Reconstitution of SEC gene product-dependent intercompartmental protein transport
| Autor: | Manfred Schleyer, David Baker, Linda A Hicke, Randy Schekman, M F Rexach |
|---|---|
| Rok vydání: | 1988 |
| Předmět: |
Glycosylation
Golgi Apparatus Saccharomyces cerevisiae Spheroplasts Biology Endoplasmic Reticulum General Biochemistry Genetics and Molecular Biology Gene product symbols.namesake Adenosine Triphosphate Cytosol Freezing Protein Precursors Endoplasmic reticulum Biological Transport Thionucleotides Golgi apparatus Spheroplast Transport protein Cell biology Cold Temperature Kinetics Membrane Guanosine 5'-O-(3-Thiotriphosphate) SEC31 Mutation symbols Guanosine Triphosphate Mating Factor Peptides |
| Zdroj: | Cell. 54:335-344 |
| ISSN: | 0092-8674 |
| DOI: | 10.1016/0092-8674(88)90196-1 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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