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. 1996 Sep 3;93(18):9612–9617. doi: 10.1073/pnas.93.18.9612

Split invertase polypeptides form functional complexes in the yeast periplasm in vivo.

O Schonberger 1, C Knox 1, E Bibi 1, O Pines 1
PMCID: PMC38476  PMID: 8790378

Abstract

The assembly of functional proteins from fragments in vivo has been recently described for several proteins, including the secreted maltose binding protein in Escherichia coli. Here we demonstrate for the first time that split gene products can function within the eukaryotic secretory system. Saccharomyces cerevisiae strains able to use sucrose produce the enzyme invertase, which is targeted by a signal peptide to the central secretory pathway and the periplasmic space. Using this enzyme as a model we find the following: (i) Polypeptide fragments of invertase, each containing a signal peptide, are independently translocated into the endoplasmic reticulum (ER) are modified by glycosylation, and travel the entire secretory pathway reaching the yeast periplasm. (ii) Simultaneous expression of independently translated and translocated overlapping fragments of invertase leads to the formation of an enzymatically active complex, whereas individually expressed fragments exhibit no activity. (iii) An active invertase complex is assembled in the ER, is targeted to the yeast periplasm, and is biologically functional, as judged by its ability to facilitate growth on sucrose as a single carbon source. These observation are discussed in relation to protein folding and assembly in the ER and to the trafficking of proteins through the secretory pathway.

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Selected References

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

  1. 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]
  2. Bergeron J. J., Brenner M. B., Thomas D. Y., Williams D. B. Calnexin: a membrane-bound chaperone of the endoplasmic reticulum. Trends Biochem Sci. 1994 Mar;19(3):124–128. doi: 10.1016/0968-0004(94)90205-4. [DOI] [PubMed] [Google Scholar]
  3. Berkower C., Michaelis S. Mutational analysis of the yeast a-factor transporter STE6, a member of the ATP binding cassette (ABC) protein superfamily. EMBO J. 1991 Dec;10(12):3777–3785. doi: 10.1002/j.1460-2075.1991.tb04947.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Betton J. M., Hofnung M. In vivo assembly of active maltose binding protein from independently exported protein fragments. EMBO J. 1994 Mar 1;13(5):1226–1234. doi: 10.1002/j.1460-2075.1994.tb06372.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bibi E., Kaback H. R. In vivo expression of the lacY gene in two segments leads to functional lac permease. Proc Natl Acad Sci U S A. 1990 Jun;87(11):4325–4329. doi: 10.1073/pnas.87.11.4325. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  7. 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]
  8. 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]
  9. 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]
  10. Esmon P. C., Esmon B. E., Schauer I. E., Taylor A., Schekman R. Structure, assembly, and secretion of octameric invertase. J Biol Chem. 1987 Mar 25;262(9):4387–4394. [PubMed] [Google Scholar]
  11. Geller D., Taglicht D., Edgar R., Tam A., Pines O., Michaelis S., Bibi E. Comparative topology studies in Saccharomyces cerevisiae and in Escherichia coli. The N-terminal half of the yeast ABC protein Ste6. J Biol Chem. 1996 Jun 7;271(23):13746–13753. doi: 10.1074/jbc.271.23.13746. [DOI] [PubMed] [Google Scholar]
  12. Gething M. J., Sambrook J. Protein folding in the cell. Nature. 1992 Jan 2;355(6355):33–45. doi: 10.1038/355033a0. [DOI] [PubMed] [Google Scholar]
  13. Gilmore R. Protein translocation across the endoplasmic reticulum: a tunnel with toll booths at entry and exit. Cell. 1993 Nov 19;75(4):589–592. doi: 10.1016/0092-8674(93)90476-7. [DOI] [PubMed] [Google Scholar]
  14. Haas I. G., Wabl M. Immunoglobulin heavy chain binding protein. Nature. 1983 Nov 24;306(5941):387–389. doi: 10.1038/306387a0. [DOI] [PubMed] [Google Scholar]
  15. Hammond C., Helenius A. Folding of VSV G protein: sequential interaction with BiP and calnexin. Science. 1994 Oct 21;266(5184):456–458. doi: 10.1126/science.7939687. [DOI] [PubMed] [Google Scholar]
  16. Haselbeck A., Schekman R. Interorganelle transfer and glycosylation of yeast invertase in vitro. Proc Natl Acad Sci U S A. 1986 Apr;83(7):2017–2021. doi: 10.1073/pnas.83.7.2017. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Helenius A., Marquardt T., Braakman I. The endoplasmic reticulum as a protein-folding compartment. Trends Cell Biol. 1992 Aug;2(8):227–231. doi: 10.1016/0962-8924(92)90309-b. [DOI] [PubMed] [Google Scholar]
  18. Hendershot L., Bole D., Köhler G., Kearney J. F. Assembly and secretion of heavy chains that do not associate posttranslationally with immunoglobulin heavy chain-binding protein. J Cell Biol. 1987 Mar;104(3):761–767. doi: 10.1083/jcb.104.3.761. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Hurtley S. M., Bole D. G., Hoover-Litty H., Helenius A., Copeland C. S. Interactions of misfolded influenza virus hemagglutinin with binding protein (BiP). J Cell Biol. 1989 Jun;108(6):2117–2126. doi: 10.1083/jcb.108.6.2117. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Kaur P., Rosen B. P. Complementation between nucleotide binding domains in an anion-translocating ATPase. J Bacteriol. 1993 Jan;175(2):351–357. doi: 10.1128/jb.175.2.351-357.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Kern G., Schülke N., Schmid F. X., Jaenicke R. Stability, quaternary structure, and folding of internal, external, and core-glycosylated invertase from yeast. Protein Sci. 1992 Jan;1(1):120–131. doi: 10.1002/pro.5560010112. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. 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]
  23. London A., Pines O. Specific beta-lactam antibiotics inhibit secretion of lipo-beta-lactamase in yeast. Mol Microbiol. 1990 Dec;4(12):2193–2200. doi: 10.1111/j.1365-2958.1990.tb00581.x. [DOI] [PubMed] [Google Scholar]
  24. Machamer C. E., Doms R. W., Bole D. G., Helenius A., Rose J. K. Heavy chain binding protein recognizes incompletely disulfide-bonded forms of vesicular stomatitis virus G protein. J Biol Chem. 1990 Apr 25;265(12):6879–6883. [PubMed] [Google Scholar]
  25. Mellor J., Dobson M. J., Roberts N. A., Tuite M. F., Emtage J. S., White S., Lowe P. A., Patel T., Kingsman A. J., Kingsman S. M. Efficient synthesis of enzymatically active calf chymosin in Saccharomyces cerevisiae. Gene. 1983 Sep;24(1):1–14. doi: 10.1016/0378-1119(83)90126-9. [DOI] [PubMed] [Google Scholar]
  26. 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]
  27. Pines O., London A. Expression and secretion of staphylococcal nuclease in yeast: effects of amino-terminal sequences. J Gen Microbiol. 1991 Apr;137(4):771–778. doi: 10.1099/00221287-137-4-771. [DOI] [PubMed] [Google Scholar]
  28. Pines O., Lunn C. A., Inouye M. Defective Escherichia coli signal peptides function in yeast. Mol Microbiol. 1988 Mar;2(2):209–217. doi: 10.1111/j.1365-2958.1988.tb00022.x. [DOI] [PubMed] [Google Scholar]
  29. 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]
  30. Rothman J. E. Mechanisms of intracellular protein transport. Nature. 1994 Nov 3;372(6501):55–63. doi: 10.1038/372055a0. [DOI] [PubMed] [Google Scholar]
  31. Schonberger O., Hirst T. R., Pines O. Targeting and assembly of an oligomeric bacterial enterotoxoid in the endoplasmic reticulum of Saccharomyces cerevisiae. Mol Microbiol. 1991 Nov;5(11):2663–2671. doi: 10.1111/j.1365-2958.1991.tb01975.x. [DOI] [PubMed] [Google Scholar]
  32. Shani O., Pines O. The relationship between disulphide bond formation, processing and secretion of lipo-beta-lactamase in yeast. Mol Microbiol. 1992 Jan;6(2):189–195. doi: 10.1111/j.1365-2958.1992.tb02000.x. [DOI] [PubMed] [Google Scholar]
  33. Shiba K., Schimmel P. Functional assembly of a randomly cleaved protein. Proc Natl Acad Sci U S A. 1992 Mar 1;89(5):1880–1884. doi: 10.1073/pnas.89.5.1880. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Sikorski R. S., Hieter P. A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae. Genetics. 1989 May;122(1):19–27. doi: 10.1093/genetics/122.1.19. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Tabor S., Richardson C. C. A bacteriophage T7 RNA polymerase/promoter system for controlled exclusive expression of specific genes. Proc Natl Acad Sci U S A. 1985 Feb;82(4):1074–1078. doi: 10.1073/pnas.82.4.1074. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Taniuchi H., Parr G. R., Juillerat M. A. Complementation in folding and fragment exchange. Methods Enzymol. 1986;131:185–217. doi: 10.1016/0076-6879(86)31042-5. [DOI] [PubMed] [Google Scholar]
  37. Vogel J. P., Misra L. M., Rose M. D. Loss of BiP/GRP78 function blocks translocation of secretory proteins in yeast. J Cell Biol. 1990 Jun;110(6):1885–1895. doi: 10.1083/jcb.110.6.1885. [DOI] [PMC free article] [PubMed] [Google Scholar]

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