Spongiform neurodegeneration-associated E3 ligase Mahogunin ubiquitylates TSG101 and regulates endosomal trafficking - PubMed (original) (raw)

Spongiform neurodegeneration-associated E3 ligase Mahogunin ubiquitylates TSG101 and regulates endosomal trafficking

Bong Yoon Kim et al. Mol Biol Cell. 2007 Apr.

Abstract

A null mutation in the gene encoding the putative E3 ubiquitin-protein ligase Mahogunin causes spongiform neurodegeneration, a recessively transmitted prion-like disease in mice. However, no substrates of Mahogunin have been identified, and the cellular role of Mahogunin is unknown. Here, we report the identification of TSG101, a key component of the endosomal sorting complex required for transport (ESCRT)-I, as a specific Mahogunin substrate. We find that Mahogunin interacts with the ubiquitin E2 variant (UEV) domain of TSG101 via its PSAP motif and that it catalyzes monoubiquitylation of TSG101 both in vivo and in vitro. Depletion of Mahogunin by small interfering RNAs in mammalian cells disrupts endosome-to-lysosome trafficking of epidermal growth factor receptor, resulting in prolonged activation of a downstream signaling cascade. Our findings support a role for Mahogunin in a proteasome-independent ubiquitylation pathway and suggest a link between dysregulation of endosomal trafficking and spongiform neurodegeneration.

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Figures

Figure 1.

Figure 1.

Mahogunin specifically interacts with TSG101. (A) Sequence alignment of the conserved C-terminal PSAP motif in Mahogunin homologues from different species. (B) Interaction between Mahogunin and TSG101 was tested by yeast two-hybrid assays. TomL1-VHSGAT and TomL1-VHS were used as a positive and a negative control, respectively. (C) The interactions between Mahogunin and P(S/T)AP motif-containing TSG101-binding proteins were analyzed in yeast two-hybrid assays. Growth on histidine-deficient (−His) medium is indicative of an interaction. (D) Anti-Mahogunin antibody recognizes a 64-kDa protein in rat brain and several cell lines (top). Lysate from HeLa cells expressing Myc-tagged Mahogunin was included as a positive control (lane 1). The same membrane was reprobed with anti-Myc and anti-TSG101 antibodies as indicated. The specificity of the anti-Mahogunin antibody was confirmed by preabsorption with 20 μg of GST–Mahogunin fusion protein (bottom). (E) HEK293 cell lysates were subjected to immunoprecipitation with anti-TSG101 antibody or mouse IgG, followed by immunoblotting with anti-Mahogunin and anti-TSG101 antibodies. IB, immunoblot; IP, immunoprecipitation.

Figure 2.

Figure 2.

Identification of Mahogunin and TSG101 binding sites by deletion and site-specific mutation analyses. (A and B) Schematic representation of the human Mahogunin and TSG101 constructs used in this study. The following domains of Mahogunin and TSG101 are indicated: RF, RING finger; UEV, ubiquitin E2 variant; PRD, proline-rich domain; CC, coiled-coil; SB, steadiness box. (C and D) Yeast two-hybrid analysis of the interaction between N- or C-terminal deletion mutants of Mahogunin and various deletion mutants of TSG101. (E) Binding of the Mahogunin PSAP motif mutant, Mgrn1-ASAA, to wild-type and mutant forms of TSG101. (F) Binding of wild-type Mahogunin to site-specific mutants of TSG101. AD, activation domain; BD, binding domain.

Figure 3.

Figure 3.

In vitro and in vivo interactions between Mahogunin and TSG101. (A) Coimmunoprecipitation analysis of the interaction between Mahogunin and TSG101. HeLa cells expressing the indicated Myc-tagged Mahogunin and GFP-tagged wild-type or mutant TSG101 were immunoprecipitated with anti-GFP antibody, followed by immunoblotting with anti-Myc antibody. Cell lysates were analyzed by immunoblotting with anti-GFP (middle) and anti-Myc (bottom) antibodies. IB, immunoblot; IP, immunoprecipitation. (B) GST pull-down assays were performed by incubation of GST-fused TSG101ΔC proteins or GST with lysates from COS-7 cells expressing the indicated Myc-tagged wild-type or mutant Mahogunin. Bound proteins were detected by immunoblotting using anti-Myc antibody. (C) GST-fused Mgrn1ΔN3ΔC1 proteins or GST was incubated with lysates from COS-7 cells expressing the indicated GFP-tagged wild-type TSG101 or TSG101ΔN2. Bound proteins were detected by immunoblotting using anti-GFP antibody. (D) Model illustrating the identified binding sites of Mahogunin and TSG101.

Figure 4.

Figure 4.

Endogenous Mahogunin and TSG101 colocalize. (a–i) HeLa cells were costained with antibodies against Mahogunin and TSG101 or LAMP2 as indicated. Preabsorption with GST-tagged Mahogunin protein specifically abolished the immunostaining by anti-Mahogunin antibody (a). (j–k) HeLa cells expressing Myc-tagged Mahogunin were costained with anti-Mahogunin (j) and anti-Myc (k) antibodies. Insets show threefold magnification of the boxed areas. Bar, 10 μm.

Figure 5.

Figure 5.

Colocalization of Mahogunin with TSG101 on endosomes depends on its interaction with TSG101. (A) HeLa cells coexpressing GFP-tagged TSG101 and Myc-tagged Mgrn1 were costained with anti-Myc and anti-EEA1, or anti-LAMP2 antibodies. GFP-TSG101 was directly visualized by GFP fluorescence. (B) HeLa cells coexpressing the indicated GFP-tagged wild-type or mutant TSG101 and Myc-tagged wild-type or mutant Mahogunin were immunostained with anti-Myc antibody. GFP-TSG101 was directly visualized by GFP fluorescence. Insets show twofold magnification of the boxed areas. Bar, 10 μm.

Figure 6.

Figure 6.

Mahogunin ubiquitylates TSG101 in vivo and in vitro. (A and B) Lysates of HeLa cells expressing the indicated Myc-tagged Mahogunin, GFP-tagged TSG101, and wild-type HA-tagged ubiquitin (Ub-wt) were immunoprecipitated with anti-GFP antibody. Ubiquitylated TSG101 was detected by immunoblotting with anti-HA antibodies (top). Cell lysates were subjected to Western blot analysis to detect the expression of TSG101 (middle) and Mahogunin (bottom). (C and D) Immunopurified GFP-TSG101 was subjected to in vitro ubiquitylation in the presence of E1, E2 (Ubc5a, Ubc7, or Ubc8), wild-type ubiquitin (Ub-wt), and wild-type or mutant GST-tagged Mahogunin as indicated. Ubiquitylated TSG101 was detected by immunoblotting with anti-ubiquitin antibody. (E) HeLa cells were cotransfected with Myc-tagged wild-type or mutant Mahogunin, GFP-tagged wild- type TSG101, and HA-tagged wild-type (Ub-wt) or mutant (Ub-K0) ubiquitin. After immunoprecipitation, ubiquitylated GFP-TSG101 was detected by immunoblotting with anti-HA antibodies. (F) Immunopurified GFP-TSG101 were ubiquitylated in vitro in the presence of recombinant E1, E2 (Ubc5a), wild-type ubiquitin (Ub-wt) or K0 mutant ubiquitin (Ub-K0), and wild-type or mutant of GST-tagged Mahogunin, as indicated. IB, immunoblot; IP, immunoprecipitation.

Figure 7.

Figure 7.

Mahogunin mediates multiple monoubiquitylation of TSG101. (A) Homogenates from rat brain and liver were analyzed along with an ubiquitin ladder by SDS-PAGE and immunoblotting with FL76 and FK2 anti-ubiquitin antibodies as indicated. (B) HeLa cells were transfected with GFP-tagged TSG101 and FLAG-tagged Mahogunin as indicated. Cell lysates were subjected to immunoprecipitation with anti-GFP antibodies and analyzed by SDS-PAGE and immunoblotting with FL76 and FK2 anti-ubiquitin antibodies and anti-GFP antibodies. (C) HeLa cells were transfected with GFP-tagged TSG101, HA-tagged Ub-K0, Myc-tagged Ub-K0, and FLAG-tagged Mahogunin as indicated. Cell lysates were subjected to sequential immunoprecipitations with anti-GFP, anti-HA, and anti-Myc antibodies. Precipitated proteins from the first (anti-GFP) and third (anti-Myc) immunoprecipitations were analyzed by SDS-PAGE and immunoblotting with anti-GFP, anti-HA, and anti-Myc antibodies. IB, immunoblot; IP, immunoprecipitation.

Figure 8.

Figure 8.

Mahogunin knockdown alters endosome and lysosome morphology. (A) HeLa cells were transfected with Mgrn1 siRNA, TSG101 siRNA, or mock treated for 48 h and then analyzed by immunoblotting with anti-Mahogunin, anti-TSG101, anti-EEA1, and anti- actin antibodies. (B) Mock (a and c) or Mgrn1 siRNA-treated (b and d) HeLa cells were immunostained using anti-EEA-1 (a and b) and anti-LAMP2 (c and d) antibodies and analyzed by immunofluorescence confocal microscopy. Bar, 10 μm.

Figure 9.

Figure 9.

Mahogunin knockdown disrupts endosome-to-lysosome trafficking of EGFR. (A) Mock (a–f) or Mgrn1 siRNA-transfected (g–l) HeLa cells were treated with Alexa488-EGF (green) for 1 h at 4°C. After washing, cells were incubated at 37°C for 30 min or 3 h, and then stained for EEA1 (red). Bar, 10 μm. (B) The amount of intracellular Alexa488-EGF was quantified and represented as the percentage of EGF after 30 min. The bar graph shows the results (mean ± SE) from three independent experiments. An ANOVA with a Tukey's post hoc test revealed statistically significant (p < 0.01) difference (asterisk) in the amount of Alexa488-EGF remaining in the Mock siRNA-treated cells versus that in the Mgrn1 siRNA-treated cells.

Figure 10.

Figure 10.

Depletion of Mahogunin inhibits EGFR degradation and disrupts downstream signaling of EGFR. (A) Mock or Mgrn1 siRNA-transfected HeLa cells were treated with 100 ng/ml EGF for the indicated times. Equal amounts of protein from whole cell lysates were analyzed by immunoblotting with anti-EGFR, anti-Mahogunin, and anti-actin antibodies. (B) The remaining EGFR level after EGF treatment was quantified and expressed as a percentage of the EGFR level in untreated control cells. Data are mean ± SEM (error bar) of the results from two independent experiments. (C) Mock or Mgrn1 siRNA-transfected HeLa cells were treated with 10 ng/ml EGF for 5 min followed by an acidic wash to remove cell surface EGF. Cells were then chased in serum-free medium for the indicated times and analyzed by immunoblotting using anti-phospho-ERK1/2 (top), anti-ERK 1/2 (middle), and anti-Mahogunin antibodies. (D) The level of phospho-ERK was quantified and normalized against the level of total ERK and plotted as a percentage of the relative level of phospho-ERK at chase time t = 0.

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