The yeast mating pheromone precursor prepro-alpha factor was fused to C-terminal signals for glycosyl-phosphatidylinositol (GPI) anchor attachment, based on the sequence of the Saccharomyces cerevisiae protein Gas1p. Maturation of fusion proteins expressed in vivo required the presence of both a functional GPI attachment site and the synthesis of GPI precursors. Constructs were translated in vitro for use in cell-free studies of glycolipid attachment. The radiolabelled polypeptides were post-translationally translocated into yeast microsomes, where at least one third of the molecules received a GPI anchor. This approach offers distinct advantages over anchor attachment reactions that require co-translational translocation of secretory peptide substrates.
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December 1997
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Research Article|
December 01 1997
Glycosyl-phosphatidylinositol anchor attachment in a yeast in vitro system
L. Tamara DOERING
;
L. Tamara DOERING
1
1Department of Molecular and Cell Biology, Barker Hall, Howard Hughes Research Institute, University of California, Berkeley, CA 94720-3202, U.S.A.
1To whom correspondence should be addressed, at present address: Cornell University Medical College, Department of Pharmacology, LC-218A, 1300 York Avenue, New York, NY 10021, U.S.A.
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Randy SCHEKMAN
Randy SCHEKMAN
1Department of Molecular and Cell Biology, Barker Hall, Howard Hughes Research Institute, University of California, Berkeley, CA 94720-3202, U.S.A.
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Biochem J (1997) 328 (2): 669–675.
Article history
Received:
April 21 1997
Revision Received:
August 07 1997
Accepted:
August 13 1997
Citation
L. Tamara DOERING, Randy SCHEKMAN; Glycosyl-phosphatidylinositol anchor attachment in a yeast in vitro system. Biochem J 1 December 1997; 328 (2): 669–675. doi: https://doi.org/10.1042/bj3280669
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