Activation of the endosome-associated ubiquitin isopeptidase AMSH by STAM, a component of the multivesicular body-sorting machinery.
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.
McCullough
John
J
Physiological Laboratory, School of Biomedical Sciences, University of Liverpool, Crown Street, Liverpool L69 3BX, United Kingdom.
Row
Paula E
PE
Lorenzo
Oscar
O
Doherty
Mary
M
Beynon
Robert
R
Clague
Michael J
MJ
Urbé
Sylvie
S
D048868:Adaptor Proteins, Signal Transducing / Q000032:analysis / Q000737:chemistry / Q000502:physiology*; D002460:Cell Line; D002966:Clathrin / Q000378:metabolism; D010450:Endopeptidases / Q000032:analysis / Q000378:metabolism*; D056827:Endosomal Sorting Complexes Required for Transport; D011992:Endosomes / Q000201:enzymology*; D004789:Enzyme Activation; D006801:Humans; D008954:Models, Biological; D010750:Phosphoproteins / Q000032:analysis / Q000737:chemistry / Q000502:physiology*; D017434:Protein Structure, Tertiary; D017384:Sequence Deletion; D013379:Substrate Specificity; D022161:Transport Vesicles / Q000378:metabolism; D043222:Ubiquitin Thiolesterase; D033921:Vesicular Transport Proteins / Q000378:metabolism
D048868:Adaptor Proteins, Signal Transducing; C498543:CHMP3 protein, human; D002966:Clathrin; D056827:Endosomal Sorting Complexes Required for Transport; D010750:Phosphoproteins; C109775:STAM protein, human; C486696:STAMBP protein, human; D033921:Vesicular Transport Proteins; D010450:Endopeptidases; D043222:Ubiquitin Thiolesterase