Ca2+ and N-ethylmaleimide-sensitive factor differentially regulate disassembly of SNARE complexes on early endosomes.
The endosome-associated protein Hrs inhibits the homotypic fusion of early endosomes. A helical region of Hrs containing a Q-SNARE motif mediates this effect as well as its endosomal membrane association via SNAP-25, an endosomal receptor for Hrs. Hrs inhibits formation of an early endosomal SNARE complex by displacing VAMP-2 from the complex, suggesting a mechanism by which Hrs inhibits early endosome fusion. We examined the regulation of endosomal SNARE complexes to probe how Hrs may function as a negative regulator. We show that although NSF dissociates the VAMP-2.SNAP-25.syntaxin 13 complex, it has no effect on the Hrs-containing complex. Whereas Ca(2+) dissociates the Hrs-containing complex but not the VAMP-2-containing SNARE complex. This is the first demonstration of differential regulation of R/Q-SNARE and all Q-SNARE-containing SNARE complexes. Ca(2+) also reverses the Hrs-induced inhibition of early endosome fusion in a tetanus toxin-sensitive manner and removes Hrs from early endosomal membranes. Moreover, Hrs inhibition of endosome fusion and its endosomal localization are sensitive to bafilomycin, implying a role for luminal Ca(2+). Thus, Hrs may bind a SNARE protein on early endosomal membranes negatively regulating trans-SNARE pairing and endosomal fusion. The release of Ca(2+) from the endosome lumen dissociates Hrs, allowing a VAMP-2-containing complex to form enabling fusion.
Yan
Qing
Q
Department of Neurobiology and Anatomy, University of Texas Medical School, Houston, Texas 77030, USA.
Sun
Wei
W
McNew
James A
JA
Vida
Thomas A
TA
Bean
Andrew J
AJ
D002118:Calcium / Q000494:pharmacology*; D002352:Carrier Proteins / Q000502:physiology*; D056827:Endosomal Sorting Complexes Required for Transport; D011992:Endosomes / Q000737:chemistry / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D008561:Membrane Fusion; D008565:Membrane Proteins / Q000378:metabolism*; D052066:N-Ethylmaleimide-Sensitive Proteins; D009419:Nerve Tissue Proteins / Q000378:metabolism; D010750:Phosphoproteins / Q000502:physiology*; D011485:Protein Binding / Q000187:drug effects; D050765:Qa-SNARE Proteins; D050683:R-SNARE Proteins; D050600:SNARE Proteins; D050825:Synaptosomal-Associated Protein 25; D033921:Vesicular Transport Proteins
D002352:Carrier Proteins; D056827:Endosomal Sorting Complexes Required for Transport; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D010750:Phosphoproteins; D050765:Qa-SNARE Proteins; D050683:R-SNARE Proteins; C494208:SNAP25 protein, human; D050600:SNARE Proteins; D050825:Synaptosomal-Associated Protein 25; D033921:Vesicular Transport Proteins; C096164:hepatocyte growth factor-regulated tyrosine kinase substrate; D052066:N-Ethylmaleimide-Sensitive Proteins; D002118:Calcium