Reconstitution of SEC gene product-dependent intercompartmental protein transport

TitleReconstitution of SEC gene product-dependent intercompartmental protein transport
Publication TypeJournal Article
Year of Publication1988
AuthorsBaker, D., Hicke L., Rexach M., Schleyer M., & Schekman R.
Date Published1988 Jul 29
KeywordsAdenosine Triphosphate, Biological Transport, Cold Temperature, Cytosol, Endoplasmic Reticulum, Freezing, Glycosylation, Golgi Apparatus, Guanosine 5'-O-(3-Thiotriphosphate), Guanosine Triphosphate, Kinetics, Mutation, Peptides, Primary Publication, Protein Precursors, Saccharomyces cerevisiae, Spheroplasts, Thionucleotides

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.

Alternate JournalCell
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