Abstract
ADP-ribosylation factor (ARF) is a small GTP-binding protein that has been implicated in intracellular vesicular transport. ARF regulates the budding of vesicles that mediate endoplasmic reticulum to Golgi and intra-Golgi transport. It also plays an important role in maintaining the function and morphology of the Golgi apparatus. Using a permeabilized cell system derived from GH3 cells, we provide evidence that ARF-1 regulates the formation of nascent secretory vesicles from the trans-Golgi network. Both myristoylated and non-myristoylated forms of recombinant human ARF-1 enhanced secretory vesicle budding about 2-fold. A mutant lacking the first 17 N-terminal residues, as well as one that preferentially binds GDP (T31N) did not stimulate vesicle formation. In contrast, a mutant defective in GTP hydrolysis (Q71L) promoted vesicle budding. Strikingly, a peptide corresponding to the N terminus of human ARF-1 (amino acids 2-17) also stimulated vesicle budding from the trans-Golgi network, in marked contrast to its inhibitory effect on vesicular transport from the endoplasmic reticulum to Golgi. These data demonstrate that in endocrine cells, ARF-1 and in particular its N terminus play an essential role in the formation of secretory vesicles.
Highlights
ADP-ribosylation factor (ARF)1 is a Mr 20,000 polypeptide that is a member of the Ras superfamily of small GTP-binding proteins
Other in vitro studies have shown that ARF-1 is required for the binding of ␥-adaptin to isolated Golgi membranes [10, 11], suggesting that it functions in the assembly of clathrin coats at the TGN
Experiments in which native and mutant ARF polypeptides were overexpressed in Chinese hamster ovary and normal rat kidney cells have established a key role for ARF in ER to Golgi, intra-Golgi transport, and maintenance of Golgi morphology in vivo [12, 13]
Summary
ADP-ribosylation factor (ARF)1 is a Mr 20,000 polypeptide that is a member of the Ras superfamily of small GTP-binding proteins. To determine if nascent secretory vesicle budding from the TGN might be ARF-1-regulated, we used recombinant human ARF-1 in our permeabilized cell system. Permeabilized GH3 cells were incubated in the absence and presence of the ARF-1 polypeptides, and nascent secretory vesicles were separated by brief centrifugation and analyzed for immunoprecipitable GH (Fig. 1).
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