A possible predocking attachment site for N-ethylmaleimide-sensitive fusion protein. Insights from in vitro endosome fusion.
N-Ethylmaleimide-sensitive fusion protein (NSF) is an ubiquitous protein required for multiple vesicular transport events. We have investigated the role of the two nucleotide-binding regions of NSF in endosomal fusion by analyzing NSF mutants in a cell-free system. Our results indicate that mutations on the first ATP-binding domain, that render a protein defective in either ATP binding or ATP hydrolysis, results in almost complete inhibition of endosomal fusion. A mutation in the second ATP-binding site of NSF was only slightly inhibitory. The inhibitory effect was observed only when the mutant proteins were added at early times during the fusion reaction indicating that NSF may be required for an early step during the docking/fusion process. Binding studies using Western blotting reveal that the binding of NSF mutants to endosomal membranes is differentially affected by Ca2+. Our results indicate that NSF, depending on its nucleotide state, may interact with membranes via an alternate mechanism. Our findings suggest the existence of a predocking binding site either independent of the docking complex or a site that leads to the formation of the SNAP-SNARE complex (e.g. 20 S particle).
Colombo
M I
MI
Department of Cell Biology and Physiology, Washington University, School of Medicine, St. Louis, Missouri 63110, USA.
Taddese
M
M
Whiteheart
S W
SW
Stahl
P D
PD
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D011992:Endosomes / Q000378:metabolism*; D005033:Ethylmaleimide / Q000494:pharmacology; D007150:Immunohistochemistry; D066298:In Vitro Techniques; D008561:Membrane Fusion; D051379:Mice; D052066:N-Ethylmaleimide-Sensitive Proteins; D017354:Point Mutation; D033921:Vesicular Transport Proteins
D002352:Carrier Proteins; D033921:Vesicular Transport Proteins; D000255:Adenosine Triphosphate; D052066:N-Ethylmaleimide-Sensitive Proteins; C498733:Nsf protein, mouse; D005033:Ethylmaleimide