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PMID: 9371730 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't

Glycosyl-phosphatidylinositol anchor attachment in a yeast in vitro system.

The Biochemical journal ·Vol. 328 ( Pt 2) ·1997-12-01 ·Pages 669-75

Doering TL, Schekman R

Abstract

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.

MeSH Terms
Biological Transport Cell-Free System Fungal Proteins/genetics,metabolism Glycosylphosphatidylinositols/metabolism Membrane Glycoproteins/genetics,metabolism Protein Precursors/genetics,metabolism Recombinant Fusion Proteins/metabolism Research Design Saccharomyces cerevisiae/genetics,metabolism Saccharomyces cerevisiae Proteins
Chemicals
Fungal Proteins GAS1 protein, S cerevisiae Glycosylphosphatidylinositols MF(ALPHA)1 protein, S cerevisiae Membrane Glycoproteins Protein Precursors Recombinant Fusion Proteins Saccharomyces cerevisiae Proteins
Authors & Affiliations
2 authors, click to expand affiliations / ORCID
Doering T L
Department of Molecular and Cell Biology, Barker Hall, Howard Hughes Research Institute, University of California, Berkeley, CA 94720-3202, USA.
Schekman R
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Article Info
Journal
The Biochemical journal
Abbr.
Biochem J
ISSN
0264-6021
DOI
10.1042/bj3280669
Published
1997-12-01
Pages
669-75
Language
English
Region
England
NLM ID
2984726R
PMCID
PMC1218970
Subset
IM
External Links
PubMed source DOI: 10.1042/bj3280669 PMC Full Text View journal details
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