GTP-binding Ypt1 protein and Ca2+ function independently in a cell-free protein transport reaction.
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.
Baker
D
D
Division of Biochemistry and Molecular Biology, University of California, Berkeley 94720.
Wuestehube
L
L
Schekman
R
R
Botstein
D
D
Segev
N
N
D000906:Antibodies; D000936:Antigen-Antibody Complex; D001692:Biological Transport; D002118:Calcium / Q000378:metabolism* / Q000494:pharmacology; D002474:Cell-Free System; D004533:Egtazic Acid / Q000494:pharmacology; D004721:Endoplasmic Reticulum / Q000378:metabolism; D005656:Fungal Proteins / Q000378:metabolism*; D006056:Golgi Apparatus / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism*; D007700:Kinetics; D008345:Manganese / Q000378:metabolism / Q000494:pharmacology; D008861:Microsomes / Q000378:metabolism; D010957:Plasmids; D011506:Proteins / Q000276:immunology / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins; D020691:rab GTP-Binding Proteins
D000906:Antibodies; D000936:Antigen-Antibody Complex; D005656:Fungal Proteins; D011506:Proteins; D029701:Saccharomyces cerevisiae Proteins; D008345:Manganese; D004533:Egtazic Acid; D006160:Guanosine Triphosphate; C060755:YPT1 protein, S cerevisiae; D020691:rab GTP-Binding Proteins; D002118:Calcium