Activation of the Endosome-Associated Ubiquitin Isopeptidase AMSH by STAM, a Component of the Multivesicular Body-Sorting Machinery

Abstract

AMSH is an endosomal ubiquitin isopeptidase that can limit EGF receptor downregulation . It directly binds to the SH3 domain of STAM, which is constitutively associated with Hrs, a component of clathrin-coated structures on endosomes. This clathrin coat has been implicated in the recruitment of ubiquitinated growth factor receptors prior to their incorporation into internal vesicles of the multivesicular body (MVB) , through the concerted action of ESCRT complexes I, II, and III . We now show that AMSH is embedded within a network of interactions with components of the MVB-sorting machinery. AMSH and STAM, like Hrs , both bind directly to clathrin. AMSH also interacts with mVps24/CHMP3, a component of ESCRT III complex, and this interaction is reinforced through simultaneous STAM binding. We have explored the effect of interacting components on the in vitro enzymatic activity of AMSH. The enzyme shows specificity for K63- over K48-linked polyubiquitin chains in vitro and is markedly stimulated by coincubation with STAM, indicating that activation of AMSH is coupled to its association with the MVB-sorting machinery. Other interacting factors do not directly stimulate AMSH but may serve to orient the enzyme with respect to substrates on the endosomal membrane.

AMSH, an ESCRT-III Associated Enzyme, Deubiquitinates Cargo on MVB/Late Endosomes

“…As was expected from its high homology to yeast Vps24p, CHMP3 is now known to be a mammalian ESCRT-III subunit (Howard et al, 2001). Our present conclusion that AMSH binds CHMP3 is consistent with reports by others (Agromayor and Martin-Serrano, 2006;McCullough et al, 2006), and similar binding has been identified between Drosophila orthologues of these molecules (Giot et al, 2003). Although genetic evidence has already proven that ESCRT-III functions downstream of ESCRT-II in sorting and concentrating the ubiquitinated cargo of the MVB/late endosome (Bowers et al, 2004), the involvement of a DUB in mammalian MVB sorting had not been clarified previously.…”

“…1D). Because earlier reports indicated that AMSH 1-103 , which includes the MIT-like domain (AMSH 34-97 ), is required for CHMP3 binding (McCullough et al, 2006;Tsang et al, 2006), our present study has further refined the responsible region. However, we found that the localization of AMSH to endosomes does not depend on the CHMP3 association (Fig.…”

“…Including AMSH in the complex could result in efficient cargo deubiquitination, since ESCRT-III has access to the ubiquitin moieties on the cargo proteins. Interestingly, a recent report demonstrated that the in vitro DUB activity of AMSH is augmented when AMSH is bound to STAM (McCullough et al, 2006). However, binding CHMP3 did not strengthen AMSH's DUB activity (McCullough et al, 2006).…”

“…2B). Nonetheless, clathrin heavy chain (CHC) is also known to bind AMSH (McCullough et al, 2006;Nakamura et al, 2006), and therefore may play some role in its localization to late endosomes, as well as its reported localization to early endosomes. Furthermore, because we repeatedly observed an increased recruitment of AMSH to endosomes in cells expressing CHMP3 (data not shown), we speculate that, although CHMP3 is not required, it may have at least an auxiliary function to support the recruitment of AMSH to endosomes (Fig.…”

“…Interestingly, a recent report demonstrated that the in vitro DUB activity of AMSH is augmented when AMSH is bound to STAM (McCullough et al, 2006). However, binding CHMP3 did not strengthen AMSH's DUB activity (McCullough et al, 2006). In addition to cargo proteins, it is also possible that ESCRT proteins themselves could be included in the vesicle as ubiquitinated proteins, because ubiquitination of Hrs is reported (Hoeller et al, 2006).…”