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J. Biol. Chem., Vol. 278, Issue 16, 13959-13967, April 18, 2003
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From the Department of Immunoregulation, Research Institute for
Microbial Diseases, Osaka University, Osaka 565-0871, Japan
Many eukaryotic proteins are tethered to the
plasma membrane via glycosylphosphatidylinositol (GPI). GPI
transamidase is localized in the endoplasmic reticulum and mediates
post-translational transfer of preformed GPI to proteins bearing a
carboxyl-terminal GPI attachment signal. Mammalian GPI transamidase is
a multimeric complex consisting of at least five subunits. Here we
report that two subunits of mammalian GPI transamidase, GPI8 and PIG-T,
form a functionally important disulfide bond between conserved cysteine
residues. GPI8 and PIG-T mutants in which relevant cysteines were
replaced with serines were unable to fully restore the surface
expression of GPI-anchored proteins upon transfection into their
respective mutant cells. Microsomal membranes of these transfectants
had markedly decreased activities in an in vitro
transamidase assay. The formation of this disulfide bond is not
essential but required for full transamidase activity. Antibodies
against GPI8 and PIG-T revealed that endogenous as well as exogenous
proteins formed a disulfide bond. Furthermore trypanosome GPI8 forms a
similar intermolecular disulfide bond via its conserved cysteine
residue, suggesting that the trypanosome GPI transamidase is also a
multimeric complex likely containing the orthologue of PIG-T. We also
demonstrate that an inactive human GPI transamidase complex that
consists of non-functional GPI8 and four other components was
co-purified with the proform of substrate proteins, indicating that
these five components are sufficient to hold the substrate proteins.
Two Subunits of Glycosylphosphatidylinositol Transamidase,
GPI8 and PIG-T, Form a Functionally Important Intermolecular
Disulfide Bridge*
,
*
This work was supported by grants from the Ministry of
Education, Culture, Sports, Science and Technology of Japan.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.
Present address: Advanced Medical Discovery Inst. of the
University Health Network and Ontario Cancer Inst., University of Toronto, Toronto, Ontario M5G 2C1, Canada.
§
To whom correspondence should be addressed: Dept. of
Immunoregulation, Research Inst. for Microbial Diseases, Osaka
University, 3-1 Yamada-oka, Suita, Osaka 565-0871, Japan. Tel.:
81-6-6879-8328; Fax: 81-6-6875-5233; E-mail:
tkinoshi@biken.osaka-u.ac.jp.
Copyright © 2003 by The American Society for Biochemistry and Molecular Biology, Inc.
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