Hrs regulates early endosome fusion by inhibiting formation of an endosomal SNARE complex

Abstract

Movement through the endocytic pathway occurs principally via a series of membrane fusion and fission reactions that allow sorting of molecules to be recycled from those to be degraded. Endosome fusion is dependent on SNARE proteins, although the nature of the proteins involved and their regulation has not been fully elucidated. We found that the endosome-associated hepatocyte responsive serum phosphoprotein (Hrs) inhibited the homotypic fusion of early endosomes. A region of Hrs predicted to form a coiled coil required for binding the Q-SNARE, SNAP-25, mimicked the inhibition of endosome fusion produced by full-length Hrs, and was sufficient for endosome binding. SNAP-25, syntaxin 13, and VAMP2 were bound from rat brain membranes to the Hrs coiled-coil domain. Syntaxin 13 inhibited early endosomal fusion and botulinum toxin/E inhibition of early endosomal fusion was reversed by addition of SNAP-25(150–206), confirming a role for syntaxin 13, and establishing a role for SNAP-25 in endosomal fusion. Hrs inhibited formation of the syntaxin 13–SNAP-25–VAMP2 complex by displacing VAMP2 from the complex. These data suggest that SNAP-25 is a receptor for Hrs on early endosomal membranes and that the binding of Hrs to SNAP-25 on endosomal membranes inhibits formation of a SNARE complex required for homotypic endosome fusion.

CART: An Hrs/Actinin-4/BERP/Myosin V Protein Complex Required for Efficient Receptor Recycling

“…The role of hrs in endosomal recycling is not inconsistent with a hypothesis suggesting that hrs or Vps27p functions in endocytic protein sorting (Katzmann et al, 2001;Bilodeau et al, 2002;Lloyd et al, 2002) and in early endosome fusion (Sun et al, 2003). These studies have suggested that hrs/Vps27p is linked with proteins required for the ubiquitination and sorting of cargo (Katzmann et al, 2001;Bilodeau et al, 2002) and that hrs inhibits homotypic early endosome fusion (Sun et al, 2003).…”

“…The role of hrs in endosomal recycling is not inconsistent with a hypothesis suggesting that hrs or Vps27p functions in endocytic protein sorting (Katzmann et al, 2001;Bilodeau et al, 2002;Lloyd et al, 2002) and in early endosome fusion (Sun et al, 2003). These studies have suggested that hrs/Vps27p is linked with proteins required for the ubiquitination and sorting of cargo (Katzmann et al, 2001;Bilodeau et al, 2002) and that hrs inhibits homotypic early endosome fusion (Sun et al, 2003). Hrs/Vps27p may bind ubiquitinated cargo with its UIM domain (Bilodeau et al, 2002;Polo et al, 2002;Shih et al, 2002), which is required for its cargo sorting function because mutation of that domain blocks sorting of ubiquitinated cargo proteins (Bilodeau et al, 2002;Shih et al, 2002).…”

“…The role of hrs in recruiting sorting or signaling components to the endosomal membrane likely is a function of a number of factors, including its oligomerization (Bean et al, 1997) and/or competition among binding proteins . Therefore, hrs may bind to an endosomal receptor, SNAP-25, by using its Q-SNARE domain and inhibit endosomal fusion (Sun et al, 2003), whereas it is involved in cargo sorting and/or endosome motility using N-terminal VHS, FYVE, or UIM domains or via actinin-4 interactions. Thus, a sorting step might occur before, or coincident with, the inhibition of fusion.…”

“…(A) BERP and actinin-4 are enriched with early endosomal membranes. HeLa cell lysate and endosomal membranes were isolated as described previously (Sun et al, 2003;Yan et al, 2004), separated on SDS-PAGE, and examined for the presence of BERP and actinin-4. The endosomal fractions do not contain detectable Na ϩ /K ϩ ATPase, calnexin, LAMP1, or rab 7, but they are enriched for the early endosomal marker protein, EEA1.…”

Role of protein ubiquitylation in regulating endocytosis of receptor tyrosine kinases

“…Hgs interacts with Eps15, and negatively regulates its function by affecting the interaction of Eps15 with AP2 (Asao et al, 1997;Takata et al, 2000;Bache et al, 2003b). Hgs also binds SNAP25, thereby inhibiting formation of the SNARE complex, and regulating endosomal membrane fusion (Sun et al, 2003), as well as binds a deubiquitylating enzyme, UBPY .…”