Abstract
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.
Highlights
Endocytosis is a fundamental process essential for all eukaryotic cells
Hrs may bind to SNAP-25 using its Q-SNARE domain and inhibit endosomal fusion while it is involved in cargo sorting or endosome motility using NH2-terminal VHS (Vps27, sensitive factor attachment protein receptors; SNAP-25, synaptosomal associated protein of 25 kDa; VAMP, vesicle-associated membrane protein; Nethylmaleimide-sensitive factor (NSF), N-ethylmaleimide-sensitive factor; GST, glutathione S-transferase; EEA1, early endosomal antigen-1; TeTx, tetanus toxin; ATP␥S, adenosine 5Ј-O-(thiotriphosphate); ER, endoplasmic reticulum
We have examined the regulation of Hrs- and VAMP-2-containing endosomal SNARE complexes and find that NSF regulates the dissociation of the VAMP-21⁄7SNAP-251⁄7syntaxin 13 complex, it has no effect on the Hrs-containing complex
Summary
Materials—Hrs was expressed in insect cells as previously described [16]. Syntaxin 13 [20] and VAMP-2 were expressed in Escherichia coli as previously described (17, 20 –23). TeTx treatment was performed by isolating the early endosomes as described above and resuspending the vesicles with varying concentrations of TeTx (5– 6400 nM in a final volume of 30 l) [9, 24] followed by incubation at 37 °C for 30 or 60 min. SNARE Complex Disassembly—A constant amount of GST-syntaxin 13 (2 g/reaction) bound to glutathione-agarose was incubated with constant amounts of SNAP-25 (1 g), VAMP-2 (1 g), or Hrs (2 g) in the presence or absence of NSF/␣SNAP (0.25 mg/ml), MgATP, or ATP␥S (0.5 mM) or EDTA in phosphate-buffered saline binding buffer to a final reaction volume of 50 l. A constant amount of His-tagged Hrs (180 nM) was added to reactions containing purified endosomal membranes (as in a fusion reaction), along with various concentration of free Ca2ϩ (0, 0.003, 0.03, 0.1, 0.3, 1, 3, 10, and 30 mM) and 1 mM of Cu2ϩ, Ba2ϩ, and Mn2ϩ.
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