Peptides Glycosylated in the Endoplasmic Reticulum of Yeast Are Subsequently Deglycosylated by a Soluble Peptide: N-Glycanase Activity*
- From the ¶Department of Applied Molecular Biosciences, Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya 464-01, Japan and the ‡Department of Biochemistry and Cell Biology and the Institute for Cell and Developmental Biology, SUNY at Stony Brook, Stony Brook, New York 11794
Abstract
Several lines of evidence suggest that soluble peptide:N-glycanase (PNGase) is involved in the quality control system for newly synthesized glycoproteins in mammalian cells. Here we report the occurrence of a soluble PNGase activity inSaccharomyces cerevisiae. The enzyme, which was recovered in the cytosolic fraction, has a neutral pH optimum, and dithiothreitol is required for activity. All of these properties were similar to those of earlier described for mammalian PNGases. Interestingly, the yeast enzyme activity was found to be present almost exclusively in cells in stationary phase; little activity was detected in logarithmic growth phase cells. Upon incubation of a glycosylatable peptide R-Asn-X-Thr-R′ with permeabilized yeast spheroplasts, we detected formation of both glycosylated peptide and the peptide product expected from PNGase-mediated deglycosylation of this glycopeptide, namely, R-Asp-X-Thr-R′. Recent findings that yeast have an active system for the retrograde transport of unfolded (glyco)proteins and glycopeptides out of the endoplasmic reticulum (ER) into the cytosol raise the possibility that this PNGase may participate in an early step in degradation of these molecules following their export from the ER.
Footnotes
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↵* This work was supported by National Institutes of Health Grant GM33184 (to W. J. L.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵§ Japan Society for the Promotion of Science (JSPS) Postdoctoral Fellow for research abroad.
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↵‖ To whom correspondence should be addressed. Tel.: 516-632-8560; Fax: 516-632-8575; E-mail:wlennarz{at}life.bio.sunysb.edu.
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↵2 Strains used are as follows: PS593 (MATα leu2 ura3 trp1 his3), A364A (MATa ade1 his7 lys2 tyr1 ura1 gal1-1), L40 (MATa ade2-leu2 his3 trp1 LYS2::lexAop-HIS3 URA3::lexAop-lacZ), and Y153 (MATa ade2 leu2 his3 trp1 gal4 gal80 LYS2::UAS G -HIS3 URA3::UAS G lacZ).
- Abbreviations:
- PNGase
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peptide-N 4-(N-acetyl-β-d-glucosaminyl)asparagine amidase (peptide:N-glycanase (EC 3.5.1.52))
- BPhe
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p-benzoylphenylalanine
- CHO
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N-linked carbohydrate chain
- ConA
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concanavalin A
- DTT
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dithiothreitol
- Mes
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2-(N-morpholino)ethanesulfonic acid
- NEM
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N-ethylmaleimide
- ER
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endoplasmic reticulum
- Endo H
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endo-β-N-acetylglucosaminidase H.
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- Received May 4, 1998.
- Revision received June 26, 1998.
- The American Society for Biochemistry and Molecular Biology, Inc.
