Summary
The co-translational glycosylation of nascent polypeptide chains in the endoplasmic reticulum (ER) is catalyzed by oligosaccharyl transferase (OT). Recent studies in yeast by others have identified two proteins that are non-limiting components of OT. In an effort to identify other, limiting components of OT we have devised a sensitive assay for OT activity in lysed yeast protoplasts. This assay has been used to screen a collection of temperature sensitive yeast mutants. This approach has yielded several mutants that have defects in glycosylation, one of which may contain a limiting, defective component of OT. Efforts to characterize these mutants and to clone and sequence this putative OT subunit are in progress.
During earlier efforts to identify OT, hydrophobic peptides containing a glycosylation site for OT and a photoreactive, radiolabeled probe were developed. However, these probes were found to label a lumenal protein of the ER rather than OT. This protein was identified as protein disulfide isomerase (PDI). In yeast, deletion of the gene encoding this protein was lethal, demonstrating that PDI is encoded by an essential gene. C-terminal deletion and active site mutants of PDI were studied with respect to: 1) cell viability, 2) processing of secretory proteins, and 3) in vitro catalytic activity in disulfide bond formation. The results revealed that PDI does, in fact, catalyze disulfide bond formation in vivo. Furthermore, it is clear that the catalytic activity depends on the presence of the sequence -CGHC-. However, the essential role of PDI may be more closely related to its ability to bind and stabilize polypeptides than to its disulfide bond-forming function.
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References
Bulleid, N. J., and Freedman, R. B. (1988). Defective co-translational formation of disulfide bonds in protein disulfide-isomerase-deficient microsomes. Nature 335, 649–651.
Creighton, T. E., Hillson, D. A., and Freedman, R. B. (1980). Catalysis by protein-disulphide isomerase of the unfolding and refolding of proteins with disulphide bonds. J. Mol. Biol. 142, 43–62.
Freedman, R. B., Bulleid, N. J., Hawkins, H. C., and Paver, P. L. (1989). Role of protein disulfide-isomerase in the expression of native proteins. Biochem. Soc. Symp. 55, 167–192.
Geetha-Habib, M., Park, H. and Lennarz, W. J. (1990). In vivo N-glycosylation and fate of Asn-X-Ser/Thr tripeptides. J. Biol. Chem. 265, 13655–13660.
Givol, D., DeLorenzo, F., Goldberger, R. F., and Anfinsen, C. B. (1965). Disulfide interchange and the three-dimensional structure of proteins. Proc. Natl. Acad. Sci. USA 552, 676–684.
Goldberger, R. F., Epstein, C. J., and Anfinsen, C. B. (1963). Acceleration of reactivation of reduced bovine pancreatice ribonuclease by a microsomal system from rat liver. J. Biol. Chem. 238, 628–635.
Günther, R., Brauer, C. Janetzky, B., Forster, H.-H., Ehbrecht, E.-M. Lehle, L., and Kuntzel, H. (1991). The Saccharomyces cerevisiae TRG1gene is essential for growth and encodes a lumenal endoplasmic reticulum glycoprotein involved in the maturation of vacuolar carboxypeptidase. J. Biol. Chem. 266, 24557–24563.
Gurd, F. R. N. (1972). Carboxymethylation. Methods Enzymol. 21 424–438.
Hardwick, K. G., Lewis, M. J., Semenza, J., Dean, N., and Pelham, H. R. B. (1990). ERD1, a yeast gene required for the retention of luminal endoplasmic reticulum proteins, affects glycoprotein processing in the Golgi apparatus. EMBO J. 9, 623–630.
Hartwell, L. (1967). Macromolecole synthesis in temperature-sensitive mutants of yeast. J. Bacteriol. 92, 1662–1670.
Huffaker, T. and Robbins, P. (1983). Yeast mutants deficient in protein glycosylation. Proc. Natl. Acad. Sci. USA 80, 7466–7470.
Kelleher, D., Kreibich, G. and Gilmore, R. (1992). Oligosaccharyltransferase activity is associated with a protein complex composed of ribophorins I and II and a 48kd protein. Cell 69, 55–65.
Koivu, J., and Myllyla, R. (1987). Interchain disulfide bond formation in types I and II procollagen. J. Biol. Chem. 262, 6159–6164.
LaMantia, M. L., Miura, T., Tachikawa, H. Kaplan, H. A., Lennarz, W. J., and Mizunaga, T. (1991). Glycosylation site binding protein and protein disulfide isomerase are identical and essential for cell viability in yeast. Proc. Natl. Acad. Sci. USA 88, 4453–4457.
Pollitt, S., and Zalkin, H. (1983). Role of primary structure and disulfide bond formation in ß-lactamase secretion. J. Bacteriol. 153, 27–32.
Scheele, G., and Jacoby, R. (1982). Conformational changes associated with proteolytic processing of presecretory proteins allow glutathione-catalyzed formation of native disulfide bonds. J. Biol. Chem. 257, 12277–12282.
Sikorski, R. S., and Boeke, J. (1991). In vitro mutagenesis and plasmid shuffling: from cloned gene to mutant yeast. Methods Enzymol. 194, 302–318.
Silberstein, S., Kelleher, D. and Gilmore, R. (1992). The 48-kDa Subunit of the Mammalian Oligosaccharyltransferase Complex Is Homologous to the Essential Yeast Prottein WBP1. J. Biol. Chem. 267, 23658–23663.
to Heesen, S., Janetzky, B., Lehle, L. and Aebi, M. (1992). The yeast WBP1is essential for oligosaccharyl transferase activity in vivo and in vitro. EMBO J. 11, 2071–2075.
to Heesen, S., Knauer, R., Lehle, L. and Aebi, M. (1993). Yeast Wbplp and Swplp form a protein complex essential for oligosaccharyl transferase activity. EMBO J. 12, 279–284.
Tachibana, C., and Stevens, T. H. (1992). The yeast EUG1gene encodes an endoplasmic reticulum protein that is functionally related to protein disulfide isomerase. Mol. Cell Biol. 1, 4601–4611.
Vuori, K, Myllyla, R., Pihlajaniemi, T., and Kivirikko, K. I. (1992). Expression and site-directed mutagenesis of human protein disulfide isomerase in Escherichia coli. J. Biol. Chem. 267, 7211–7214.
Waechter, C. J. and Lennarz, W. J. (1976). The role of polyprenyl-linked sugars in glycoprotein synthesis. Ann. Rev. Biochem., 95–112.
Welply, J., Shenbagamurthi, P., Lennarz, W. J. and Naider, F. (1983). Substrate recognition by oligosacchryl transferase: Studies on glycosylation of modified Asn-XThr/Ser tripeptides. J. Biol. Chem. 258, 11856–11863.
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Roos, J., LaMantia, M., Lennarz, W.J. (1994). Oligosaccharyl Transferase and Protein Disulfide Isomerase: Two Key Enzymes in the Endoplasmic Reticulum. In: Op den Kamp, J.A.F. (eds) Biological Membranes: Structure, Biogenesis and Dynamics. NATO ASI Series, vol 82. Springer, Berlin, Heidelberg. https://doi.org/10.1007/978-3-642-78846-8_28
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