Distinct coated vesicles labeled for p200 bud from trans-Golgi network membranes.

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Abstract
Golgi-associated cytoplasmic proteins, such as the coatomer protein complex, are required for vesicle budding and trafficking. We have previously described a cytoplasmic phosphoprotein, p200, which binds dynamically and specifically to Golgi membranes. The p200 protein is dissociated from Golgi membranes in the presence of brefeldin A and it is induced to bind to Golgi membranes by activation of guanine nucleotide binding proteins (G proteins) with guanosine 5'-[gamma-thio]triphosphate or aluminum fluoride. To establish the role of p200 in vesicle budding, we localized membrane-bound p200 in intact cells and on isolated Golgi membranes. We show that p200 is preferentially associated with vesicles in the trans-Golgi network (TGN). Activation of G proteins induced budding and accumulation of small, coated vesicles from Golgi membranes and p200 was localized on the cytoplasmic surface of some of these vesicles. Using immunogold labeling we further demonstrate that p200 and beta-COP are localized on different populations of Golgi-derived vesicles. These data establish that p200 is involved in the budding and coating of a class of Goli vesicles that are likely to be derived from the TGN. The data also show that there are distinct populations of non-clathrin-coated vesicles budded from Golgi membranes, and vesicles labeled for either beta-COP or p200 may represent transport vesicles for separate steps of protein transport.