Mass spectrometry identification of proteins

McCullough, J., Row, P.E. Lorenzo, O, Doherty, M., Beynon, R.J., Clague, M.J. and Urbé, S (2006) Activation of the Endosome-Associated Ubiquitin Isopeptidase AMSH by STAM, a Component of the Multivesicular Body-Sorting Machinery Current Biology 16, 160-165.[PUBMED][PDF]

Faculty of 1000 Member Comments Harald Stenmark, The Norwegian Radium Hospital, Norway "New Finding: This article is interesting because it shows that the deubiquitinating enzyme AMSH is activated by STAM, a component of the endosomal sorting machinery for ubiquitinated membrane proteins. The authors also found that both AMSH and STAM interact with clathrin, a coat protein known to be present in microdomains of sorting endosomes. Together with previously published data, these findings suggest that deubiquitination of ubiquitinated membrane proteins in endosome membranes is closely coupled to the machinery that sorts such proteins for degradation."

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