GTP-binding Ypt1 protein and Ca2+ function independently in a cell-free protein transport reaction

TitleGTP-binding Ypt1 protein and Ca2+ function independently in a cell-free protein transport reaction
Publication TypeJournal Article
Year of Publication1990
AuthorsBaker, D., Wuestehube L., Schekman R., Botstein D., & Segev N.
JournalProceedings of the National Academy of Sciences of the United States of America
Volume87
Issue1
Pagination355-9
Date Published1990 Jan
ISSN0027-8424
KeywordsAntibodies, Antigen-Antibody Complex, Biological Transport, Calcium, Cell-Free System, Egtazic Acid, Endoplasmic Reticulum, Fungal Proteins, Golgi Apparatus, Guanosine Triphosphate, Kinetics, Manganese, Microsomes, Plasmids, Primary Publication, Proteins, rab GTP-Binding Proteins, Saccharomyces cerevisiae, Saccharomyces cerevisiae Proteins
Abstract

The 21-kDa GTP-binding Ypt1 protein (Ypt1p) is required for protein transport from the endoplasmic reticulum to the Golgi complex in yeast extracts. Ypt1 antibodies block transport; this inhibition is alleviated by competition with excess purified Ypt1p produced in bacteria. Furthermore, extracts of cells carrying the mutation ypt1-1 are defective in transport, but transport is restored if a cytosolic fraction from wild-type cells is provided. The in vitro transport reaction also requires physiological levels of Ca2+. However, Ypt1p functions independently of Ca2+. First, buffering the free Ca2+ at concentrations ranging from 1 nM to 10 microM does not relieve inhibition by Ypt1 antibodies. Second, consumption of a Ca2+-requiring intermediate that accumulates in Ca2+-deficient incubations is not inhibited by anti-Ypt1 antibodies, although completion of transport requires ATP and an N-ethylmaleimide-sensitive factor. Thus, Ypt1p and Ca2+ are required at distinct steps.

Alternate JournalProc. Natl. Acad. Sci. U.S.A.
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