Abstract
Coatomer, a complex of seven proteins, appears to be the precursor of the coat structure of non-clathrin-coated Golgi-derived vesicles. Another component of this vesicle coat is the cytosolic protein ADP-ribosylation factor (ARF). Like coatomer, ARF appears to reversibly associate with Golgi membranes. We now report that ARF is required for coatomer binding to Golgi membranes and that myristoylated, but not non-myristoylated, ARF is the required species. We utilize an antibody directed against the beta-subunit of coatomer (beta-COP) to follow coatomer binding. ARF and beta-COP bind stoichiometrically to Golgi membranes. ARF-dependent beta-COP binding requires a membrane-associated protein, is saturable, and is enhanced in the presence of stable GTP analogues like guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S). ARF and beta-COP bind sequentially to Golgi membranes, since beta-COP can be bound to reisolated membranes that had been previously incubated with ARF and GTP gamma S. We conclude that membrane-bound ARF confers to Golgi membranes all of the requirements for specific beta-COP binding.
Highlights
Coatomer, a complex of seven proteins, appears to acterization of the Golgi-derived vesicle coat revealed the be the precursorof the coat structure of non-clathrin- presence of stoichiometric amounts of low molecular mass coated Golgi-derived vesicles
We report that ADP-ribosylation factor (ARF) is required for coatomer cytosol, the mechanism by which they bind to Golgi membinding to Golgi membranes and that myristoylated, but notnon-myristoylated,ARF is the requiredspecies
ARF Gilman, 1984).The localization of ARF to theGolgi complex and @-COP bind sequentially to Golgi memsbinrcaenes,in mammalian cells and the secretion phenotype in yeast of &COP can be bound to reisolated membranes that haadn arfl mutation (Stearns et al, 1990), as well as its subsebeen previously incubatedwith ARF and GTPrS
Summary
Incubation of purified coatomer with Golgi membranes resulted in the binding of a small amount of &COP to Characterization of Antibodies against Coatomer and ARF membranes (Fig. Pa). Immunoprecipitationof Coatomerfrom Bovine Brain Cytoin the presence of GTP or GTPyS.The level of P-COP binding was significantly increased when cytosol that had sol with CMIA10"CMlAlO is a monoclonal IgG2a produced been immunodepleted of coatomer was included in incubaafter immunization of mice with native coatomer. This antibody does not react with coatomer subunits in a Western blot a analysis, so the antigenic subunit is not known. The coatomer-depleted cytosol contained approximately 15% of the original level of coatomer, as determined by Western blot analysis for @-COP (Orci et al, 1993);the remaining coatomer did not appear to contribute a significant amount of @-COPin these experiments (Fig. 2a). ARF added.pmd incubated with coatomer, membranes, and GTPyS (Fig. 2a)
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