J. Cell Biol., Volume 140, Number 3, February 9, 1998 511-523

Cell-free Transport to Distinct Golgi Cisternae Is Compartment Specific and ARF Independent

Scott Happe, and Peggy Weidman

Department of Biochemistry and Molecular Biology, St. Louis University School of Medicine, St. Louis, Missouri 63104

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) (GTPS) 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.

CiteULike    Complore    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this?

This article has been cited by other articles:

• Xiang, Y., Seemann, J., Bisel, B., Punthambaker, S., Wang, Y. (2007). Active ADP-ribosylation Factor-1 (ARF1) Is Required for Mitotic Golgi Fragmentation. J. Biol. Chem. 282: 21829-21837 [Abstract] [Full Text]  
• Sztul, E., Lupashin, V. (2006). Role of tethering factors in secretory membrane traffic. Am. J. Physiol. Cell Physiol. 290: C11-C26 [Abstract] [Full Text]  
• Ellis, M. A., Miedel, M. T., Guerriero, C. J., Weisz, O. A. (2004). ADP-ribosylation Factor 1-independent Protein Sorting and Export from the trans-Golgi Network. J. Biol. Chem. 279: 52735-52743 [Abstract] [Full Text]  
• Pullikuth, A. K., Weidman, P. J. (2002). In Vitro Transport on Cis and Trans Sides of the Golgi Involves Two Distinct Types of Coatomer and ADP-ribosylation Factor-independent Transport Intermediates. J. Biol. Chem. 277: 50355-50364 [Abstract] [Full Text]  
• Boman, A. L., Zhang, C.-j., Zhu, X., Kahn, R. A. (2000). A Family of ADP-Ribosylation Factor Effectors That Can Alter Membrane Transport through the trans-Golgi. Mol. Biol. Cell 11: 1241-1255 [Abstract] [Full Text]  
• Kuai, J., Boman, A. L., Arnold, R. S., Zhu, X., Kahn, R. A. (2000). Effects of Activated ADP-ribosylation Factors on Golgi Morphology Require neither Activation of Phospholipase D1 nor Recruitment of Coatomer. J. Biol. Chem. 275: 4022-4032 [Abstract] [Full Text]  
• Lin, C.-C., Love, H. D., Gushue, J. N., Bergeron, J. J.M., Ostermann, J. (1999). ER/Golgi Intermediates Acquire Golgi Enzymes by Brefeldin A-Sensitive Retrograde Transport In Vitro. JCB 147: 1457-1472 [Abstract] [Full Text]  
• Dominguez, M., Fazel, A., Dahan, S., Lovell, J., Hermo, L., Claude, A., Melancon, P., Bergeron, J.J.M. (1999). Fusogenic Domains of Golgi Membranes Are Sequestered into Specialized Regions of the Stack that Can Be Released by Mechanical Fragmentation. JCB 145: 673-688 [Abstract] [Full Text]  
• Jones, A., Spiro, D., Kirchhausen, T, Melancon, P, Wessling-Resnick, M (1999). Studies on the inhibition of endosome fusion by GTPgammaS-bound ARF. J. Cell Sci. 112: 3477-3485 [Abstract]  
• Walter, D. M., Paul, K. S., Waters, M. G. (1998). Purification and Characterization of a Novel 13 S Hetero-oligomeric Protein Complex That Stimulates in Vitro Golgi Transport. J. Biol. Chem. 273: 29565-29576 [Abstract] [Full Text]  

Home | Help | Feedback | Subscriptions | Archive | Search | Table of Contents