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Journal of Cell Science, Vol 111, Issue 17 2625-2633, Copyright © 1998 by Company of Biologists
JOURNAL ARTICLES |
F Peter, SH Wong, VN Subramaniam, BL Tang and W Hong
Membrane Biology Laboratory, Institute of Molecular and Cell Biology, Singapore 117609, Singapore.
N-ethylmaleimide-sensitive factor (NSF) and soluble NSF attachment proteins (SNAPs) have been implicated in diverse vesicular transport events; yet their exact role and site of action remain to be established. Using an established in vitro system, we show that antibodies against alpha-SNAP inhibit vesicle transport from the ER to the cis-Golgi and that recombinant alpha-SNAP enhances/stimulates the process. Cytosol immunodepleted of alpha-SNAP does not support normal transport unless supplemented with recombinant alpha-SNAP but not gamma-SNAP. In marked contrast, cytosol immunodepleted of gamma-SNAP supports ER-Golgi transport to the normal level. Neither antibodies against gamma-SNAP nor recombinant gamma-SNAP have any effect on ER-Golgi transport. These results clearly establish an essential role for alpha-SNAP but not gamma-SNAP in ER-Golgi transport. When the transport assay is performed with cytosol immunodepleted of alpha-SNAP, followed by incubation with cytosol immunodepleted of a COPII subunit, normal transport is achieved. In marked contrast, no transport is detected when the assay is first performed with cytosol depleted of the COPII subunit followed by alpha-SNAP-depleted cytosol, suggesting that alpha-SNAP is required after a step that requires COPII (the budding step). In combination with cytosol immunodepleted of Rab1, it is seen that alpha-SNAP is required after a Rab1-requiring step. It has been shown previously that EGTA blocks ER-Golgi transport at a step after vesicle docking but before fusion and we show here that alpha-SNAP acts before the step that is blocked by EGTA. Our results suggest that alpha-SNAP may be involved in the pre-docking or docking but not the fusion process.
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 K. Tani, M. Shibata, K. Kawase, H. Kawashima, K. Hatsuzawa, M. Nagahama, and M. Tagaya Mapping of Functional Domains of gamma -SNAP J. Biol. Chem., April 4, 2003; 278(15): 13531 - 13538. [Abstract] [Full Text] [PDF]
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H. Horstmann, C. P. Ng, B. L. Tang, and W. Hong Ultrastructural characterization of endoplasmic reticulum -- Golgi transport containers (EGTC) J. Cell Sci., November 15, 2002; 115(22): 4263 - 4273. [Abstract] [Full Text] [PDF]
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W Hong Protein transport from the endoplasmic reticulum to the Golgi apparatus J. Cell Sci., January 10, 1998; 111(19): 2831 - 2839. [Abstract] [PDF]
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D. Chen, W. Xu, P. He, E. E. Medrano, and S. W. Whiteheart Gaf-1, a gamma -SNAP-binding Protein Associated with the Mitochondria J. Biol. Chem., April 13, 2001; 276(16): 13127 - 13135. [Abstract] [Full Text] [PDF]
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