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. 1991 Jan;173(2):627–635. doi: 10.1128/jb.173.2.627-635.1991

Glycosylation and structure of the yeast MF alpha 1 alpha-factor precursor is important for efficient transport through the secretory pathway.

S Caplan 1, R Green 1, J Rocco 1, J Kurjan 1
PMCID: PMC207053  PMID: 1987155

Abstract

The MF alpha 1 gene encodes a precursor, prepro-alpha-factor, that undergoes several proteolytic processing steps within the classical secretory pathway to produce the mature peptide pheromone, alpha-factor. To investigate the role of structural features of the MF alpha 1 precursor in alpha-factor production, we analyzed the effect of mf alpha 1 mutations that alter precursor structure in a number of ways. These mutations resulted in decreased alpha-factor secretion and intracellular accumulation of pro-alpha-factor. With the exception of the mutant lacking all three N glycosylation sites, the pro-alpha-factor forms that accumulated were core glycosylated but had not yet undergone the addition of outer chain carbohydrate. The delay, therefore, occurred at a step prior to the first proteolytic processing step involved in maturation of the precursor and was probably due to inefficient endoplasmic reticulum-to-Golgi transport. Elimination of all three N-glycosylation sites caused a delay in disappearance of intracellular precursor, and alpha-factor secretion was also slowed. These data indicate that N glycosylation is important but not essential for transport of the precursor through the secretory pathway. The decreased alpha-factor secretion and increased precursor accumulation seen with many different structural changes of pro-alpha-factor indicate that the secretory pathway is extremely sensitive to changes in precursor structure. This sensitivity could cause inefficient secretion of heterologous proteins and hybrids between MF alpha 1 and heterologous proteins in yeast cells.

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Selected References

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  1. Achstetter T., Wolf D. H. Hormone processing and membrane-bound proteinases in yeast. EMBO J. 1985 Jan;4(1):173–177. doi: 10.1002/j.1460-2075.1985.tb02333.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Allison D. S., Young E. T. Single-amino-acid substitutions within the signal sequence of yeast prepro-alpha-factor affect membrane translocation. Mol Cell Biol. 1988 May;8(5):1915–1922. doi: 10.1128/mcb.8.5.1915. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Brake A. J., Julius D. J., Thorner J. A functional prepro-alpha-factor gene in Saccharomyces yeasts can contain three, four, or five repeats of the mature pheromone sequence. Mol Cell Biol. 1983 Aug;3(8):1440–1450. doi: 10.1128/mcb.3.8.1440. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Brake A. J. Secretion of heterologous proteins directed by the yeast alpha-factor leader. Biotechnology. 1989;13:269–280. [PubMed] [Google Scholar]
  5. Bücking-Throm E., Duntze W., Hartwell L. H., Manney T. R. Reversible arrest of haploid yeast cells in the initiation of DNA synthesis by a diffusible sex factor. Exp Cell Res. 1973 Jan;76(1):99–110. doi: 10.1016/0014-4827(73)90424-2. [DOI] [PubMed] [Google Scholar]
  6. Chu F. K., Trimble R. B., Maley F. The effect of carbohydrate depletion on the properties of yeast external invertase. J Biol Chem. 1978 Dec 25;253(24):8691–8693. [PubMed] [Google Scholar]
  7. Cross F., Hartwell L. H., Jackson C., Konopka J. B. Conjugation in Saccharomyces cerevisiae. Annu Rev Cell Biol. 1988;4:429–457. doi: 10.1146/annurev.cb.04.110188.002241. [DOI] [PubMed] [Google Scholar]
  8. 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]
  9. Dorner A. J., Bole D. G., Kaufman R. J. The relationship of N-linked glycosylation and heavy chain-binding protein association with the secretion of glycoproteins. J Cell Biol. 1987 Dec;105(6 Pt 1):2665–2674. doi: 10.1083/jcb.105.6.2665. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Duntze W., MacKay V., Manney T. R. Saccharomyces cerevisiae: a diffusible sex factor. Science. 1970 Jun 19;168(3938):1472–1473. doi: 10.1126/science.168.3938.1472. [DOI] [PubMed] [Google Scholar]
  11. Emter O., Mechler B., Achstetter T., Müller H., Wolf D. H. Yeast pheromone alpha-factor is synthesized as a high molecular weight precursor. Biochem Biophys Res Commun. 1983 Nov 15;116(3):822–829. doi: 10.1016/s0006-291x(83)80216-2. [DOI] [PubMed] [Google Scholar]
  12. Gething M. J., McCammon K., Sambrook J. Expression of wild-type and mutant forms of influenza hemagglutinin: the role of folding in intracellular transport. Cell. 1986 Sep 12;46(6):939–950. doi: 10.1016/0092-8674(86)90076-0. [DOI] [PubMed] [Google Scholar]
  13. Gibson R., Schlesinger S., Kornfeld S. The nonglycosylated glycoprotein of vesicular stomatitis virus is temperature-sensitive and undergoes intracellular aggregation at elevated temperatures. J Biol Chem. 1979 May 10;254(9):3600–3607. [PubMed] [Google Scholar]
  14. Green R., Kramer R. A., Shields D. Misplacement of the amino-terminal positive charge in the prepro-alpha-factor signal peptide disrupts membrane translocation in vivo. J Biol Chem. 1989 Feb 15;264(5):2963–2968. [PubMed] [Google Scholar]
  15. Green R., Schaber M. D., Shields D., Kramer R. Secretion of somatostatin by Saccharomyces cerevisiae. Correct processing of an alpha-factor-somatostatin hybrid. J Biol Chem. 1986 Jun 5;261(16):7558–7565. [PubMed] [Google Scholar]
  16. Hubbard S. C., Ivatt R. J. Synthesis and processing of asparagine-linked oligosaccharides. Annu Rev Biochem. 1981;50:555–583. doi: 10.1146/annurev.bi.50.070181.003011. [DOI] [PubMed] [Google Scholar]
  17. Julius D., Blair L., Brake A., Sprague G., Thorner J. Yeast alpha factor is processed from a larger precursor polypeptide: the essential role of a membrane-bound dipeptidyl aminopeptidase. Cell. 1983 Mar;32(3):839–852. doi: 10.1016/0092-8674(83)90070-3. [DOI] [PubMed] [Google Scholar]
  18. 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]
  19. Julius D., Schekman R., Thorner J. Glycosylation and processing of prepro-alpha-factor through the yeast secretory pathway. Cell. 1984 Feb;36(2):309–318. doi: 10.1016/0092-8674(84)90224-1. [DOI] [PubMed] [Google Scholar]
  20. Kozutsumi Y., Segal M., Normington K., Gething M. J., Sambrook J. The presence of malfolded proteins in the endoplasmic reticulum signals the induction of glucose-regulated proteins. Nature. 1988 Mar 31;332(6163):462–464. doi: 10.1038/332462a0. [DOI] [PubMed] [Google Scholar]
  21. 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]
  22. 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]
  23. Kurjan J. Alpha-factor structural gene mutations in Saccharomyces cerevisiae: effects on alpha-factor production and mating. Mol Cell Biol. 1985 Apr;5(4):787–796. doi: 10.1128/mcb.5.4.787. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Kurjan J., Herskowitz I. Structure of a yeast pheromone gene (MF alpha): a putative alpha-factor precursor contains four tandem copies of mature alpha-factor. Cell. 1982 Oct;30(3):933–943. doi: 10.1016/0092-8674(82)90298-7. [DOI] [PubMed] [Google Scholar]
  25. 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]
  26. Lodish H. F. Transport of secretory and membrane glycoproteins from the rough endoplasmic reticulum to the Golgi. A rate-limiting step in protein maturation and secretion. J Biol Chem. 1988 Feb 15;263(5):2107–2110. [PubMed] [Google Scholar]
  27. Machamer C. E., Florkiewicz R. Z., Rose J. K. A single N-linked oligosaccharide at either of the two normal sites is sufficient for transport of vesicular stomatitis virus G protein to the cell surface. Mol Cell Biol. 1985 Nov;5(11):3074–3083. doi: 10.1128/mcb.5.11.3074. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Matzuk M. M., Boime I. The role of the asparagine-linked oligosaccharides of the alpha subunit in the secretion and assembly of human chorionic gonadotrophin. J Cell Biol. 1988 Apr;106(4):1049–1059. doi: 10.1083/jcb.106.4.1049. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Miyajima A., Arai K. Use of a cDNA expression-cloning vector and a secretion vector for mammalian gene expression in Saccharomyces cerevisiae. Biotechnology. 1989;13:281–304. [PubMed] [Google Scholar]
  30. Normington K., Kohno K., Kozutsumi Y., Gething M. J., Sambrook J. S. cerevisiae encodes an essential protein homologous in sequence and function to mammalian BiP. Cell. 1989 Jun 30;57(7):1223–1236. doi: 10.1016/0092-8674(89)90059-7. [DOI] [PubMed] [Google Scholar]
  31. Parker R. C., Watson R. M., Vinograd J. Mapping of closed circular DNAs by cleavage with restriction endonucleases and calibration by agarose gel electrophoresis. Proc Natl Acad Sci U S A. 1977 Mar;74(3):851–855. doi: 10.1073/pnas.74.3.851. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Payne G. S., Schekman R. Clathrin: a role in the intracellular retention of a Golgi membrane protein. Science. 1989 Sep 22;245(4924):1358–1365. doi: 10.1126/science.2675311. [DOI] [PubMed] [Google Scholar]
  33. Rose M. D., Misra L. M., Vogel J. P. KAR2, a karyogamy gene, is the yeast homolog of the mammalian BiP/GRP78 gene. Cell. 1989 Jun 30;57(7):1211–1221. doi: 10.1016/0092-8674(89)90058-5. [DOI] [PubMed] [Google Scholar]
  34. Rothblatt J. A., Webb J. R., Ammerer G., Meyer D. I. Secretion in yeast: structural features influencing the post-translational translocation of prepro-alpha-factor in vitro. EMBO J. 1987 Nov;6(11):3455–3463. doi: 10.1002/j.1460-2075.1987.tb02669.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Rothstein R. J. One-step gene disruption in yeast. Methods Enzymol. 1983;101:202–211. doi: 10.1016/0076-6879(83)01015-0. [DOI] [PubMed] [Google Scholar]
  36. 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]
  37. Singh A., Chen E. Y., Lugovoy J. M., Chang C. N., Hitzeman R. A., Seeburg P. H. Saccharomyces cerevisiae contains two discrete genes coding for the alpha-factor pheromone. Nucleic Acids Res. 1983 Jun 25;11(12):4049–4063. doi: 10.1093/nar/11.12.4049. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Southern E. M. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol. 1975 Nov 5;98(3):503–517. doi: 10.1016/s0022-2836(75)80083-0. [DOI] [PubMed] [Google Scholar]
  39. Tarentino A. L., Plummer T. H., Jr, Maley F. The release of intact oligosaccharides from specific glycoproteins by endo-beta-N-acetylglucosaminidase H. J Biol Chem. 1974 Feb 10;249(3):818–824. [PubMed] [Google Scholar]
  40. Waters M. G., Evans E. A., Blobel G. Prepro-alpha-factor has a cleavable signal sequence. J Biol Chem. 1988 May 5;263(13):6209–6214. [PubMed] [Google Scholar]
  41. Weinstock G. M., ap Rhys C., Berman M. L., Hampar B., Jackson D., Silhavy T. J., Weisemann J., Zweig M. Open reading frame expression vectors: a general method for antigen production in Escherichia coli using protein fusions to beta-galactosidase. Proc Natl Acad Sci U S A. 1983 Jul;80(14):4432–4436. doi: 10.1073/pnas.80.14.4432. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Zoller M. J., Smith M. Oligonucleotide-directed mutagenesis: a simple method using two oligonucleotide primers and a single-stranded DNA template. DNA. 1984 Dec;3(6):479–488. doi: 10.1089/dna.1.1984.3.479. [DOI] [PubMed] [Google Scholar]

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