|
|
|
|
|
|
|
||
© The Rockefeller University Press,
0021-9525/1998//511 $5.00
The Journal of Cell Biology, Volume 140, Number 3,
, 1998 511-523
Article |
Cell-free Transport to Distinct Golgi Cisternae Is Compartment Specific and ARF Independent
The small GTPase ADP-ribosylation factor (ARF) is absolutely required for coatomer vesicle formation on Golgi membranes but not for anterograde transport to the medial-Golgi in a mammalian in vitro transport system. This might indicate that the in vivo mechanism of intra-Golgi transport is not faithfully reproduced in vitro, or that intra-Golgi transport occurs by a nonvesicular mechanism. As one approach to distinguishing between these possibilities, we have characterized two additional cell-free systems that reconstitute transport to the trans-Golgi (trans assay) and trans-Golgi network (TGN assay). Like in vitro transport to the medial-Golgi (medial assay), transport to the trans-Golgi and TGN requires cytosol, ATP, and N-ethylmaleimide–sensitive fusion protein (NSF). However, each assay has its own distinct characteristics of transport. The kinetics of transport to late compartments are slower, and less cytosol is needed for guanosine-5'-O-(3-thiotriphosphate) (GTP
S) to inhibit transport, suggesting that each assay reconstitutes a distinct transport event. Depletion of ARF from cytosol abolishes vesicle formation and inhibition by GTPS, but transport in all assays is otherwise unaffected. Purified recombinant myristoylated ARF1 restores inhibition by GTPS, indicating that the GTP-sensitive component in all assays is ARF. We also show that asymmetry in donor and acceptor membrane properties in the medial assay is a unique feature of this assay that is unrelated to the production of vesicles. These findings demonstrate that characteristics specific to transport between different Golgi compartments are reconstituted in the cell-free system and that vesicle formation is not required for in vitro transport at any level of the stack.
Abbreviations used in this paper: ARF, ADP-ribosylation factor; ERGIC, endoplasmic reticulum–Golgi intermediate compartment; GlcNAc, N-acetylglucosamine; GTPS, guanosine-5'-O-(3-thiotriphosphate); MESNA, 2-mercaptoethanesulfonic acid; myr-rARF1, recombinant myristoylated ARF1; NEM, N-ethylmaleimide; non-myr-rARF1, recombinant nonmyristoylated ARF1; NSF, NEM-sensitive fusion protein; STI, soybean trypsin inhibitor; VSV-G protein, vesicular stomatitis viral glycoprotein.
The authors are indebted to Dr. John Heuser, Robyn Roth, and Mike Morgan for their generous assistance in the electron microscopic portion of these studies. Drs. Paul Melançon, Ben Glick, Maurine Linder, and Steve Scholnick provided helpful comments and suggestions during the preparation of the manuscript. S. Happe also thanks Dr. C. Klein, Dr. J. Corbett, and B. Hunter for their input and encouragement during course of this work. Special thanks go to M. Cairns for preparing many of the materials used in these studies, to R. Kahn for providing ARF antibodies and myr-rARF1, and to S. Berger and P. Melançon for providing non-myr-rARF1.
This research was supported by National Institutes of Health (NIH) grant GM54428 to P. Weidman. S. Happe received one year of support from NIH Research Training Grant HL07050.
Address all correspondence to Peggy Weidman, Department of Biochemistry and Molecular Biology, Saint Louis University School of Medicine, St. Louis, MO 63104. Tel.: 314-577-8179. Fax: 314-577-8156. E-mail: weidmanp{at}wpogate.slu.edu
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Facebook 
Reddit 
Technorati 
Twitter What's this?
This article has been cited by other articles:
|
|
