A Deubiquitinating Enzyme UBPY Regulates the Level of Protein Ubiquitination on Endosomes

Monoubiquitination of endocytosed cell surface receptors serves as a sorting signal for their trafficking from endosomes to lysosomes. The sorting of ubiquitinated proteins is executed by concerted actions of class E vacuolar protein sorting (Vps) proteins. Some proteins in the sorting machinery undergo monoubiquitination, suggesting that their functions are also regulated by ubiquitination. The Hrs–STAM complex, a class E Vps protein complex essential for the initial step of the sorting pathway, binds two deubiquitinating enzymes, UBPY and AMSH. Here we examined the effects of inactivating UBPY on protein ubiquitination at endosomes. Overexpression of a catalytically inactive UBPY mutant or depletion of UBPY by RNA interference resulted in the accumulation of ubiquitinated proteins on morphologically aberrant endosomes. Electron microscopy showed that they are aggregates of multivesicular endosomes. Among the sorting machinery proteins that undergo ubiquitination, Eps15 was monoubiquitinated at an elevated level in UBPY‐inactivated cells. UBPY also deubiquitinated Eps15 in vitro, suggesting that Eps15 is a cellular substrate for UBPY. Furthermore, inactivation of UBPY caused the accumulation of Eps15 on the endosomal aggregates. These results suggest that UBPY regulates the level of protein ubiquitination on endosomes, which is required for maintaining the morphology of the organelle.

[1]  I. Prior,et al.  The Ubiquitin Isopeptidase UBPY Regulates Endosomal Ubiquitin Dynamics and Is Essential for Receptor Down-regulation* , 2006, Journal of Biological Chemistry.

[2]  P. Lehner,et al.  Degradation of Endocytosed Epidermal Growth Factor and Virally Ubiquitinated Major Histocompatibility Complex Class I Is Independent of Mammalian ESCRTII* , 2006, Journal of Biological Chemistry.

[3]  Sebastian A. Wagner,et al.  Regulation of ubiquitin-binding proteins by monoubiquitination , 2006, Nature Cell Biology.

[4]  René Bernards,et al.  A Genomic and Functional Inventory of Deubiquitinating Enzymes , 2005, Cell.

[5]  M. Komada,et al.  Regulation of epidermal growth factor receptor down-regulation by UBPY-mediated deubiquitination at endosomes. , 2005, Molecular biology of the cell.

[6]  A. Amerik,et al.  Mechanism and function of deubiquitinating enzymes. , 2004, Biochimica et biophysica acta.

[7]  Xiuli Wu,et al.  Stabilization of the E3 Ubiquitin Ligase Nrdp1 by the Deubiquitinating Enzyme USP8 , 2004, Molecular and Cellular Biology.

[8]  J. McCullough,et al.  AMSH is an endosome-associated ubiquitin isopeptidase , 2004, The Journal of cell biology.

[9]  I. Amit,et al.  Tal, a Tsg101-specific E3 ubiquitin ligase, regulates receptor endocytosis and retrovirus budding. , 2004, Genes & development.

[10]  M. Komada,et al.  A role for Hrs in endosomal sorting of ligand-stimulated and unstimulated epidermal growth factor receptor. , 2004, Experimental cell research.

[11]  Jean Gruenberg,et al.  The biogenesis of multivesicular endosomes , 2004, Nature Reviews Molecular Cell Biology.

[12]  M. Komada,et al.  Association with Hrs is required for the early endosomal localization, stability, and function of STAM. , 2004, Journal of biochemistry.

[13]  A. J. Koster,et al.  Endosomal compartmentalization in three dimensions: Implications for membrane fusion , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[14]  M. Komada,et al.  STAM proteins bind ubiquitinated proteins on the early endosome via the VHS domain and ubiquitin-interacting motif. , 2003, Molecular biology of the cell.

[15]  R. D. Fisher,et al.  HIV Gag mimics the Tsg101-recruiting activity of the human Hrs protein , 2003, The Journal of cell biology.

[16]  A. Brech,et al.  Hrs regulates multivesicular body formation via ESCRT recruitment to endosomes , 2003, The Journal of cell biology.

[17]  Yosef Yarden,et al.  Endocytosis of Receptor Tyrosine Kinases Is Driven by Monoubiquitylation, Not Polyubiquitylation* , 2003, Journal of Biological Chemistry.

[18]  Stanley N Cohen,et al.  TSG101 interaction with HRS mediates endosomal trafficking and receptor down-regulation , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[19]  Pier Paolo Di Fiore,et al.  Multiple monoubiquitination of RTKs is sufficient for their endocytosis and degradation , 2003, Nature Cell Biology.

[20]  H. Stenmark,et al.  STAM and Hrs Are Subunits of a Multivalent Ubiquitin-binding Complex on Early Endosomes* , 2003, The Journal of Biological Chemistry.

[21]  S. Emr,et al.  Receptor downregulation and multivesicular-body sorting , 2002, Nature Reviews Molecular Cell Biology.

[22]  Y. Yarden,et al.  Ligand‐Independent Degradation of Epidermal Growth Factor Receptor Involves Receptor Ubiquitylation and Hgs, an Adaptor Whose Ubiquitin‐Interacting Motif Targets Ubiquitylation by Nedd4 , 2002, Traffic.

[23]  I. Madshus,et al.  Hrs sorts ubiquitinated proteins into clathrin-coated microdomains of early endosomes , 2002, Nature Cell Biology.

[24]  J. Klumperman,et al.  Bilayered clathrin coats on endosomal vacuoles are involved in protein sorting toward lysosomes. , 2002, Molecular biology of the cell.

[25]  P. Woodman,et al.  Mammalian class E vps proteins recognize ubiquitin and act in the removal of endosomal protein–ubiquitin conjugates , 2002, The Journal of cell biology.

[26]  Pier Paolo Di Fiore,et al.  A single motif responsible for ubiquitin recognition and monoubiquitination in endocytic proteins , 2002, Nature.

[27]  E. Martegani,et al.  Cloning and Characterization of Mouse UBPy, a Deubiquitinating Enzyme That Interacts with the Ras Guanine Nucleotide Exchange Factor CDC25Mm/Ras-GRF1* , 2001, The Journal of Biological Chemistry.

[28]  H. Stenmark,et al.  Hrs recruits clathrin to early endosomes , 2001, The EMBO journal.

[29]  R. Piper,et al.  Ubiquitin Sorts Proteins into the Intralumenal Degradative Compartment of the Late‐Endosome/Vacuole , 2001, Traffic.

[30]  S. Emr,et al.  Ubiquitin-Dependent Sorting into the Multivesicular Body Pathway Requires the Function of a Conserved Endosomal Protein Sorting Complex, ESCRT-I , 2001, Cell.

[31]  W. Langdon,et al.  Cbl: many adaptations to regulate protein tyrosine kinases , 2001, Nature Reviews Molecular Cell Biology.

[32]  K. Miyazawa,et al.  A Deubiquitinating Enzyme UBPY Interacts with the Src Homology 3 Domain of Hrs-binding Protein via a Novel Binding Motif PX(V/I)(D/N)RXXKP* , 2000, The Journal of Biological Chemistry.

[33]  A. Amerik,et al.  The Doa4 deubiquitinating enzyme is functionally linked to the vacuolar protein-sorting and endocytic pathways. , 2000, Molecular biology of the cell.

[34]  R. Scheller,et al.  The cellular and developmental expression of hrs‐2 in rat , 1999, The European journal of neuroscience.

[35]  N. Tanaka,et al.  Possible Involvement of a Novel STAM-associated Molecule “AMSH” in Intracellular Signal Transduction Mediated by Cytokines* , 1999, The Journal of Biological Chemistry.

[36]  S. Kellokumpu,et al.  EAST, an Epidermal Growth Factor Receptor- and Eps15-associated Protein with Src Homology 3 and Tyrosine-based Activation Motif Domains* , 1998, The Journal of Biological Chemistry.

[37]  N. Nomura,et al.  UBPY: a growth‐regulated human ubiquitin isopeptidase , 1998, The EMBO journal.

[38]  M. Komada,et al.  Hrs, a Tyrosine Kinase Substrate with a Conserved Double Zinc Finger Domain, Is Localized to the Cytoplasmic Surface of Early Endosomes* , 1997, The Journal of Biological Chemistry.

[39]  A. Verkleij,et al.  Epidermal Growth Factor Induces Ubiquitination of Eps15* , 1997, The Journal of Biological Chemistry.

[40]  A. Verkleij,et al.  Association and Colocalization of Eps15 with Adaptor Protein-2 and Clathrin , 1997, The Journal of cell biology.

[41]  C. Futter,et al.  Multivesicular endosomes containing internalized EGF-EGF receptor complexes mature and then fuse directly with lysosomes , 1996, The Journal of cell biology.

[42]  A. Dautry‐Varsat,et al.  The tyrosine kinase substrate eps15 is constitutively associated with the plasma membrane adaptor AP-2 , 1995, The Journal of cell biology.

[43]  M. Komada,et al.  Growth factor-induced tyrosine phosphorylation of Hrs, a novel 115-kilodalton protein with a structurally conserved putative zinc finger domain , 1995, Molecular and cellular biology.

[44]  M. Fujimuro,et al.  Production and characterization of monoclonal antibodies specific to multi‐ubiquitin chains of polyubiquitinated proteins , 1994, FEBS letters.

[45]  M. Fukuda,et al.  Isolation and characterization of human lysosomal membrane glycoproteins, h-lamp-1 and h-lamp-2. Major sialoglycoproteins carrying polylactosaminoglycan. , 1988, The Journal of biological chemistry.

[46]  M. Komada,et al.  The Hrs/STAM complex in the downregulation of receptor tyrosine kinases. , 2005, Journal of biochemistry.

[47]  Patricia A. Lovelace,et al.  Two isoforms of otubain 1 regulate T cell anergy via GRAIL , 2004, Nature Immunology.

[48]  S. Carlsson,et al.  Isolation and Characterization of Human Lysosomal Membrane Glycoproteins , h-lamp-1 and h-lamp-2 , 2001 .