Cytosolic ARFs are required for vesicle formation but not for cell-free intra-Golgi transport: evidence for coated vesicle-independent transport.
We investigated the role of ADP-ribosylation factors (ARFs) in Golgi function using biochemical and morphological cell-free assays. An ARF-free cytosol produced from soluble Chinese hamster ovary (CHO) extracts supports intra-Golgi transport by a mechanism that is biochemically indistinguishable from control transport reactions: ARF-free transport reactions are NSF-dependent, remain sensitive to the donor Golgi-specific inhibitor ilimaquinone, and exhibit kinetics that are identical to that of control reactions containing ARFs. In contrast, ARF-free cytosol does not support the formation of coated vesicles on Golgi cisternae. However, vesicle formation is reconstituted upon the addition of ARF1. These data suggest that neither soluble ARFs nor coated vesicle formation are essential for transport. We conclude that cell-free intra-Golgi transport proceeds via a coated vesicle-independent mechanism regardless of vesicle formation on Golgi cisternae.
Department of Chemistry and Biochemistry, University of Colorado, Boulder 80303-0215.
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Taylor
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Kanstein
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Weidman
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Melançon
D020823:ADP-Ribosylation Factor 1; D020727:ADP-Ribosylation Factors; D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D016466:CHO Cells; D002474:Cell-Free System; D003034:Coated Pits, Cell-Membrane / Q000378:metabolism*; D006224:Cricetinae; D003412:Cricetulus; D003600:Cytosol / Q000378:metabolism; D005033:Ethylmaleimide / Q000494:pharmacology; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006031:Glycosylation; D006056:Golgi Apparatus / Q000378:metabolism* / Q000648:ultrastructure; D007425:Intracellular Membranes / Q000378:metabolism / Q000648:ultrastructure; D007700:Kinetics; D011809:Quinones / Q000494:pharmacology
D011809:Quinones; C067346:illimaquinone; D019204:GTP-Binding Proteins; D020823:ADP-Ribosylation Factor 1; D020727:ADP-Ribosylation Factors; D005033:Ethylmaleimide