The activity of Golgi transport vesicles depends on the presence of the N-ethylmaleimide-sensitive factor (NSF) and a soluble NSF attachment protein (alpha SNAP) during vesicle formation.
An assay designed to measure the formation of functional transport vesicles was constructed by modifying a cell-free assay for protein transport between compartments of the Golgi (Balch, W. E., W. G. Dunphy, W. A. Braell, and J. E. Rothman. 1984. Cell. 39:405-416). A 35-kD cytosolic protein that is immunologically and functionally indistinguishable from alpha SNAP (soluble NSF attachment protein) was found to be required during vesicle formation. SNAP, together with the N-ethylmaleimide-sensitive factor (NSF) have previously been implicated in the attachment and/or fusion of vesicles with their target membrane. We show that NSF is also required during the formation of functional vesicles. Strikingly, we found that after vesicle formation, the NEM-sensitive function of NSF was no longer required for transport to proceed through the ensuing steps of vesicle attachment and fusion. In contrast to these functional tests of vesicle formation, SNAP was not required for the morphological appearance of vesicular structures on the Golgi membranes. If SNAP and NSF have a direct role in transport vesicle attachment and/or fusion, as previously suggested, these results indicate that these proteins become incorporated into the vesicle membranes during vesicle formation and are brought to the fusion site on the transport vesicles.
Wattenberg
B W
BW
Cell Biology Unit, Upjohn Company, Kalamazoo, Michigan 49007.
Raub
T J
TJ
Hiebsch
R R
RR
Weidman
P J
PJ
D000818:Animals; D001693:Biological Transport, Active / Q000187:drug effects / Q000502:physiology; D016466:CHO Cells; D002352:Carrier Proteins / Q000502:physiology*; D002462:Cell Membrane / Q000187:drug effects / Q000502:physiology; D002845:Chromatography; D006224:Cricetinae; D003600:Cytosol / Q000187:drug effects / Q000502:physiology; D005033:Ethylmaleimide / Q000494:pharmacology; D006056:Golgi Apparatus / Q000502:physiology*; D015151:Immunoblotting; D008565:Membrane Proteins / Q000502:physiology*; D052066:N-Ethylmaleimide-Sensitive Proteins; D011319:Primaquine / Q000494:pharmacology; D052067:Soluble N-Ethylmaleimide-Sensitive Factor Attachment Proteins; D033921:Vesicular Transport Proteins
D002352:Carrier Proteins; D008565:Membrane Proteins; D052067:Soluble N-Ethylmaleimide-Sensitive Factor Attachment Proteins; D033921:Vesicular Transport Proteins; D052066:N-Ethylmaleimide-Sensitive Proteins; D011319:Primaquine; D005033:Ethylmaleimide