Alpha-SNAP but not gamma-SNAP is required for ER-Golgi transport after vesicle budding and the Rab1-requiring step but before the EGTA-sensitive step.
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.
Peter
F
F
Membrane Biology Laboratory, Institute of Molecular and Cell Biology, Singapore 117609, Singapore.
Wong
S H
SH
Subramaniam
V N
VN
Tang
B L
BL
Hong
W
W
D000906:Antibodies / Q000378:metabolism; D001692:Biological Transport / Q000187:drug effects / Q000502:physiology; D002352:Carrier Proteins / Q000378:metabolism / Q000502:physiology*; D004533:Egtazic Acid / Q000494:pharmacology*; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D019204:GTP-Binding Proteins / Q000502:physiology; D006056:Golgi Apparatus / Q000378:metabolism*; D006801:Humans; D008565:Membrane Proteins / Q000378:metabolism / Q000502:physiology*; D008954:Models, Biological; D015388:Organelles / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism / Q000494:pharmacology; D052067:Soluble N-Ethylmaleimide-Sensitive Factor Attachment Proteins; D033921:Vesicular Transport Proteins; D020761:rab1 GTP-Binding Proteins
D000906:Antibodies; D002352:Carrier Proteins; D008565:Membrane Proteins; D011994:Recombinant Proteins; D052067:Soluble N-Ethylmaleimide-Sensitive Factor Attachment Proteins; D033921:Vesicular Transport Proteins; D004533:Egtazic Acid; D019204:GTP-Binding Proteins; D020761:rab1 GTP-Binding Proteins