutants defective in the delivery and processing of multiple vacuolar hydrolases. Mol Cell Biol. 1988 Nov;8(11):4936–4948. doi: 10.1128/mcb.8.11.4936. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Rothman J. H., Howald I., Stevens T. H. Characterization of genes required for protein sorting and vacuolar function in the yeast Saccharomyces cerevisiae. EMBO J. 1989 Jul;8(7):2057–2065. doi: 10.1002/j.1460-2075.1989.tb03614.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Sharp P. M., Tuohy T. M., Mosurski K. R. Codon usage in yeast: cluster analysis clearly differentiates highly and lowly expressed genes. Nucleic Acids Res. 1986 Jul 11;14(13):5125–5143. doi: 10.1093/nar/14.13.5125. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Struhl K., Stinchcomb D. T., Scherer S., Davis R. W. High-frequency transformation of yeast: autonomous replication of hybrid DNA molecules. Proc Natl Acad Sci U S A. 1979 Mar;76(3):1035–1039. doi: 10.1073/pnas.76.3.1035. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. To-E A., Ueda Y., Kakimoto S. I., Oshima Y. Isolation and characterization of acid phosphatase mutants in Saccharomyces cerevisiae. J Bacteriol. 1973 Feb;113(2):727–738. doi: 10.1128/jb.113.2.727-738.1973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Wada Y., Kitamoto K., Kanbe T., Tanaka K., Anraku Y. The SLP1 gene of Saccharomyces cerevisiae is essential for vacuolar morphogenesis and function. Mol Cell Biol. 1990 May;10(5):2214–2223. doi: 10.1128/mcb.10.5.2214. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Wiemken A., Matile P., Moor H. Vacuolar dynamics in synchronously budding yeast. Arch Mikrobiol. 1970;70(2):89–103. doi: 10.1007/BF00412200. [DOI] [PubMed] [Google Scholar]
  44. Woolford C. A., Daniels L. B., Park F. J., Jones E. W., Van Arsdell J. N., Innis M. A. The PEP4 gene encodes an aspartyl protease implicated in the posttranslational regulation of Saccharomyces cerevisiae vacuolar hydrolases. Mol Cell Biol. 1986 Jul;6(7):2500–2510. doi: 10.1128/mcb.6.7.2500. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Woolford C. A., Dixon C. K., Manolson M. F., Wright R., Jones E. W. Isolation and characterization of PEP5, a gene essential for vacuolar biogenesis in Saccharomyces cerevisiae. Genetics. 1990 Aug;125(4):739–752. doi: 10.1093/genetics/125.4.739. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. 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. doi: 10.1016/0378-1119(85)90120-9. [DOI] [PubMed] [Google Scholar]
  47. Yoshihisa T., Anraku Y. A novel pathway of import of alpha-mannosidase, a marker enzyme of vacuolar membrane, in Saccharomyces cerevisiae. J Biol Chem. 1990 Dec 25;265(36):22418–22425. [PubMed] [Google Scholar]
  48. Yoshihisa T., Anraku Y. Nucleotide sequence of AMS1, the structure gene of vacuolar alpha-mannosidase of Saccharomyces cerevisiae. Biochem Biophys Res Commun. 1989 Sep 15;163(2):908–915. doi: 10.1016/0006-291x(89)92308-5. [DOI] [PubMed] [Google Scholar]
  49. Yoshihisa T., Ohsumi Y., Anraku Y. Solubilization and purification of alpha-mannosidase, a marker enzyme of vacuolar membranes in Saccharomyces cerevisiae. J Biol Chem. 1988 Apr 15;263(11):5158–5163. [PubMed] [Google Scholar]
  50. Zubenko G. S., Jones E. W. Protein degradation, meiosis and sporulation in proteinase-deficient mutants of Saccharomyces cerevisiae. Genetics. 1981 Jan;97(1):45–64. doi: 10.1093/genetics/97.1.45. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Molecular and Cellular Biology are provided here courtesy of Taylor & Francis

RESOURCES