UBPY is a ubiquitin-specific protease that can deubiquitinate monoubiquitinated receptor tyrosine kinases, as well as process Lys-48-and Lys-63-linked polyubiquitin to lower denomination forms in vitro. Catalytically inactive UBPY localizes to endosomes, which accumulate ubiquitinated proteins. We have explored the sequelae of short interfering RNA-mediated knockdown of UBPY. Global levels of ubiquitinated protein increase and ubiquitin accumulates on endosomes, although free ubiquitin levels are unchanged. UBPY-depleted cells have more and larger multivesicular endosomal structures that are frequently associated through extended contact areas, characterized by regularly spaced, electron-dense, bridging profiles. Degradation of acutely stimulated receptor tyrosine kinases, epidermal growth factor receptor and Met, is strongly inhibited in UBPY knockdown cells suggesting that UBPY function is essential for growth factor receptor down-regulation. In contrast, stability of the UBPY binding partner STAM is dramatically compromised in UBPY knockdown cells. The cellular functions of UBPY are complex but clearly distinct from those of the Lys-63-ubiquitin-specific protease, AMSH, with which it shares a binding site on the SH3 domain of STAM.

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.

We have identified and characterized a Microtubule Interacting and Transport (MIT) domain at the N terminus of the deubiquitinating enzyme UBPY/USP8. In common with other MITcontaining proteins such as AMSH and VPS4, UBPY can interact with CHMP proteins, which are known to regulate endosomal sorting of ubiquitinated receptors. Comparison of binding preferences for the 11 members of the human CHMP family between the UBPY MIT domain and another ubiquitin isopeptidase, AMSH, reveals common interactions with CHMP1A and CHMP1B but a distinct selectivity of AMSH for CHMP3/VPS24, a core subunit of the ESCRT-III complex, and UBPY for CHMP7. We also show that in common with AMSH, UBPY deubiquitinating enzyme activity can be stimulated by STAM but is unresponsive to its cognate CHMPs. The UBPY MIT domain is dispensable for its catalytic activity but is essential for its localization to endosomes. This is functionally significant as an MIT-deleted UBPY mutant is unable to rescue its binding partner STAM from proteasomal degradation or reverse a block to epidermal growth factor receptor degradation imposed by small interfering RNA-mediated depletion of UBPY.Lysosomal degradation rates determine the levels of cell surface receptor tyrosine kinases, an important parameter in the control of cell growth (1, 2). Activated receptors are internalized and consequently committed to the lysosomal pathway by budding from the limiting membrane of the early endosome into lumenal vesicles, which define the multivesicular body (MVB).3 Ubiquitination of receptors is required for their sorting into MVBs, which precludes their recycling to the plasma membrane (3,4). The constituents of the endosomal sorting machinery were initially identified as class E mutants in screens for vacuolar protein-sorting (VPS) defects in Saccharomyces cerevisiae (5,6), characterized by an expanded pre-vacuolar compartment (7). These engage in a complex set of protein-protein interactions, which link four core complexes (endosomal sorting complexes required for transport, ESCRTs-0, I, II and III), and somehow impart directionality to the process (5, 8 -11). It has been proposed that ESCRT-0 (comprising Hrs (hepatocyte growth factor-regulated tyrosine kinase substrate) and STAM (signal-transducing adaptor molecule)) may provide the first means of engagement with ubiquitinated receptor (12-15) through ubiquitin interaction motifs in both proteins, although both ESCRT-I and -II also contain ubiquitin-binding proteins (10,16). In yeast, ESCRT-III is composed of four subunits (VPS2/Chm2, VPS24/Chm3, Snf7/Chm4, VPS20/Chm6), whereas mammals possess an expanded complement of isoforms, CHMP2A/B, CHMP3, CHMP4A/B/C, and CHMP6, providing the possibility for distinct ESCRT-III functions through combinatorial coding of core components. Two related proteins, Did2/VPS46/Chm1 and VPS60/MOS10/Chm5 (CHMP1A/B and CHMP5 in mammals), have poorly defined auxiliary roles in MVB sorting (17)(18)(19), and a further mammalian CHMP protein (CHMP7) has no yeast orthologue (20). All the CHMPs...

Hepatocyte growth factor regulated tyrosine kinase substrate (Hrs), a main component of the `bilayered' clathrin coat on sorting endosomes, was originally identified as a substrate of activated tyrosine kinase receptors. We have analysed Hrs phosphorylation in response to epidermal growth factor (EGF) stimulation and show that the evolutionary conserved tyrosines Y329 and Y334 provide the principal phosphorylation sites. Hrs is proposed to concentrate ubiquitinated receptors within clathrin-coated regions via direct interaction with its UIM (ubiquitin interaction motif) domain. We show that the same UIM domain is necessary for EGF-stimulated tyrosine phosphorylation of Hrs. Over-expression of wild-type Hrs or a double mutant, Y329/334F, defective in EGF-dependent phosphorylation, both substantially retard EGF receptor (EGFR) degradation by inhibiting internal vesicle formation and thereby preventing EGFR incorporation into lumenal vesicles of the multivesicular bodies. In contrast, mutation or deletion of the Hrs-UIM domain strongly suppresses this effect. In addition the UIM-deletion and point mutants are also observed on internal membranes, indicating a failure to dissociate from the endosomal membrane prior to incorporation of the receptor complex into lumenal vesicles. Our data suggest a role for the UIM-domain of Hrs in actively retaining EGFR at the limiting membrane of endosomes as a prelude to lumenal vesicle formation.

The precise trafficking routes followed by newly synthesized lysosomal membrane proteins after exit from the Golgi are unclear. To study these events we created a novel chimera (YAL) having a lumenal domain comprising two tyrosine sulfation motifs fused to avidin, and the transmembrane and cytoplasmic domains of lysosome associated membrane protein 1 (Lamp1). The newly synthesized protein rapidly transited from the transGolgi Network (TGN) to lysosomes (t 1/2~3 0 min after a lag of 15-20 min). However, labeled chimera was captured by biotinylated probes endocytosed for only 5 min, indicating that the initial site of entry into the endocytic pathway was early endosomes. Capture required export of YAL from the TGN, and endocytosis of the biotinylated reagent, and was essentially quantitative within 2 h of chase, suggesting that all molecules were following an identical route. There was no evidence of YAL trafficking via the cell surface. Fusion of TGN-derived vesicles with 5 min endosomes could be recapitulated in vitro, but neither late endosomes nor lysosomes could serve as acceptor compartments. This suggests that contrary to previous conclusions, most if not all newly synthesized Lamp1 traffics from the TGN to early endosomes prior to delivery to late endosomes and lysosomes.