Rexach et al 1994

Rexach et al 1994 - Published September 1, 1994...

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Characteristics of Endoplasmic Reticulum-derived Transport Vesicles Michael E Rexach, Martin Latterich, and Randy W. Schekman Department of Molecular and Cell Biology and Howard Hughes Medical Research Institute, University of California, Berkeley, Berkeley, California 94720 Abstract. We have isolated vesicles that mediate pro- tein transport from the ER to Golgi membranes in perforated yeast. These vesicles, which form de novo during in vitro incubations, carry lumenal and mem- brane proteins that include core-glycosylated pro- ~x-factor, Betl, Sec22, and Bosl, but not ER-resident Kar2 or Sec61 proteins. Thus, lumenal and membrane proteins in the ER are sorted prior to transport vesicle scission. Inhibition of Yptlp-function, which prevents newly formed vesicles from docking to cis-Golgi membranes, was used to block transport. Vesicles that accumulate are competent for fusion with cis-Golgi membranes, but not with ER membranes, and thus are functionally committed to vectorial transport. A 900- fold enrichment was developed using differential cen- trifugation and a series of velocity and equilibrium density gradients. Electron microscopic analysis shows a uniform population of 60 nm vesicles that lack pe- ripheral protein coats. Quantitative Western blot anal- ysis indicates that protein markers of cytosol and cel- lular membranes are depleted throughout the purification, whereas the synaptobrevin-like Betl, Sec22, and Bosl proteins are highly enriched. Uncoated E___RR-derived transport vesicles (ERV) contain twelve major proteins that associate tightly with the membrane. The ERV proteins may represent abundant cargo and additional targeting molecules. M ORPHOLOGICAL analysis of eukaryotic cells led to the hypothesis that small vesicles mediate the directional transport of proteins between succes- sive organelles of the secretory pathway (Jamieson and Palade, 1967; Palade, 1975). The machinery that catalyzes vesicular transport is now being studied at the molecular level using in vitro assays that reconstitute intercompartmen- tal protein transport in perforated cells and membrane ex- tracts (Pryer et al., 1992). The characterization of vesicles that mediate intercompartmental protein transport in vitro will help address the role of integral membrane proteins that function in vesicular traffic. Although most transport vesicles are transient intermedi- ates, they can be forced to accumulate as stable intermedi- ates when a component of the vesicle targeting machinery is inactivated. Most transport vesicles are 50-100 nm in size and may be coated with a peripheral protein lattice. In coated transport vesicles, the subunits of the coat are the most abun- dant proteins in pure vesicle preparations. Clathrin com- plexes coat vesicles derived from the trans-Golgi and plasma membranes (Brodsky, 1988). Assembly of clathrin lattices M. E Rexach's present address is Laboratory of Cell Biology, Box 168, The Rockefeller University, New York, NY 10021.
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Rexach et al 1994 - Published September 1, 1994...

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