title: Ubiquilin recruits Eps15 into ubiquitin-rich cytoplasmic aggregates via a UIM-UBL interaction.
abstract: Eps15 and its related protein Eps15R are key components of the clathrin-mediated endocytic pathway. We searched for new binding partners of Eps15 using a yeast two-hybrid screen. We report here that ubiquilin (hPLIC1), a type-2 ubiquitin-like protein containing a ubiquitin-like domain (UBL) and a ubiquitin-associated domain (UBA), interacts with both Eps15 and Eps15R. Using glutathione-S-transferase pull-down experiments, we show that the first ubiquitin-interacting motif of Eps15 (UIM1) interacts directly with the UBL domain of ubiquilin, whereas it does not bind to ubiquitinated proteins. The second UIM of Eps15 (UIM2) binds poorly to the UBL domain but does bind to ubiquitinated proteins. Two other UIM-containing endocytic proteins, Hrs and Hbp, also interact with ubiquilin in a UIM-dependent manner, whereas epsin does not. Immunofluorescence analysis showed that endogenous Eps15 and Hrs, but not epsin, colocalize with green-fluorescent-protein-fused ubiquilin in cytoplasmic aggregates that are not endocytic compartments. We have characterized these green-fluorescent-protein-fused-ubiquilin aggregates as ubiquitin-rich intracytoplasmic inclusions that are recruited to aggresomes upon proteasome inhibition. Moreover, we show that endogenous Eps15 and endogenous ubiquilin colocalize to cytoplasmic aggregates and aggresomes. Finally, we show that the recruitment of Eps15 into ubiquilin-positive aggregates is UIM dependent. Altogether, our data identify ubiquilin as the first common UIM-binding partner of a subset of UIM-containing endocytic proteins. We propose that this UIM/UBL-based interaction is responsible for the sequestration of certain UIM-containing endocytic proteins into cytoplasmic ubiquitin-rich protein aggregates.
authors.affiliation: Molecular Cell Biology, Institute of Biomembranes, University of Utrecht, Padualaan 8, 3584 CH Utrecht, The Netherlands.
authors.forename: Elsa
authors.initials: E
authors.lastname: Regan-Klapisz
authors.forename: Irina
authors.initials: I
authors.lastname: Sorokina
authors.forename: Jarno
authors.initials: J
authors.lastname: Voortman
authors.forename: Peter
authors.initials: P
authors.lastname: de Keizer
authors.forename: Rob C
authors.initials: RC
authors.lastname: Roovers
authors.forename: Peter
authors.initials: P
authors.lastname: Verheesen
authors.forename: Sylvie
authors.initials: S
authors.lastname: Urbé
authors.forename: Lara
authors.initials: L
authors.lastname: Fallon
authors.forename: Edward A
authors.initials: EA
authors.lastname: Fon
authors.forename: Arie
authors.initials: A
authors.lastname: Verkleij
authors.forename: Alexandre
authors.initials: A
authors.lastname: Benmerah
authors.forename: Paul M P
authors.initials: PM
authors.lastname: van Bergen en Henegouwen
mesh_terms: D048868:Adaptor Proteins, Signal Transducing; D020816:Amino Acid Motifs; D000818:Animals; D000071183:Autophagy-Related Proteins; D019556:COS Cells; D002135:Calcium-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism*; D002522:Chlorocebus aethiops; D003593:Cytoplasm / Q000378:metabolism*; D056827:Endosomal Sorting Complexes Required for Transport; D006367:HeLa Cells; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D010750:Phosphoproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology; D025801:Ubiquitin / Q000378:metabolism*
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D000071183:Autophagy-Related Proteins; D002135:Calcium-Binding Proteins; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; C486206:EPS15 protein, human; D056827:Endosomal Sorting Complexes Required for Transport; D047908:Intracellular Signaling Peptides and Proteins; D010750:Phosphoproteins; D011993:Recombinant Fusion Proteins; C107792:UBQLN1 protein, human; D025801:Ubiquitin; C096164:hepatocyte growth factor-regulated tyrosine kinase substrate; D046988:Proteasome Endopeptidase Complex