Reconstitution of SEC gene product-dependent intercompartmental protein transport.
Transport of alpha-factor precursor from the endoplasmic reticulum to the Golgi apparatus has been reconstituted in gently lysed yeast spheroplasts. Transport is measured through the coupled addition of outer-chain carbohydrate to [35S]methionine-labeled alpha-factor precursor translocated into the endoplasmic reticulum of broken spheroplasts. The reaction is absolutely dependent on ATP, stimulated 6-fold by cytosol, and occurs between physically separable sealed compartments. Transport is inhibited by the guanine nucleotide analog GTP gamma S. sec23 mutant cells have a temperature-sensitive defect in endoplasmic reticulum-to-Golgi transport in vivo. This defect has been reproduced in vitro using sec23 membranes and cytosol. Transport at 30 degrees C with sec23 membranes requires addition of cytosol containing the SEC23 (wild-type) gene product. This demonstrates that an in vitro inter-organelle transport reaction depends on a factor required for transport in vivo. Complementation of sec mutations in vitro provides a functional assay for the purification of individual intercompartmental transport factors.
Baker
D
D
Department of Biochemistry, University of California, Berkeley 94720.
Hicke
L
L
Rexach
M
M
Schleyer
M
M
Schekman
R
R
D000255:Adenosine Triphosphate / Q000494:pharmacology; D001692:Biological Transport; D003080:Cold Temperature; D003600:Cytosol / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D005615:Freezing; D006031:Glycosylation; D006056:Golgi Apparatus / Q000378:metabolism*; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006160:Guanosine Triphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D007700:Kinetics; D000072235:Mating Factor; D009154:Mutation; D010455:Peptides / Q000378:metabolism*; D011498:Protein Precursors / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism / Q000648:ultrastructure; D013104:Spheroplasts / Q000378:metabolism*; D013873:Thionucleotides / Q000494:pharmacology
D010455:Peptides; D011498:Protein Precursors; D013873:Thionucleotides; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D000072235:Mating Factor; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate