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J. Biol. Chem., Vol. 275, Issue 43, 33222-33230, October 27, 2000
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From the Bidirectional transport of proteins via the
Sec61p translocon across the endoplasmic reticulum (ER) membrane is a
recognized component of the ER quality control machinery. Following
translocation and engagement by the luminal quality control system,
misfolded and unassembled proteins are exported from the ER lumen back
to the cytosol for degradation by the proteasome. Additionally, other ER contents, including oligosaccharides, oligopeptides, and
glycopeptides, are efficiently exported from mammalian and yeast
systems, indicating that bidirectional transport across ER membranes is
a general eukaryotic phenomenon. Glycopeptide and protein export from
the ER in in vitro systems is both ATP- and
cytosol-dependent. Using a well established system to study
glycopeptide export and conventional liquid chromatography, we isolated
a single polypeptide species of 23 kDa from rat liver cytosol that was
capable of fully supporting glycopeptide export from rat microsomes in
the presence of an ATP-regenerating system. The protein was identified
by mass spectrometric sequence analysis as guanylate kinase (GK), a
housekeeping enzyme critical in the regulation of cellular GTP levels.
We confirmed the ability of GK to substitute for complete cytosol by
reconstitution of glycopeptide export from rat liver microsomes using
highly purified recombinant GK from Saccharomyces
cerevisiae. Most significantly, we found that the GK (and hence
the cytosolic component) requirement was fully bypassed by low
micromolar concentrations of GDP or GTP. Similarly, export was
inhibited by non-hydrolyzable analogues of GDP and GTP, indicating a
requirement for GTP hydrolysis. Membrane integrity was fully maintained
under assay conditions, as no ER luminal proteins were released.
Competence for glycopeptide export was abolished by very mild protease
treatment of microsomes, indicating the presence of an essential
protein on the cytosolic face of the ER membrane. These data
demonstrate that export of glycopeptide export is controlled by a
microsomal GTPase and is independent of cytosolic protein factors.
A Microsomal GTPase Is Required for Glycopeptide Export from the
Mammalian Endoplasmic Reticulum*
,
Wellcome Trust Laboratories for Molecular
Parasitology, Department of Biochemistry, Imperial College of
Science, Technology, and Medicine, Exhibition Road, London SW7 2AY,
United Kingdom and the § Laboratory of Biological Mass
Spectrometry and Gaseous Ion Chemistry, Rockefeller University,
New York, New York 10021
*
This work was supported by Biotechnology and
Biological Sciences Research Council Project Grant 28/C09893 (to
M. C. F.) and National Institutes of Health Grant RR00862 (to
B. T. C.).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.
To whom correspondence should be addressed. Tel.:
44-171-5945277; Fax: 44-171-5945207; E-mail: mfield@ic.ac.uk.
Copyright © 2000 by The American Society for Biochemistry and Molecular Biology, Inc.
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