Rubicon controls endosome maturation as a Rab7 effector

Abstract

The activation and recruitment of the small GTPase Rab7 to early endosome is a critical step for early to late endosome maturation, a process that requires the class III phosphatidylinositol 3-kinase (PI3KC3) and GTPase regulators. However, the molecular mechanism underlying Rab7 activation and endosome maturation is still poorly defined. Here we report that Rubicon, a component of the PI3KC3 complex, prevents endosome maturation through differential interactions with Rab7 and UVRAG. UVRAG activates PI3KC3 and C-VPS/HOPS, a guanine nucleotide exchange factor that catalyzes the exchange of GDP for GTP on Rab7. We demonstrate that Rubicon sequesters UVRAG from C-VPS/HOPS. Active GTP-bound Rab7 competes for Rubicon binding and releases UVRAG to associate with C-VPS/HOPS, which in turn promotes further loading of Rab7 with GTP. This feed-forward loop ensures rapid amplification of GTP-bound Rab7 and consequent stimulation of endosome maturation. Hence, Rubicon serves as a previously unknown Rab7 effector to ensure the proper progression of the endocytic pathway.

Fig. 1.

Rubicon interacts with Rab7 via its C terminus. (A) Rubicon interacts with Rab7 but not Rab5. Rubicon-Flag vector was cotransfected with either Rab5-Myc (lanes 1–3) or Rab7-Myc vectors (lanes 4–6). The whole 293T cell lysates (lanes 1 and 4) were immunoprecipitated with anti-Flag antibody (lanes 3 and 6) or control IgG (lanes 2 and 5), followed by immunoblotting with anti-Flag antibody for Rubicon and anti-Myc antibody for Rabs. (B) Rubicon interacts with UVRAG and Rab7 in different complexes. Myc tagged Vps34, Beclin 1, Vps16, Rab7, and Rubicon were expressed in HEK293T cells and immunoprecipitated (IP) with anti-Myc antibody. The proteins indicated were detected by immunoblotting (IB) in the bound (B) and input fractions. (C) The CT domain of Rubicon is required for Rab7 binding. A vector expressing HA-tagged Rab7 was cotransfected with EGFP-Rubicon-Flag (WT) vector or the vectors expressing different mutants, followed by immunoprecipitation with anti-HA antibody (Rab7) and immunoblotting with the antibodies indicated.

Fig. 2.

Rubicon preferentially binds to GTP-bound Rab7. (A) Rubicon binds to GTP-bound Rab7 in vivo. Rubicon-Flag vector was cotransfected in HEK293T cells with Rab7-Myc vectors expressing Rab7-WT, GTP-bound Rab7 Q67L mutant, or GDP-bound Rab7 T22N mutant, followed by immunoprecipitation with anti-Myc antibody (Rab7) and immunoblotting with the antibodies indicated. (B) Rab7 differentially interacts with Rubicon and p150. Purified recombinant HA-tagged Rab7 (0.1 μM) was incubated with recombinant Flag-tagged p150, Vps34, Beclin 1, UVRAG, Barkor/Atg14(L), or Rubicon (0.1 μM) in an in vitro pull-down assay using HA affinity beads. The bound and input proteins were detected by anti-Flag or HA antibodies. (C) Rubicon directly binds GTP-bound Rab7. Recombinant full-length Flag-tagged p150, Vps34, or Rubicon (0.1 μM) were incubated with HA affinity beads prebound with recombinant HA-tagged Rab7 WT, T22N, or Q67L mutant proteins (0.1 μM). The bound proteins were resolved by 7.5% SDS/PAGE and analyzed by Western blot using anti-Flag antibody.

Fig. 3.

GTP-bound Rab7 competes with UVRAG for Rubicon binding. (A) Overexpression of UVRAG compromises the Rab7–Rubicon interaction. Different concentrations of UVRAG were overexpressed in HEK293T cells (Top), and the resulting cell lysates were immunoprecipitated with anti-Rab7 antibody (Middle) or anti-Rubicon antibody (Bottom). The Rab7 or Rubicon immunocomplex was then probed for the proteins indicated. The normalized binding of Rubicon and Rab7 was quantified as the relative ratio of bound/input (B/I). The B/I in the control cells was set as 1.0. (B) Overexpression of Rab7 decreases UVRAG–Rubicon interaction. Rab7 was expressed in HEK293T cells. The resulting cell lysate was then immunoprecipitated with anti-Rubicon antibody. The bound and input fractions were probed for the proteins indicated. (C) GTP–Rab7 competes Rubicon from UVRAG in vivo. Different forms of Rab7 (WT, T22N, Q67L) were expressed and the resulting cell lysate was immunoprecipitated with anti-UVRAG antibody. The bound and input were probed for the proteins indicated. (D) Recombinant purified GST-tagged Rab7 WT and mutants from E. coli were analyzed by 10% SDS/PAGE followed by Coomassie blue staining. (E) In vitro competition among Rubicon, UVRAG, and different forms of Rab7. Rubicon-Flag-His (0.1 μM) was bound to Ni column first and then incubated with UVRAG-Flag (0.1 μM) to form a Rubicon–UVRAG complex. Different concentrations (0.1, 0.5, 2 μM) of Rab7 proteins were incubated with the Rubicon–UVRAG complex. The bound proteins were analyzed by Western blotting using anti-Flag and anti-Rab7 antibodies.

Fig. 4.

Rubicon localizes to maturing early endosomes. (A) Rubicon localizes to maturing early endosomes. U₂OS cells expressing Rubicon-Flag were transfected with GFP-Rab5-WT, GFP-Rab5-S34N, or GFP-Rab5-Q79L and observed under a fluorescence microscope. Cy3-conjugated M2 antibody was used to label Rubicon-Flag. (Scale bar: 5 μm.) Framed areas of Rab5 Q79L-expressing cells are enlarged (Bottom). (B) Rab7 GTP-bound form disrupts the overlap of Rubicon–UVRAG. Mock transfection: GDP-bound Rab7 T22N or GTP-bound Rab7 Q67L were coexpressed with GFP–UVRAG and Rubicon-Flag in U₂OS cells. The subcellular localization of UVRAG (green) and Rubicon (red) was observed under a fluorescence microscope. (C) The number of cellular puncta positive for UVRAG and Rubicon was counted and plotted in cells described in B.

Fig. 5.

Rubicon sequesters UVRAG from C-VPS/HOPS and negatively regulates Rab7 activation. (A) Rubicon depletion promotes the interaction between UVRAG and C-VPS/HOPS. Cell lysates from Rubicon WT or knockdown (KD) U₂OS cells were immunoprecipitated with anti-UVRAG antibody. The indicated proteins were detected. (B) Rubicon overexpression decreases the binding of UVRAG to C-VPS/HOPS. HEK293T cells were transfected with Rubicon-Myc or vector alone. The cell lysates were immunoprecipitated with anti-UVRAG antibody. The indicated proteins were detected. (C) Depletion of Rubicon stimulates Rab7-RILP interaction. In cell lysates prepared from WT or Rubicon RNAi-depleted cells, Rab7 was immunoprecipitated and the resulting immunocomplexes were probed for Rab7 and RILP. 3-MA (10 mM) was used to treat both Rubicon WT and depleted cells for 8 h before collection. (D) Depletion of Rubicon promotes Rab7–Vps41 interaction. In cell lysates prepared from WT or Rubicon RNAi-depleted cells, Rab7 was immunoprecipitated and the resulting immunocomplex was probed for Rab7 and Vps41.

Fig. 6.

Depletion of Rubicon alters EGFR degradation via endocytic pathway. (A) EGFR degradation is accelerated in Rubicon-depleted cells. EGFR degradation was examined in Rubicon RNAi-depleted 293T cells. Cells were serum-starved in DMEM-only medium for 10 h, followed by 200 ng/mL EGF addition to initiate EGFR degradation. Cells were collected at indicated time points and tested for EGFR level by Western Blotting. (B, D, and F) EGFR levels in A, C, and E were quantified by a Phosphorimager and normalized by calculating as the percentage of the initial receptor content. (C) EGFR degradation was tested in Rubicon overexpression cells. (E) EGFR degradation was examined in Rubicon-depleted 293T cells expressing vector alone, Rubicon WT, or Rubicon-ΔCT. (G) EGFR accumulates in large vacuoles in Rubicon-overexpressing cells. In U₂OS cells expressing vector alone or Rubicon, the endogenous EGFR localization was detected by immunostaining using anti-EGFR antibody. (Scale bar: 5 μm.) (H) Number of vacuoles greater than 800 nm in diameter was counted and plotted in cells (G).