Beclin1-binding UVRAG targets the class C Vps complex to coordinate autophagosome maturation and endocytic trafficking - PubMed (original) (raw)

Beclin1-binding UVRAG targets the class C Vps complex to coordinate autophagosome maturation and endocytic trafficking

Chengyu Liang et al. Nat Cell Biol. 2008 Jul.

Abstract

Autophagic and endocytic pathways are tightly regulated membrane rearrangement processes that are crucial for homeostasis, development and disease. Autophagic cargo is delivered from autophagosomes to lysosomes for degradation through a complex process that topologically resembles endosomal maturation. Here, we report that a Beclin1-binding autophagic tumour suppressor, UVRAG, interacts with the class C Vps complex, a key component of the endosomal fusion machinery. This interaction stimulates Rab7 GTPase activity and autophagosome fusion with late endosomes/lysosomes, thereby enhancing delivery and degradation of autophagic cargo. Furthermore, the UVRAG-class-C-Vps complex accelerates endosome-endosome fusion, resulting in rapid degradation of endocytic cargo. Remarkably, autophagosome/endosome maturation mediated by the UVRAG-class-C-Vps complex is genetically separable from UVRAG-Beclin1-mediated autophagosome formation. This result indicates that UVRAG functions as a multivalent trafficking effector that regulates not only two important steps of autophagy - autophagosome formation and maturation - but also endosomal fusion, which concomitantly promotes transport of autophagic and endocytic cargo to the degradative compartments.

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Figures

Figure 1

Figure 1. Interaction and colocalization of UVRAG with C-Vps

(a) UVRAG interaction with C-Vps. 293T cells were co-transfected with Flag–UVRAG and HA–Vps16, HA–Vps11, HA–Vps18, HA–Vps33 or HA–Vps39. Whole-cell lysates (WCLs) were used for immunoprecipitation (IP) with an anti-Flag antibody, followed by immunoblotting (IB) with an anti-HA antibody. 1% WCL was used as the input. (b) Interaction between endogenous UVRAG and C-Vps subunits. WCLs of 293T cells were used for IP with control serum (control) or an anti-UVRAG antibody, followed by IB with the indicated antibodies. The right panel shows endogenous protein expression. (c) Confocal microscopy analysis of the colocalization of endogenous UVRAG and Vps16 or Vps18 in HeLa cells. Insets highlight colocalization. (d) Confocal microscopy analysis of the colocalization of UVRAG with C-Vps subunits in HeLa cells transfected with Flag–UVRAG, and epitope-tagged C-Vps subunits as indicated. (e) Colocalization of UVRAG–C-Vps in early endosomes. HeLa cells were co-transfected with EGFP–Vps16 and Flag–UVRAG, stained with antibodies to LAMP1, LAMP2, EEA1 and Rab5, followed by confocal microscopy. All images are representative of at least three independent experiments. Scale bars, 5 μm. The raw data for a and b are shown in Supplementary Information, Fig. S6

Figure 2

Figure 2. Mapping of Vps16 binding domains of UVRAG

(a) The N-terminal C2 domain or the C-terminal region of UVRAG interacts individually with Vps16. 293T cells were co-transfected with HA–Vps16, together with vector, Flag–UVRAG or Flag–UVRAG mutants. WCLs were immunoprecipitated with anti-Flag followed by immunoblotting with an anti-HA antibody. (b) HeLa cells were transfected with EGFP–Vps16 together with Flag–UVRAG mutants as indicated and stained with anti-Flag (red), followed by confocal microscopy. Scale bars, 5 μm. (c) Interactions of the UVRAG C-terminal truncated mutants with Vps16. At 48 h post-transfection with HA–Vps16 and Flag–UVRAG C-terminal mutants, 293T WCLs were immunoprecipitated with anti-Flag, followed by immunoblotting with anti-HA. (d) UVRAG C2 domain or C-terminal region deletion abolishes Vps16 binding. At 48 h post-transfection with HA–Vps16 together with Flag–UVRAG or its mutants, 293T WCLs were immunoprecipitated with anti-Flag followed by immunoblotting with an anti-HA antibody. (e) Schematic representation of UVRAG and its deletion mutants. ++, strong binding; +, weak binding; −, no binding. The raw data for a, c and d are shown in Supplementary Information, Fig. S6

Figure 3

Figure 3. UVRAG recruits C-Vps protein to the GFP–LC3-labelled autophagosomes

(a) HeLa.Vec and HeLa.UVRAG cells transfected with HA–Vps16 and GFP–LC3 were treated with 2 μM rapamycin for 2 h, and processed for confocal microscopy (left panel; scale bars, 5 μm). The percentage of GFP–LC3 punctae positive for HA–Vps16 staining was quantified (right panel; data are mean ± s.e.m., n = 60, *P < 0.01). (b) Effects of Vps16 on UVRAG-mediated autophagosome formation. Light microscopic quantification of autophagosomes in HeLa.Vec and HeLa.UVRAG cells (upper left panel) or in HCT116.Vec and HCT116.UVRAG cells (bottom left panel) when transfected with GFP–LC3 together with control siRNA or Vps16 siRNA (data are mean ± s.e.m., n = 60 for HeLa cells; n = 450 for HCT116 cells, *P < 0.01; **P < 0.001). Immunoblotting of Vps16 and tubulin are shown in the right panel.

Figure 4

Figure 4. Autophagosome maturation in UVRAG-expressing HeLa cells

(a) UVRAG enhances the colocalization efficiency of GFP–LC3 with LAMP1. HeLa.Vec and HeLa.UVRAG cells transfected with GFP–LC3 were either untreated or treated with rapamycin (2 μM) in the absence or presence of bafilomycin A1 (0.1 μM) and stained for LAMP1. Insets highlight the colocalization. The percentage of LAMP1-positive autophagosomes was calculated in each setting (data are mean ± s.e.m., n = 200; 5 independent experiments, **P < 0.01; *P < 0.05). (b) UVRAG enhances the acquisition of CD63 by autophagosomes. The GFP–LC3-transfected HeLa.Vec and HeLa.UVRAG cells were treated as described in a and stained for CD63 and the percentage of CD63-positive autophagosomes was calculated (data are mean ± s.e.m., n = 200, **P < 0.01; *P < 0.05). (c) UVRAG promotes autophagic degradation of long-lived proteins. HeLa.Vec and HeLa.UVRAG cells were incubated for 16 h with

l

-3H-Leu (1 μCi ml−1). The degradation of long-lived proteins was measured at the indicated time points in complete medium (NC), EBSS alone (Starv.), or EBSS + 0.1 μM bafilomycin A1 (Starv.+BalfA1). Results (data are mean ± s.e.m. of triplicates) are representative of 2 independent experiments. Scale bars, 5 μm.

Figure 5

Figure 5. UVRAG-mediated autophagosome maturation is dependent on C-Vps, but not Beclin1

(a) Colocalization of GFP–LC3 and LAMP1. HeLa cells expressing wild-type or mutant UVRAG were transfected with GFP–LC3 and incubated under normal conditions or treated with rapamycin (2 μM). Autophagosome fusion with LAMP+ structures was analysed by confocal microscopy (left panel; rapamycin-treated) and quantified (right upper panel; data are mean ± s.e.m., n = 100). Arrows and insets show the LAMP1+ autophagosomes. The number of GFP–LC3-positive dots per cell was counted using a fluorescence microscope (right bottom panel; data are mean ± s.e.m., n = 60, *P < 0.05; **P < 0.0001). (b) Vps16 or Vps18 knockdown inhibits UVRAG-mediated autophagosome maturation. HeLa.Vec (left) and HeLa.UVRAG (right) cells were transfected with GFP–LC3 together with control siRNA, Vps16 siRNA, or Vps18 siRNA, followed by confocal microscopy. Arrows in the top panel denote the autophagosomes with LAMP1 staining. Immunoblotting of Vps16, or Vps18 and tubulin are shown in the middle panel. The percentage of LAMP1+ autophagosomes was calculated (data are mean ± s.e.m., n = 50, *P < 0.05). Scale bars, 5 μm. The raw data of the immunoblots are shown in Supplementary Information, Fig. S6.

Figure 6

Figure 6. Rab7 acquisition and activation by UVRAG

(a) UVRAG expression promotes the recruitment of Rab7 GTPase to autophagosomes. HeLa. Vec and HeLa.UVRAG cells transfected with GFP–Rab7 and RFP–LC3 were maintained either under normal conditions (NC) or treated with rapamycin, followed by confocal microscopy. Insets highlight the Rab7 acquisition of autophagosomes. The percentage of Rab7+-autophagosomes was quantified (right panel; data are mean ± s.e.m., n = 60, ***P < 0.001; **P < 0.01). Scale bars, 5 μm. (b) Rab7 GTPase activity is increased with UVRAG expression. At 48 h post-transfection with GFP–Rab7 together with vector, UVRAG or UVRAGΔN(1–147) vector, 293T WCLs were used for immunoprecipitation with anti-GFP, followed by the Rab7 GTPase activity assay. Left panel: Autoradiographs of the GTP hydrolysis products analysed by TLC. Right panel: Quantification of the percentage of GTP hydrolyzed by Rab7 (data are mean n = 2 independent experiments). (c) Increased Rab7–RILP interaction UVRAG is expressed. HCT116.vector, HCT116.UVRAG and HCT116.UVRAGΔN(1–147) cells were transfected with GFP–Rab7 together with the GST-tagged Rab7 binding domain of RILP (GST–RILPRBD, residues 243–318). WCLs were used for GST pulldown, followed by immunoblotting with an anti-GFP antibody. The raw data of the immunoblots are shown in Supplementary Information, Fig. S6.

Figure 7

Figure 7. UVRAG promotes endocytic trafficking

(a) EGF-stimulated endocytosis and post-endocytic trafficking. Uptake of Alexa Fluor 488–EGF (green) by HCT116.Vec (left panel) and HCT116.UVRAG (right panel) cells was followed by confocal microscopy. At the indicated times, cells were fixed and stained with antibodies to LAMP1 (red), and Topro-3 (blue, to stain the nucleus). Magnified images of the insets (fourth row) highlight trafficking of EGF towards LAMP1-positive structures. (b) EGFR degradation. HCT116. Vec, HCT116.UVRAG and HCT116.UVRAGΔN(1–147) cells were treated with EGF (200 ng ml−1) at 37 °C for the indicated times. WCLs were subjected to immunoblotting with an anti-EGFR antibody. EGFR at each time point was quantified and indicated as a percentage relative to that at time zero (right panel; data are mean ± s.e.m., n = 3). (c) DQ-green BSA dequenching assay. Representative confocal microscopy images of DQ-BSA proteolysis (left panel) and flow cytometry analysis (right panel) of RAW264.7 cells expressing an empty vector (RAW264.7.Vec; dotted black line), wild-type UVRAG (red line) or UVRAGΔN(1–147) mutant (green line) loaded with 10 μg ml−1 DQ green–BSA for the indicated times. Background (grey line) represents samples without the BSA load. (d) Effect of UVRAG on endosome fusion. RAW264.7.Vec and RAW264.7.UVRAG cells were pulse-labelled with Oregon green 514–avidin and dextran-568 for 10 min and chased for 30 min. Cells were then labelled with biotin–BSA for 10 min, and processed for confocal microscopy (left panel). In the absence of biotin–BSA, Oregon green 514–avidin showed very low fluorescence (first row of confocal image). The percentage of dextran-568-labelled endosomes with visible Oregon green staining (denoted by arrows in the left panel) was calculated from approximately 20 different cells in 2 independent experiments (mean, right panel). Scale bars, 5 μm. The raw data of the immunoblots in b are shown in the Supplementary Information, Fig. S6.

Figure 8

Figure 8

Model of the role of UVRAG as a coordinator of the autophagosomal and endosomal machineries. At an early stage of autophagy, UVRAG targets Beclin1 to facilitate autophagosome formation, whereas at the later stages, UVRAG interacts with C-Vps to promote autophagosome maturation. UVRAG–C-Vps is also involved in the endosome–lysosome transition by activation of Rab7.

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