The second step in glycosylphosphatidylinositol (GPI) biosynthesis, the deacetylation of GlcNAc-phosphatidylinositol (GlcNAc-PI), has been shown to be stimulated by GTP hydrolysis [Stevens (1993) J. Biol. Chem. 268, 9718-9724]. We have now developed a system to study this regulation that uses microsomes from cells defective in the first step in GPI biosynthesis (class A, C and H lymphoma mutants) and the second reaction in the pathway (G9PLAP.85). With this mixed-microsome system, the deacetylation of GlcNAc-PI was almost completely dependent on GTP hydrolysis. Because GlcNAc-PI synthesized by the G9PLAP.85 microsomes cannot readily move to the first-step-mutant microsomes to be deacetylated, this result indicated that the role of GTP was to facilitate the ‘apparent’ transfer of this substrate between membrane vesicles. The microsomes could be stably preactivated by pretreatment with GTP before GPI biosynthesis was initiated, indicating that fusion was the most likely mechanism for this regulation. GlcNAc-PI deacetylation could also be stably preactivated in EL4 microsomes, suggesting that fusion also occurred in wild-type membranes. Some differential localization of the GlcNAc-PI synthetic and deacetylation activities with the endoplasmic reticulum was found. Therefore fusion seems to stimulate GPI biosynthesis in mammalian microsomes by bringing together the first two enzymes in the pathway in the same membrane vesicle.
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Research Article|
July 26 1999
Stimulation of glycosylphosphatidylinositol biosynthesis in mammaliancell-free systems by GTP hydrolysis: evidence for the involvement of membrane fusion
Victoria L. STEVENS;
Victoria L. STEVENS
1
*Division of Cancer Biology, Department of Radiation Oncology, Emory University School of Medicine Atlanta, Loughlin Radiation Oncology Center, 145 Edgewood Avenue, S.E., Atlanta, GA 30335, U.S.A.
†Department of Biochemistry, Emory University School of Medicine Atlanta, GA 30335, U.S.A.
1To whom correspondence should be addressed at the Department of Radiation Oncology (e-mail vlsteve@emory.edu).
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Hui ZHANG;
Hui ZHANG
*Division of Cancer Biology, Department of Radiation Oncology, Emory University School of Medicine Atlanta, Loughlin Radiation Oncology Center, 145 Edgewood Avenue, S.E., Atlanta, GA 30335, U.S.A.
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Eva Szucs KRISTYANNE
Eva Szucs KRISTYANNE
*Division of Cancer Biology, Department of Radiation Oncology, Emory University School of Medicine Atlanta, Loughlin Radiation Oncology Center, 145 Edgewood Avenue, S.E., Atlanta, GA 30335, U.S.A.
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Publisher: Portland Press Ltd
Received:
December 18 1998
Revision Received:
April 13 1999
Accepted:
May 17 1999
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London © 1999
1999
Biochem J (1999) 341 (3): 577–584.
Article history
Received:
December 18 1998
Revision Received:
April 13 1999
Accepted:
May 17 1999
Citation
Victoria L. STEVENS, Hui ZHANG, Eva Szucs KRISTYANNE; Stimulation of glycosylphosphatidylinositol biosynthesis in mammaliancell-free systems by GTP hydrolysis: evidence for the involvement of membrane fusion. Biochem J 1 August 1999; 341 (3): 577–584. doi: https://doi.org/10.1042/bj3410577
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