Skip to main content
NCBI home page
Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1984 Nov;4(11):2347–2355. doi: 10.1128/mcb.4.11.2347

Invertase beta-galactosidase hybrid proteins fail to be transported from the endoplasmic reticulum in Saccharomyces cerevisiae.

S D Emr, I Schauer, W Hansen, P Esmon, R Schekman
PMCID: PMC369064  PMID: 6440005

Abstract

The yeast SUC2 gene codes for the secreted enzyme invertase. A series of 16 different-sized gene fusions have been constructed between this yeast gene and the Escherichia coli lacZ gene, which codes for the cytoplasmic enzyme beta-galactosidase. Various amounts of SUC2 NH2-terminal coding sequence have been fused in frame to a constant COOH-terminal coding segment of the lacZ gene, resulting in the synthesis of hybrid invertase-beta-galactosidase proteins in Saccharomyces cerevisiae. The hybrid proteins exhibit beta-galactosidase activity, and they are recognized specifically by antisera directed against either invertase or beta-galactosidase. Expression of beta-galactosidase activity is regulated in a manner similar to that observed for invertase activity expressed from a wild-type SUC2 gene: repressed in high-glucose medium and derepressed in low-glucose medium. Unlike wild-type invertase, however, the invertase-beta-galactosidase hybrid proteins are not secreted. Rather, they appear to remain trapped at a very early stage of secretory protein transit: insertion into the endoplasmic reticulum (ER). The hybrid proteins appear only to have undergone core glycosylation, an ER process, and do not receive the additional glycosyl modifications that take place in the Golgi complex. Even those hybrid proteins containing only a short segment of invertase sequences at the NH2 terminus are glycosylated, suggesting that no extensive folding of the invertase polypeptide is required before initiation of transmembrane transfer. beta-Galactosidase activity expressed by the SUC2-lacZ gene fusions cofractionates on Percoll density gradients with ER marker enzymes and not with other organelles. In addition, the hybrid proteins are not accessible to cell-surface labeling by 125I. Accumulation of the invertase-beta-galactosidase hybrid proteins within the ER does not appear to confer a growth-defective phenotype to yeast cells. In this location, however, the hybrid proteins and the beta-galactosidase activity they exhibit could provide a useful biochemical tag for yeast ER membranes.

Full text

PDF
2347

Images in this article

2351Image
on p.2351
2351Image
on p.2351
2352Image
on p.2352

Selected References

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

  1. Bassford P. J., Jr, Silhavy T. J., Beckwith J. R. Use of gene fusion to study secretion of maltose-binding protein into Escherichia coli periplasm. J Bacteriol. 1979 Jul;139(1):19–31. doi: 10.1128/jb.139.1.19-31.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bassford P., Beckwith J. Escherichia coli mutants accumulating the precursor of a secreted protein in the cytoplasm. Nature. 1979 Feb 15;277(5697):538–541. doi: 10.1038/277538a0. [DOI] [PubMed] [Google Scholar]
  3. Benson S. A., Silhavy T. J. Information within the mature LamB protein necessary for localization to the outer membrane of E coli K12. Cell. 1983 Apr;32(4):1325–1335. doi: 10.1016/0092-8674(83)90313-6. [DOI] [PubMed] [Google Scholar]
  4. Blobel G., Dobberstein B. Transfer of proteins across membranes. II. Reconstitution of functional rough microsomes from heterologous components. J Cell Biol. 1975 Dec;67(3):852–862. doi: 10.1083/jcb.67.3.852. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Blobel G., Walter P., Chang C. N., Goldman B. M., Erickson A. H., Lingappa V. R. Translocation of proteins across membranes: the signal hypothesis and beyond. Symp Soc Exp Biol. 1979;33:9–36. [PubMed] [Google Scholar]
  6. 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. doi: 10.1016/0092-8674(82)90384-1. [DOI] [PubMed] [Google Scholar]
  7. Carlson M., Osmond B. C., Botstein D. Genetic evidence for a silent SUC gene in yeast. Genetics. 1981 May;98(1):41–54. doi: 10.1093/genetics/98.1.41. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Carlson M., Taussig R., Kustu S., Botstein D. The secreted form of invertase in Saccharomyces cerevisiae is synthesized from mRNA encoding a signal sequence. Mol Cell Biol. 1983 Mar;3(3):439–447. doi: 10.1128/mcb.3.3.439. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Casadaban M. J., Cohen S. N. Analysis of gene control signals by DNA fusion and cloning in Escherichia coli. J Mol Biol. 1980 Apr;138(2):179–207. doi: 10.1016/0022-2836(80)90283-1. [DOI] [PubMed] [Google Scholar]
  10. Douglas M. G., Geller B. L., Emr S. D. Intracellular targeting and import of an F1-ATPase beta-subunit-beta-galactosidase hybrid protein into yeast mitochondria. Proc Natl Acad Sci U S A. 1984 Jul;81(13):3983–3987. doi: 10.1073/pnas.81.13.3983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Emr S. D., Schekman R., Flessel M. C., 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. doi: 10.1073/pnas.80.23.7080. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Emr S. D., Schwartz M., Silhavy T. J. Mutations altering the cellular localization of the phage lambda receptor, an Escherichia coli outer membrane protein. Proc Natl Acad Sci U S A. 1978 Dec;75(12):5802–5806. doi: 10.1073/pnas.75.12.5802. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Emr S. D., Silhavy T. J. Mutations affecting localization of an Escherichia coli outer membrane protein, the bacteriophage lambda receptor. J Mol Biol. 1980 Jul 25;141(1):63–90. doi: 10.1016/s0022-2836(80)80029-5. [DOI] [PubMed] [Google Scholar]
  14. 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]
  15. Ferro-Novick S., Novick P., Field C., Schekman R. Yeast secretory mutants that block the formation of active cell surface enzymes. J Cell Biol. 1984 Jan;98(1):35–43. doi: 10.1083/jcb.98.1.35. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Finkelstein D. B., Strausberg S. Heat shock-regulated production of Escherichia coli beta-galactosidase in Saccharomyces cerevisiae. Mol Cell Biol. 1983 Sep;3(9):1625–1633. doi: 10.1128/mcb.3.9.1625. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Fowler A. V., Zabin I. The amino acid sequence of beta-galactosidase of Escherichia coli. Proc Natl Acad Sci U S A. 1977 Apr;74(4):1507–1510. doi: 10.1073/pnas.74.4.1507. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. 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]
  19. Guarente L., Ptashne M. Fusion of Escherichia coli lacZ to the cytochrome c gene of Saccharomyces cerevisiae. Proc Natl Acad Sci U S A. 1981 Apr;78(4):2199–2203. doi: 10.1073/pnas.78.4.2199. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. 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]
  21. Kubota S., Yoshida Y., Kumaoka H., Furumichi A. Studies on the microsomal electron-transport system of anaerobically grown yeast. V. Purification and characterization of NADPH-cytochrome c reductase. J Biochem. 1977 Jan;81(1):197–205. doi: 10.1093/oxfordjournals.jbchem.a131436. [DOI] [PubMed] [Google Scholar]
  22. 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]
  23. Lehle L., Cohen R. E., Ballou C. E. Carbohydrate structure of yeast invertase. Demonstration of a form with only core oligosaccharides and a form with completed polysaccharide chains. J Biol Chem. 1979 Dec 10;254(23):12209–12218. [PubMed] [Google Scholar]
  24. Martinez-Arias A. E., Casadaban M. J. Fusion of the Saccharomyces cerevisiae leu2 gene to an Escherichia coli beta-galactosidase gene. Mol Cell Biol. 1983 Apr;3(4):580–586. doi: 10.1128/mcb.3.4.580. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Michaelis S., Inouye H., Oliver D., Beckwith J. Mutations that alter the signal sequence of alkaline phosphatase in Escherichia coli. J Bacteriol. 1983 Apr;154(1):366–374. doi: 10.1128/jb.154.1.366-374.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. 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]
  27. Novick P., Field C., Schekman R. Identification of 23 complementation groups required for post-translational events in the yeast secretory pathway. Cell. 1980 Aug;21(1):205–215. doi: 10.1016/0092-8674(80)90128-2. [DOI] [PubMed] [Google Scholar]
  28. 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]
  29. Osley M. A., Hereford L. Identification of a sequence responsible for periodic synthesis of yeast histone 2A mRNA. Proc Natl Acad Sci U S A. 1982 Dec;79(24):7689–7693. doi: 10.1073/pnas.79.24.7689. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Palade G. Intracellular aspects of the process of protein synthesis. Science. 1975 Aug 1;189(4200):347–358. doi: 10.1126/science.1096303. [DOI] [PubMed] [Google Scholar]
  31. Perlman D., Halvorson H. O., Cannon L. E. Presecretory and cytoplasmic invertase polypeptides encoded by distinct mRNAs derived from the same structural gene differ by a signal sequence. Proc Natl Acad Sci U S A. 1982 Feb;79(3):781–785. doi: 10.1073/pnas.79.3.781. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Rose M., Casadaban M. J., Botstein D. Yeast genes fused to beta-galactosidase in Escherichia coli can be expressed normally in yeast. Proc Natl Acad Sci U S A. 1981 Apr;78(4):2460–2464. doi: 10.1073/pnas.78.4.2460. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Silhavy T. J., Benson S. A., Emr S. D. Mechanisms of protein localization. Microbiol Rev. 1983 Sep;47(3):313–344. doi: 10.1128/mr.47.3.313-344.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Silverman S. J., Rose M., Botstein D., Fink G. R. Regulation of HIS4-lacZ fusions in Saccharomyces cerevisiae. Mol Cell Biol. 1982 Oct;2(10):1212–1219. doi: 10.1128/mcb.2.10.1212. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. 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]
  36. Struhl K. Regulatory sites for his3 gene expression in yeast. Nature. 1982 Nov 18;300(5889):285–286. doi: 10.1038/300284a0. [DOI] [PubMed] [Google Scholar]
  37. Taussig R., Carlson M. Nucleotide sequence of the yeast SUC2 gene for invertase. Nucleic Acids Res. 1983 Mar 25;11(6):1943–1954. doi: 10.1093/nar/11.6.1943. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Trimble R. B., Maley F. Subunit structure of external invertase from Saccharomyces cerevisiae. J Biol Chem. 1977 Jun 25;252(12):4409–4412. [PubMed] [Google Scholar]
  39. Wickner W. The assembly of proteins into biological membranes: The membrane trigger hypothesis. Annu Rev Biochem. 1979;48:23–45. doi: 10.1146/annurev.bi.48.070179.000323. [DOI] [PubMed] [Google Scholar]

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

RESOURCES