The small GTP-binding protein, ADP-ribosylation factor (ARF), has previously been shown to mediate the binding to Golgi membranes of the coatomer of non-cathrin-coated (COP-coated) vesicles. We now report that ARF is also required for the binding of the AP-1 adaptor protein of clathrin-coated vesicles to Golgi membranes. The binding of coat proteins from both clathrin- and COP-coated vesicles requires an additional Golgi membrane-associated factor. These results suggest that a mechanistic similarity underlies diverse types of vesicle coats.
Electron microscope immunocytochemistry reveals that both anterograde-directed (proinsulin and VSV G protein) and retrograde-directed (the KDEL receptor) cargo are present in COPI-coated vesicles budding from every level of the Golgi stack in whole cells; however, they comprise two distinct populations that together can account for at least 80% of the vesicles budding from Golgi cisternae. Segregation of anterograde- from retrograde-directed cargo into distinct sets of COPI-coated vesicles is faithfully reproduced in the cell-free Golgi transport system, in which VSV G protein and KDEL receptor are packaged into separable vesicles, even when budding is driven by highly purified coatomer and a recombinant ARF protein.
Cytoplasmic domains of members of the p24 family of putative cargo receptors were shown to bind to coatomer, the coat protein of COPI-coated transport vesicles. Domains that contained dilysine endoplasmic reticulum retrieval signals bound the alpha-, beta'-, and epsilon-COP subunits of coatomer, whereas other p24 domains bound the beta-, gamma-, and zeta-COP subunits and required a phenylalanine-containing motif. Transit of a CD8-p24 chimera from the endoplasmic reticulum through the Golgi complex was slowed when the phenylalanine motif was mutated, suggesting that this motif may function as an anterograde transport signal. The either-or bimodal binding of coatomer to p24 tails suggests models for how coatomer can potentially package retrograde-directed and anterograde-directed cargo into distinct COPI-coated vesicles.
We have isolated a major integral membrane protein from Golgi-derived coatomer-coated vesicles. This 24-kDa protein, p24, defines a family of integral membrane proteins with homologs present in yeast and humans. In addition to sequence similarity, all p24 family members contain a motif with the characteristic heptad repeats found in coiled coils. When the yeast p24 isoform, yp24A, is knocked out in a strain defective for vesicle fusion, a dramatic reduction in the accumulation of transport vesicles is observed.
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