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First published online 13 September 2005
doi: 10.1242/jcs.02571

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Ubiquilin recruits Eps15 into ubiquitin-rich cytoplasmic aggregates via a UIM-UBL interaction

¹ Molecular Cell Biology, Institute of Biomembranes, University of Utrecht, Padualaan 8, 3584 CH Utrecht, The Netherlands
² Physiological Laboratory, University of Liverpool, Crown St, Liverpool, L69 3BX, UK
³ Centre for Neuronal Survival, Neurological Institute, McGill University, 3801 University Street, Montreal, Quebec, H3A 2B4, Canada
⁴ Departement Maladies Infectieuses, Institut Cochin-U567 INSERM/UMR8104 CNRS, Pavillon G. Roussy, 27 rue de Fbg St Jacques, 75015 Paris, France

View larger version (35K): [in a new window]   Fig. 1. Identification of an interaction between Eps15 and ubiquilin. (A) The pACT2 construct encoding ubiquilin (amino acids 14-589) fused to the GAL4 transactivation domain was co-transformed with pGBT8 constructs expressing the GAL4 DNA binding domain alone (empty vector) or fused with the EH domains of Eps15 or Eps15R (Eps15-EH or Eps15R-EH) in S. cerevisiae strain AH109. Yeast was grown on selective medium (SC -Trp -Leu -Ade) for 4 days. (B) Cos-1 cells were transiently transfected with constructs encoding GFP-ubiquilin and FLAG-Eps15, Myc-Eps15R or empty vector (-), as indicated. Immunoprecipitation (IP) was performed with anti-FLAG (-FLAG) or anti-Myc (-Myc) antibody. The immunoprecipitates were separated by SDS-PAGE followed by immunoblotting (IB) with anti-GFP antibody (-GFP). The membranes were stripped and reprobed with either anti-FLAG or anti-Myc antibody. Total lysates immunoblotted with anti-GFP antibody are shown as a control for GFP-ubiquilin expression. Notice the ladder-like pattern of ubiquilin in the immunoprecipitates but not in the total lysates.

View larger version (22K): [in a new window]   Fig. 2. Ubiquitinated GFP-ubiquilin interacts with Eps15 but ubiquitination is not essential for this interaction. (A) Cos-1 cells were transiently transfected with a vector encoding GFP-ubiquilin or GFP alone, or mock transfected. Cell extracts were subjected to immunoprecipitation (IP) with anti-GFP antibody (-GFP) in RIPA buffer. Immunoprecipitates were separated by SDS-PAGE and immunoblotted (IB) with anti-ubiquitin antibody (-ubiquitin). The membrane was stripped and reprobed with anti-GFP antibody. (B) Cos-1 cells were transiently co-transfected with FLAG-Eps15 and GFP-ubiquilin or empty vector (-), as indicated. Immunoprecipitation (IP) was performed with anti-FLAG antibody (-FLAG). The immunoprecipitates were separated by SDS-PAGE followed by immunoblotting (IB) with anti-ubiquitin antibody. The membrane was stripped and reprobed with anti-GFP antibody. After a second stripping, the membrane was reprobed with anti-FLAG antibody. (C) Equal amounts of Cos-1 total cell lysate expressing FLAG-Eps15 (TL; 5% of the total input is shown) was incubated with GST or GST-ubiquilin immobilized on glutathione-Sepharose beads. The beads were washed and subjected to SDS-PAGE followed by immunoblotting (IB) with anti-FLAG antibody. The lower panel shows a Coomassie staining of the membrane. The positions of GST and GST-ubiquilin are indicated.

View larger version (36K): [in a new window]   Fig. 3. The coiled-coil domain and the third domain of Eps15 are necessary for the interaction with ubiquilin. (A) The FLAG-tagged Eps15 deletion mutants. The three structural domains of Eps15 (I, II and III) were deleted as indicated. The FLAG tag (black box) is located at the N-terminus of the constructs. AP-2, AP-2 binding sites; PRM, proline-rich motif; UIMs, ubiquitin-interacting motifs. (B) Cos-1 cells were transiently co-transfected with GFP-ubiquilin and the indicated FLAG-Eps15 constructs (WT, wild type) or empty vector (-). Immunoprecipitation (IP) was performed with anti-FLAG antibody (-FLAG) and samples were subjected to immunoblotting with anti-GFP antibody (-GFP). The membrane was stripped and reprobed with anti-FLAG antibody. Notice that the FLAG-Eps15 constructs are expressed at similar levels. (bottom) Total cell lysates immunoblotted with anti-GFP antibody as a control for GFP-ubiquilin expression.

View larger version (32K): [in a new window]   Fig. 4. Interaction between the UIMs of Eps15 and Ubiquilin. (A) Cos-1 cells were transiently transfected with GFP-ubiquilin and FLAG-Eps15 wild-type (WT), UIM1mut (E863A/S864A/E865A) or UIM2mut (L883A/L885A). Immunoprecipitation (IP) was performed with anti-FLAG antibody (-FLAG). The immunoprecipitates were subjected to immunoblotting (IB) with anti-GFP antibody (-GFP). The membrane was stripped and reprobed with anti-FLAG antibody. Notice that equal amounts of Eps15 were present in each immunoprecipitate. (bottom) An anti-GFP immunoblot of total cell lysates as a control for GFP-ubiquilin expression. (B) Equal amounts of GST alone, GST-UIM1 (amino acids 847-877 of mEps15) or GST-UIM2 (amino acids 872-897 of mEps15) immobilized on glutathione-Sepharose beads was incubated with total cell lysates (TL, 5% of the total input is shown) of mock-transfected or GFP-ubiquilin transfected Cos-1 cells (Cos-1 + GFP-ubiquilin). The beads were washed and subjected to SDS-PAGE followed by immunoblotting (IB) with anti-ubiquitin antibody (-ubiquitin). In the experiment using GFP/ubiquilin-transfected cells, the membrane was stripped and reprobed with anti-GFP antibody. Notice that the non-ubiquitinated GFP-ubiquilin (100 kDa) was precipitated by GST-UIM1 and, to a lesser extent, by GST-UIM2. (bottom) Coomassie staining of the membranes.

View larger version (33K): [in a new window]   Fig. 5. The UBL domain of ubiquilin is sufficient to promote the interaction with Eps15 UIMs. (A) GFP-tagged ubiquilin constructs. UBA, ubiquitin-associated domain (amino acids 493-589); UBL, ubiquitin-like domain (amino acids 14-129). (B) Cos-1 cells were transfected with FLAG-Eps15 and GFP alone, GFP-ubiquilin, GFP-UBL or GFP-UBA. Cell lysates were subjected to immunoprecipitation (IP) with anti-GFP antibody (-GFP) followed by immunoblotting (IB) with anti-FLAG antibody (-FLAG). The membrane was stripped and reprobed with anti-GFP antibody to check the expression level of each GFP construct. Degradation products of the GFP-UBL, GFP-UBA and GFP-ubiquilin are marked with asterisks (*). (bottom) Anti-FLAG immunoblot of total cell lysates as a control for FLAG-Eps15 expression. (C) Equal amount of GST alone, GST-UIM1, GST-UIM2 or GST-UIM1+2 immobilized on glutathione-Sepharose beads was incubated with purified recombinant His6-Myc-Ubiquilin or His6-Myc-UBL (2% and 10%, respectively, of the total input are shown). The beads were washed and subjected to SDS-PAGE followed by immunoblotting (IB) with anti-Myc antibody. (bottom) Representative Coomassie staining of the membrane for both experiments. Notice that both His6-Myc-Ubiquilin and His6-Myc-UBL were precipitated more efficiently by GST-UIM1 than by GST-UIM2.

View larger version (26K): [in a new window]   Fig. 6. Hrs and Hbp interact with ubiquilin in a UIM-dependent manner, whereas epsin does not interact with ubiquilin. (A) Equal amounts of GST or GST-ubiquilin immobilized on glutathione-Sepharose beads were incubated with total cell lysates of Cos-1 cells transfected with GFP-Hrs or His6-epsin (5% of the initial input is shown). The beads were washed and subjected to SDS-PAGE followed by immunoblotting (IB) with anti-ubiquitin anti-GFP antibody (-GFP). The membrane was stripped and reprobed with anti-His6 antibody (-His6). (bottom) Coomassie staining of the membrane. The positions of GST and GST-ubiquilin are indicated. (B) GST-ubiquilin immobilized on glutathione-Sepharose beads was incubated with total cell lysates of Cos-1 cells transfected with GFP-Hrs wild type (WT), GFP-Hrs-UIM or GFP-Hrs-LSAA in which the two essential UIM residues L269 and S270 are mutated to alanine. The beads were washed and subjected to SDS-PAGE followed by immunoblotting (IB) with anti-GFP antibody (top, GST pull down). (bottom) Anti-GFP immunoblot of total cell lysates (5% of the initial input) showing similar expression levels of the GFP-Hrs constructs. (C) GST-ubiquilin immobilized on glutathione-Sepharose beads was incubated with total cell lysates of Cos-1 cells transfected with HA-Hbp wild type (WT) or HA-Hbp-LSAA in which the two essential UIM residues L176 and S177 are mutated to alanine. The beads were washed and subjected to SDS-PAGE followed by immunoblotting (IB) with anti-HA (top, GST pull down). (bottom) Anti-HA immunoblot of total cell lysates (5% of the initial input) showing similar expression levels of the HA-Hbp constructs. GST alone did not bind the HA-Hbp constructs (not shown). (D) Equal amounts of GST, GST-Hrs-CT (amino acids 1-454), GST-Hrs-CT-UIM or GST-Eps15-UIM 1+2 (amino acids 847-897) was incubated with total cell lysates of Cos-1 cells transfected with GFP-UBL. The beads were washed and subjected to SDS-PAGE followed by immunoblotting (IB) with anti-GFP antibody. The pulled-down GFP-UBL is indicated with an arrow. The asterisk (*) indicates a degradation product of the UBL. (bottom) Coomassie staining of the membrane. The GST-Hrs-CT and GST-Hrs-CT-UIM (at the top of the membrane) displayed several degradation or incompletely translated products.

View larger version (111K): [in a new window]   Fig. 7. Ubiquilin colocalizes with Eps15 and Hrs, but not with epsin, and it is not found in clathrin-coated pits or in endosomes. (A) Colocalization of GFP-ubiquilin with endogenous Eps15 and Hrs but not with epsin. HeLa cells transiently transfected with the GFP-ubiquilin construct (a-f) were processed for fluorescence microscopy using the rabbit anti-Eps15 (a,b) and anti-Hrs (e,f) polyclonal antibodies, and the goat anti-epsin (c,d) polyclonal antibody. The first antibodies were revealed by Alexa-594-labelled goat anti-rabbit and donkey anti-goat immunoglobulin secondary antibodies. (a,c,e) Green fluorescence emitted by GFP. (b,d,f) Red fluorescence emitted by Alexa 594. Insets show higher magnifications of representative areas in which arrows stress GFP-ubiquilin dots. (B) Ubiquilin is found in neither CCPs nor endosomes. HeLa cells transiently transfected with the GFP/ubiquilin-encoding construct (a-c) were processed for fluorescence microscopy using the goat anti-CALM polyclonal antibody (a) and the mouse anti-CD63 monoclonal antibody (c) revealed with Alexa-594-labelled donkey anti-goat and goat anti-mouse immunoglobulins secondary antibodies, respectively. For transferrin staining (b), the GFP/ubiquilin-transfected cells were allowed to internalize Alexa-594-labelled transferrin for 30 minutes. GFP staining and Alexa-594 staining are shown in green and red, respectively.

View larger version (41K): [in a new window]   Fig. 8. GFP-ubiquilin is localized to aggresomes upon proteasome inhibition. HeLa cells transiently transfected with the GFP-ubiquilin construct (a-f) or with HA-ubiquilin and the GFP-CFTR constructs (g,h) were mock treated (0.1% DMSO) (a,c,d) or incubated overnight with 5 µM MG132 (b,e-h). Cells were fixed, permeabilized and processed for fluorescence microscopy using the FK2 anti-ubiquitin antibody (c-f) or the anti-HA antibody (g-h). The first antibodies were revealed by Cy3-labelled goat anti-mouse immunoglobulin secondary antibodies. (a-c,e,h) Green fluorescence emitted by GFP. (d,f,g) Red fluorescence emitted by Cy3.

View larger version (65K): [in a new window]   Fig. 9. Colocalization of endogenous Eps15 and endogenous ubiquilin to aggresomes. (A) GFP-ubiquilin and FLAG-Eps15 colocalize to aggresomes. HeLa cells transiently co-transfected with constructs encoding GFP-ubiquilin and the FLAG-Eps15 were incubated overnight with 5 µM MG132 and processed for fluorescence microscopy using the anti-FLAG antibody followed by a Cy3-labelled goat anti-mouse immunoglobulin secondary antibody. (left) Green fluorescence emitted by GFP. (right) Red fluorescence emitted by Cy3. (B) Characterization of the anti-Eps15 nanobody by western blot. Cos-1 cells were transiently co-transfected with wild-type FLAG-Eps15 and with FLAG-Eps15-I (lacking the EH 1-3 domains). Total cellular lysates were subjected to SDS-PAGE and blotted onto PVDF membrane. (left) Immunodetection with the anti-Eps15 nanobody (IB: VHH--Eps15) that was detected using the anti-Myc antibody. (right) Immunodetection with the anti-FLAG antibody (-FLAG). Notice that the llama VHH against Eps15 that was raised against domain I (EH1-3) of Eps15 does not recognize FLAG-Esp15-I as expected. (C) Characterization of the anti-Eps15 nanobody by immunoprecipitation. Biotinylated anti-Eps15 nanobody was added to HeLa cellular lysates and precipitated using streptavidin-Sepharose beads (IP: VHH--Eps15). As a control, HeLa cellular lysates were precipitated with streptavidin-Sepharose beads in the absence of antibody (Control). The beads were subjected to SDS-PAGE and immunoblotting was performed with a rabbit anti-Eps15 antibody (IB: -Eps15). Cellular lysates from HeLa were also loaded on the SDS-PAGE (Lysate). (D) HeLa cells were mocked treated (0.1% DMSO) (Control, top) or incubated overnight with 5 µM MG132 (bottom) and fixed with ice-cold methanol. Endogenous ubiquilin was detected using a rabbit anti-ubiquilin antibody revealed with Alexa-488-labelled goat anti-rabbit immunoglobulins secondary antibody (left). Endogenous Eps15 was detected using the VHH against Eps15, followed by mouse anti-Myc antibody revealed with Alexa-555-labelled goat anti-mouse immunoglobulin secondary antibody (centre). (right) Merged pictures, with yellow indicating colocalization of Eps15 and ubiquilin. The arrow indicates an aggregate in which Eps15 and ubiquilin colocalize.

View larger version (98K): [in a new window]   Fig. 10. UIM-dependent localization of Eps15 into ubiquilin-containing cytoplasmic aggregates. HeLa cells transiently co-transfected with the GFP-ubiquilin construct and with the FLAG-Eps15 (a,b) or the FLAG-Eps15-UIM1+2 (c,d) construct were fixed, permeabilized and processed for fluorescence microscopy using the anti-FLAG antibody followed by Cy3-labelled goat anti-mouse immunoglobulin secondary antibodies. (a,c) Green fluorescence emitted by GFP. (b,d) Red fluorescence emitted by Cy3. Insets show higher magnifications of representative areas.