Traffic and expression of membrane proteins regulated by an ubiquitin-lysosome system

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Although the significance of ubiquitin-signals for both endocytosis from the plasma membrane (PM) and sorting at trans-Golgi network (TGN) and endosomes/multivesicular body has been postulated, the proteins whose trafficking is regulated by ubiquitylation are poorly understood. SKD1 (E235Q), an ATPase-deficient form of SKD1/Vps4B, abrogates the recycling of ubiquitin from the ubiquitylated proteins accumulated in aberrant endosomes(EQ compartment; mammalian class E vps compartment). We have taken advantage of this to identify substrates for ubiquitylation using a combination of affinity purification and LC-MS/MS. A variety of receptors, transporters, cell adhesion and signaling molecules localized to the PM are identified, and most of them are accumulated in EQ compartment in an ubiquitylation-dependent manner. Moreover, integral membrane protein 2B and Nedd4-interacting protein 2 are the first examples in mammalian cells, of proteins whose trafficking between the Golgi/TGN and endosomes is regulated by ubiquitylation. Intriguingly trafficking of transferrin receptor (TfR) is highly correlated with its ubiquitylation state, since free ubiquitin depletion inhibits ubiquitylation of TfR and resumes its accumulation in EQ compartments. Taken together, the identified proteins are the best candidates for the membrane-associated proteins whose trafficking is regulated by ubiquitylation. These data provide us forward steps to identify the mechanistic basis for ubiquitin-dependent protein sorting.