Characterization of vesicular membrane-bound alpha-SNAP and NSF in adrenal chromaffin cells.
alpha-SNAP and NSF are thought to act as soluble factors, which transiently bind to a complex formed between syntaxin and SNAP-25 located at the plasma membrane and synaptobrevin at the secretory vesicle membrane, at the moment of exocytosis. Here we present data which permit the novel conclusion that alpha-SNAP and NSF are not soluble in adrenal chromaffin cells but are rather membrane-bound in particular to undocked chromaffin vesicles. Evidence for this new paradigm is derived from several experimental approaches. First, alpha-SNAP and NSF were found predominantly at cellular membranes and not in the cytosol of cracked chromaffin cells. Second, alpha-SNAP and NSF were not released from membranes by Mg2+ATP, which causes priming of vesicles. Third, immune electron microscopy and immunoblotting of chromaffin vesicles purified by immunoisolation or density gradient centrifugation revealed the presence of alpha-SNAP and NSF together with typical vesicular proteins such as synaptobrevin and synaptotagmin. In the sucrose gradient 30% alpha-SNAP and 27% NSF were recovered with chromaffin vesicles. Bound alpha-SNAP was quantified (14 molecules/vesicle), and binding was characterized with recombinant his6-tagged alpha-SNAP. Overlay blots revealed that alpha-SNAP is bound to vesicular SNAP-25 and endogenous NSF. Our data show that mature chromaffin vesicles already contain specifically bound alpha-SNAP and NSF before docking at the plasmalemma.
Banaschewski
C
C
Anatomisches Institut der Technischen Universität München, Germany.
Höhne-Zell
B
B
Ovtscharoff
W
W
Gratzl
M
M
D000255:Adenosine Triphosphate / Q000502:physiology; D000313:Adrenal Medulla; D000818:Animals; D001665:Binding Sites; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D002417:Cattle; D002499:Centrifugation, Density Gradient; D019439:Chromaffin Cells / Q000378:metabolism; D003594:Cytoplasmic Granules / Q000378:metabolism; D007425:Intracellular Membranes / Q000378:metabolism*; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D052066:N-Ethylmaleimide-Sensitive Proteins; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012995:Solubility; D052067:Soluble N-Ethylmaleimide-Sensitive Factor Attachment Proteins; D033921:Vesicular Transport Proteins
D002352:Carrier Proteins; D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; D052067:Soluble N-Ethylmaleimide-Sensitive Factor Attachment Proteins; D033921:Vesicular Transport Proteins; D000255:Adenosine Triphosphate; D052066:N-Ethylmaleimide-Sensitive Proteins