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. 1989 Feb 1;108(2):309–325. doi: 10.1083/jcb.108.2.309

Processing pathway for protease B of Saccharomyces cerevisiae

PMCID: PMC2115423  PMID: 2645294

Abstract

The vacuolar protease B of Saccharomyces cerevisiae is a subtilisin- like protease encoded by the PRB1 gene. Antibodies raised against a synthetic peptide and an Escherichia coli-derived PRB1 open reading frame (ORF) protein cross-react with authentic protease B from yeast. By using these antibodies, the posttranslational biosynthetic pathway of protease B has been elucidated. Preproprotease B is a 76-kD unglycosylated precursor that enters the endoplasmic reticulum (ER), where it receives one asparagine-linked (Asn-linked) and an undetermined number of non-Asn-linked carbohydrate side chains. The large glycosylated intermediate is proteolytically processed to a 39-kD form before exiting the ER. In the Golgi complex, the 39-kD form becomes 40 kD, due to elaboration of the Asn-linked side chain. The carboxyterminal end of the 40-kD proprotease B undergoes protease A- mediated processing to a 37-kD intermediate, which in turn is quickly processed to 31-kD mature protease B. The ultimate processing step removes a peptide containing the Asn-linked chain; mature PrB has only non-Asn-linked carbohydrate.

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Selected References

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  1. Abbott R. J., Marzluf G. A. Major extracellular protease of Neurospora crassa. J Bacteriol. 1984 Aug;159(2):505–510. doi: 10.1128/jb.159.2.505-510.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. 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]
  3. Baker R. K., Lively M. O. Purification and characterization of hen oviduct microsomal signal peptidase. Biochemistry. 1987 Dec 29;26(26):8561–8567. doi: 10.1021/bi00400a010. [DOI] [PubMed] [Google Scholar]
  4. Ballou D. L. Genetic control of yeast mannan structure: mapping genes mnn2 and mnn4 in Saccharomyces cerevisiae. J Bacteriol. 1975 Aug;123(2):616–619. doi: 10.1128/jb.123.2.616-619.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bankaitis V. A., Johnson L. M., Emr S. D. Isolation of yeast mutants defective in protein targeting to the vacuole. Proc Natl Acad Sci U S A. 1986 Dec;83(23):9075–9079. doi: 10.1073/pnas.83.23.9075. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Burnette W. N. "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. doi: 10.1016/0003-2697(81)90281-5. [DOI] [PubMed] [Google Scholar]
  7. Böhni P. C., Deshaies R. J., Schekman R. W. SEC11 is required for signal peptide processing and yeast cell growth. J Cell Biol. 1988 Apr;106(4):1035–1042. doi: 10.1083/jcb.106.4.1035. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Cohen R. E., Zhang W., Ballou C. E. Effects of mannoprotein mutations on Saccharomyces cerevisiae core oligosaccharide structure. J Biol Chem. 1982 May 25;257(10):5730–5737. [PubMed] [Google Scholar]
  9. Cummings R. D., Kornfeld S., Schneider W. J., Hobgood K. K., Tolleshaug H., Brown M. S., Goldstein J. L. Biosynthesis of N- and O-linked oligosaccharides of the low density lipoprotein receptor. J Biol Chem. 1983 Dec 25;258(24):15261–15273. [PubMed] [Google Scholar]
  10. Deshaies R. J., Schekman R. A yeast mutant defective at an early stage in import of secretory protein precursors into the endoplasmic reticulum. J Cell Biol. 1987 Aug;105(2):633–645. doi: 10.1083/jcb.105.2.633. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Dmochowska A., Dignard D., Henning D., Thomas D. Y., Bussey H. Yeast KEX1 gene encodes a putative protease with a carboxypeptidase B-like function involved in killer toxin and alpha-factor precursor processing. Cell. 1987 Aug 14;50(4):573–584. doi: 10.1016/0092-8674(87)90030-4. [DOI] [PubMed] [Google Scholar]
  12. Esmon B., Esmon P. C., Schekman R. Early steps in processing of yeast glycoproteins. J Biol Chem. 1984 Aug 25;259(16):10322–10327. [PubMed] [Google Scholar]
  13. Esmon B., Novick P., Schekman R. Compartmentalized assembly of oligosaccharides on exported glycoproteins in yeast. Cell. 1981 Aug;25(2):451–460. doi: 10.1016/0092-8674(81)90063-5. [DOI] [PubMed] [Google Scholar]
  14. Evans E. A., Gilmore R., Blobel G. Purification of microsomal signal peptidase as a complex. Proc Natl Acad Sci U S A. 1986 Feb;83(3):581–585. doi: 10.1073/pnas.83.3.581. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Feldman R. I., Bernstein M., Schekman R. Product of SEC53 is required for folding and glycosylation of secretory proteins in the lumen of the yeast endoplasmic reticulum. J Biol Chem. 1987 Jul 5;262(19):9332–9339. [PubMed] [Google Scholar]
  16. Friis J., Ottolenghi P. The genetically determined binding of alcian blue by a minor fraction of yeast cell walls. C R Trav Lab Carlsberg. 1970;37(15):327–341. [PubMed] [Google Scholar]
  17. Green N., Alexander H., Olson A., Alexander S., Shinnick T. M., Sutcliffe J. G., Lerner R. A. Immunogenic structure of the influenza virus hemagglutinin. Cell. 1982 Mar;28(3):477–487. doi: 10.1016/0092-8674(82)90202-1. [DOI] [PubMed] [Google Scholar]
  18. Haselbeck A., Tanner W. O-glycosylation in Saccharomyces cerevisiae is initiated at the endoplasmic reticulum. FEBS Lett. 1983 Jul 25;158(2):335–338. doi: 10.1016/0014-5793(83)80608-5. [DOI] [PubMed] [Google Scholar]
  19. Hashimoto C., Cohen R. E., Zhang W. J., Ballou C. E. Carbohydrate chains on yeast carboxypeptidase Y are phosphorylated. Proc Natl Acad Sci U S A. 1981 Apr;78(4):2244–2248. doi: 10.1073/pnas.78.4.2244. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Hawthorne D C, Mortimer R K. Chromosome Mapping in Saccharomyces: Centromere-Linked Genes. Genetics. 1960 Aug;45(8):1085–1110. doi: 10.1093/genetics/45.8.1085. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Hemmings B. A., Zubenko G. S., Hasilik A., Jones E. W. 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. doi: 10.1073/pnas.78.1.435. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Huffaker T. C., Robbins P. W. Yeast mutants deficient in protein glycosylation. Proc Natl Acad Sci U S A. 1983 Dec;80(24):7466–7470. doi: 10.1073/pnas.80.24.7466. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Hunkapiller M. W., Lujan E., Ostrander F., Hood L. E. Isolation of microgram quantities of proteins from polyacrylamide gels for amino acid sequence analysis. Methods Enzymol. 1983;91:227–236. doi: 10.1016/s0076-6879(83)91019-4. [DOI] [PubMed] [Google Scholar]
  24. 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. doi: 10.1128/jb.153.1.163-168.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. 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]
  26. 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]
  27. 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]
  28. Julius D., Brake A., Blair L., Kunisawa R., Thorner J. Isolation of the putative structural gene for the lysine-arginine-cleaving endopeptidase required for processing of yeast prepro-alpha-factor. Cell. 1984 Jul;37(3):1075–1089. doi: 10.1016/0092-8674(84)90442-2. [DOI] [PubMed] [Google Scholar]
  29. Klionsky D. J., Banta L. M., Emr S. D. Intracellular sorting and processing of a yeast vacuolar hydrolase: proteinase A propeptide contains vacuolar targeting information. Mol Cell Biol. 1988 May;8(5):2105–2116. doi: 10.1128/mcb.8.5.2105. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Kominami E., Hoffschulte H., Holzer H. Purification and properties of proteinase B from yeast. Biochim Biophys Acta. 1981 Sep 15;661(1):124–135. doi: 10.1016/0005-2744(81)90091-7. [DOI] [PubMed] [Google Scholar]
  31. Kukuruzinska M. A., Bergh M. L., Jackson B. J. Protein glycosylation in yeast. Annu Rev Biochem. 1987;56:915–944. doi: 10.1146/annurev.bi.56.070187.004411. [DOI] [PubMed] [Google Scholar]
  32. 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]
  33. Kuo S. C., Lampen J. O. Tunicamycin--an inhibitor of yeast glycoprotein synthesis. Biochem Biophys Res Commun. 1974 May 7;58(1):287–295. doi: 10.1016/0006-291x(74)90925-5. [DOI] [PubMed] [Google Scholar]
  34. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  35. Lai C. Y. Detection of peptides by fluorescence methods. Methods Enzymol. 1977;47:236–243. doi: 10.1016/0076-6879(77)47028-9. [DOI] [PubMed] [Google Scholar]
  36. Lehle L. Biosynthesis of the core region of yeast mannoproteins. Formation of a glucosylated dolichol-bound oligosaccharide precursor, its transfer to protein and subsequent modification. Eur J Biochem. 1980 Aug;109(2):589–601. doi: 10.1111/j.1432-1033.1980.tb04832.x. [DOI] [PubMed] [Google Scholar]
  37. Lenney J. F., Matile P., Wiemken A., Schellenberg M., Meyer J. Activities and cellular localization of yeast proteases and their inhibitors. Biochem Biophys Res Commun. 1974 Oct 23;60(4):1378–1383. doi: 10.1016/0006-291x(74)90350-7. [DOI] [PubMed] [Google Scholar]
  38. Liu F. T., Zinnecker M., Hamaoka T., Katz D. H. New procedures for preparation and isolation of conjugates of proteins and a synthetic copolymer of D-amino acids and immunochemical characterization of such conjugates. Biochemistry. 1979 Feb 20;18(4):690–693. doi: 10.1021/bi00571a022. [DOI] [PubMed] [Google Scholar]
  39. Mechler B., Hirsch H. H., Müller H., Wolf D. H. Biogenesis of the yeast lysosome (vacuole): biosynthesis and maturation of proteinase yscB. EMBO J. 1988 Jun;7(6):1705–1710. doi: 10.1002/j.1460-2075.1988.tb02999.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Mechler B., Müller M., Müller H., Meussdoerffer F., Wolf D. H. 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] [Google Scholar]
  41. Mechler B., Müller M., Müller H., Wolf D. H. In vivo biosynthesis of vacuolar proteinases in proteinase mutants of Saccharomyces cerevisiae. Biochem Biophys Res Commun. 1982 Aug;107(3):770–778. doi: 10.1016/0006-291x(82)90590-3. [DOI] [PubMed] [Google Scholar]
  42. Moehle C. M., Aynardi M. W., Kolodny M. R., Park F. J., Jones E. W. Protease B of Saccharomyces cerevisiae: isolation and regulation of the PRB1 structural gene. Genetics. 1987 Feb;115(2):255–263. doi: 10.1093/genetics/115.2.255. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Moehle C. M., Tizard R., Lemmon S. K., Smart J., Jones E. W. Protease B of the lysosomelike vacuole of the yeast Saccharomyces cerevisiae is homologous to the subtilisin family of serine proteases. Mol Cell Biol. 1987 Dec;7(12):4390–4399. doi: 10.1128/mcb.7.12.4390. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. 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]
  45. Nakajima T., Ballou C. E. Characterization of the carbohydrate fragments obtained from Saccharomyces cerevisiae mannan by alkaline degradation. J Biol Chem. 1974 Dec 10;249(23):7679–7684. [PubMed] [Google Scholar]
  46. Novick P., Ferro S., Schekman R. Order of events in the yeast secretory pathway. Cell. 1981 Aug;25(2):461–469. doi: 10.1016/0092-8674(81)90064-7. [DOI] [PubMed] [Google Scholar]
  47. Novick P., Schekman R. Export of major cell surface proteins is blocked in yeast secretory mutants. J Cell Biol. 1983 Feb;96(2):541–547. doi: 10.1083/jcb.96.2.541. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Raschke W. C., Kern K. A., Antalis C., Ballou C. E. Genetic control of yeast mannan structure. Isolation and characterization of mannan mutants. J Biol Chem. 1973 Jul 10;248(13):4660–4666. [PubMed] [Google Scholar]
  49. Rothman J. H., Stevens T. H. Protein sorting in yeast: mutants defective in vacuole biogenesis mislocalize vacuolar proteins into the late secretory pathway. Cell. 1986 Dec 26;47(6):1041–1051. doi: 10.1016/0092-8674(86)90819-6. [DOI] [PubMed] [Google Scholar]
  50. Runge K. W., Huffaker T. C., Robbins P. W. Two yeast mutations in glucosylation steps of the asparagine glycosylation pathway. J Biol Chem. 1984 Jan 10;259(1):412–417. [PubMed] [Google Scholar]
  51. Schekman R. Protein localization and membrane traffic in yeast. Annu Rev Cell Biol. 1985;1:115–143. doi: 10.1146/annurev.cb.01.110185.000555. [DOI] [PubMed] [Google Scholar]
  52. 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. doi: 10.1016/0092-8674(82)90241-0. [DOI] [PubMed] [Google Scholar]
  53. Tanner W., Lehle L. Protein glycosylation in yeast. Biochim Biophys Acta. 1987 Apr 27;906(1):81–99. doi: 10.1016/0304-4157(87)90006-2. [DOI] [PubMed] [Google Scholar]
  54. Tolleshaug H., Goldstein J. L., Schneider W. J., Brown M. S. Posttranslational processing of the LDL receptor and its genetic disruption in familial hypercholesterolemia. Cell. 1982 Oct;30(3):715–724. doi: 10.1016/0092-8674(82)90276-8. [DOI] [PubMed] [Google Scholar]
  55. Trimble R. B., Atkinson P. H. Structure of yeast external invertase Man8-14GlcNAc processing intermediates by 500-megahertz 1H NMR spectroscopy. J Biol Chem. 1986 Jul 25;261(21):9815–9824. [PubMed] [Google Scholar]
  56. Ulane R. E., Cabib E. The activating system of chitin synthetase from Saccharomyces cerevisiae. Purification and properties of the activating factor. J Biol Chem. 1976 Jun 10;251(11):3367–3374. [PubMed] [Google Scholar]
  57. Valls L. A., Hunter C. P., Rothman J. H., Stevens T. H. Protein sorting in yeast: the localization determinant of yeast vacuolar carboxypeptidase Y resides in the propeptide. Cell. 1987 Mar 13;48(5):887–897. doi: 10.1016/0092-8674(87)90085-7. [DOI] [PubMed] [Google Scholar]
  58. Wickner R. B. Chromosomal and nonchromosomal mutations affecting the "killer character" of Saccharomyces cerevisiae. Genetics. 1974 Mar;76(3):423–432. doi: 10.1093/genetics/76.3.423. [DOI] [PMC free article] [PubMed] [Google Scholar]
  59. 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]
  60. 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]
  61. Zubenko G. S., Mitchell A. P., Jones E. W. 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. doi: 10.1073/pnas.76.5.2395. [DOI] [PMC free article] [PubMed] [Google Scholar]
  62. von Figura K., Hasilik A. Lysosomal enzymes and their receptors. Annu Rev Biochem. 1986;55:167–193. doi: 10.1146/annurev.bi.55.070186.001123. [DOI] [PubMed] [Google Scholar]

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