Regulation of retromer recruitment to endosomes by sequential action of Rab5 and Rab7 - PubMed (original) (raw)
Regulation of retromer recruitment to endosomes by sequential action of Rab5 and Rab7
Raul Rojas et al. J Cell Biol. 2008.
Abstract
The retromer complex mediates retrograde transport of transmembrane cargo from endosomes to the trans-Golgi network (TGN). Mammalian retromer is composed of a sorting nexin (SNX) dimer that binds to phosphatidylinositol 3-phosphate-enriched endosomal membranes and a vacuolar protein sorting (Vps) 26/29/35 trimer that participates in cargo recognition. The mammalian SNX dimer is necessary but not sufficient for recruitment of the Vps26/29/35 trimer to membranes. In this study, we demonstrate that the guanosine triphosphatase Rab7 contributes to this recruitment. The Vps26/29/35 trimer specifically binds to Rab7-guanosine triphosphate (GTP) and localizes to Rab7-containing endosomal domains. Interference with Rab7 function causes dissociation of the Vps26/29/35 trimer but not the SNX dimer from membranes. This blocks retrieval of mannose 6-phosphate receptors to the TGN and impairs cathepsin D sorting. Rab5-GTP does not bind to the Vps26/29/35 trimer, but perturbation of Rab5 function causes dissociation of both the SNX and Vps26/29/35 components from membranes through inhibition of a pathway involving phosphatidylinositol 3-kinase. These findings demonstrate that Rab5 and Rab7 act in concert to regulate retromer recruitment to endosomes.
Figures
Figure 1.
Binding of the retromer Vps subcomplex to active Rab7. (A) A Triton X-100 extract of microsomes was incubated with equivalent amounts of the indicated GST-Rab proteins preloaded with GMP-PNP or GDP. Bound proteins were eluted in Laemmli sample buffer and analyzed by SDS-PAGE and immunoblotting (IB) with antibodies to the Vps26, Vps29, or Vps35 subunits of retromer (top). The first lane represents 20% of the input. The approximate molecular masses of the different proteins are indicated in kilodaltons. Asterisks indicate contaminating proteins found in all of the GST preparations. (B) COS-7 cells were transfected with plasmids encoding myc-Vps26, FLAG-Vps29, and (HA)3-Vps35, either individually or in the combinations indicated in the figure. Lysates from these cells were incubated with GST-Rab7–GMP-PNP–bound beads. Input and bound proteins were analyzed by SDS-PAGE and immunoblotting with antibodies to the myc, FLAG, and HA tags (top). (C) Immobilized GST, GST-Rab7, or GST-Rab9a preloaded with GDP or GTPγS was incubated with recombinant Vps26/29/35 trimer. Bound proteins were eluted and analyzed by SDS-PAGE and immunoblotting with antibodies to Vps29, Vps26, and Vps35 (top). The input represents 1.8% of the total recombinant Vps26/29/35 trimer used per reaction. The bottom panels show SDS-PAGE/Coomassie blue staining of the GST proteins used in these experiments.
Figure 2.
Immunofluorescence microscopy showing localization of Vps26 to Rab7- and Rab5-positive endosomes. HeLa cells were transfected with plasmids encoding GFP-Rab7 (A–C) or GFP-Rab5a (D–F), fixed, permeabilized, and immunostained with rabbit polyclonal antibody to Vps26 followed by Alexa Fluor 594–conjugated donkey anti–rabbit IgG. Cells were examined by confocal microscopy. (A and D) GFP fluorescence, green channel. (B and E) Alexa Fluor 594 fluorescence, red channel. (C and F) Merged images; yellow indicates colocalization. Arrows indicate examples of foci where proteins colocalize. Bar, 10 μm.
Figure 3.
Live cell imaging showing localization of Vps29-YFP to endosomal domains that contain CFP-Rab7 but not CFP-Rab5a. HeLa cells cotransfected with plasmids encoding Vps29-YFP (green) and either CFP-Rab7 (A–C) or CFP-Rab5a (D–F; red) were imaged by time-lapse fluorescence microscopy. Pictures in this figure were extracted from Videos 1–6 (available at
http://www.jcb.org/cgi/content/full/jcb.200804048/DC1
). Merging the green and red images generated the third picture in A and D and the images in the bottom rows of B, C, E, and F. Yellow indicates overlapping localization of green and red objects. Examples of colocalization are indicated by yellow arrows. The images in A and the series in B and C show the localization of Vps29-YFP to domains that contain CFP-Rab7 (indicated by green and red arrows in individual channel images or yellow arrows in merge images). The series in C shows an endosome in which Vps29-YFP (green arrows) is enriched on a detaching tubule that does not contain CFP-Rab7 (red arrows). The images in D and the series in E and F show that Vps29-YFP (green arrows) and CFP-Rab5a (red arrows) are localized to different endosomal domains. The series in F shows a Vps29-YFP–positive endosome with little or no CFP-Rab5 (green arrows). Time after the start of imaging (in seconds) is shown on the bottom left corner of each panel. Bars: (A and D) 5 μm; (B, C, E, and F) 2 μm.
Figure 4.
Immunoelectron microscopy localization of GFP-Rab7 and Vps26. Ultrathin cryosections of HeLa cells expressing wild-type GFP-Rab7 were immunogold labeled with antibodies to GFP (10-nm gold particles) and Vps26 (15-nm gold particles). Arrows indicate the presence of GFP-Rab7 on buds adjacent to the vacuolar part of endosomes (A) and on tubules (B). Tubules and buds are also labeled for Vps26. Bars, 200 nm.
Figure 5.
RNAi-mediated depletion of Rab7 causes Vps26 dissociation from endosomes. (A) HeLa cells were treated twice at 24-h intervals with a control, inactive siRNA, or siRNAs to Rab7, Rab4 (targeting both the a and b isoforms), or Vps26. Cell extracts were analyzed by SDS-PAGE and immunoblotting with antibodies to Rab7, Rab4, Vps26, or actin (loading control) as indicated in the figure. (B–Q) HeLa cells treated as in A with control (B, C, F, G, J, K, N, and O) or Rab7 siRNA (D, E, H, I, L, M, P, and Q) were immunostained for Vps26 (B, D, F, H, J, L, N, and P) and AP-3 (C and E), AP-1 (G and I), SNX1 (K and M), and SNX2 (O and Q) using rabbit polyclonal antibody to Vps26 and mouse monoclonal antibodies to AP-3–δ, AP-1–γ1, SNX1, or SNX2 followed by Alexa Fluor 594–conjugated donkey anti–rabbit IgG and Alexa Fluor 488–conjugated donkey anti–mouse IgG. Images were captured by epifluorescence microscopy. Bar, 10 μm.
Figure 6.
Specificity and rescue of Vps26 dissociation from membranes in RNAi-treated cells. HeLa cells were treated twice at 24-h intervals with an inactive siRNA (control; A) or siRNAs to both Rab4a and Rab4b (B) or to Rab7 (C–F). At 36 h after the second treatment with siRNA to Rab7, some cells were transfected with plasmids encoding RNAi-resistant (i.e., canine) GFP-Rab7 (C and E) or GFP (D and F). Examples of cells expressing GFP or canine GFP-Rab7 are indicated by arrows. The cellular distribution of Vps26 (A–D) was assessed by indirect immunofluorescent staining using rabbit polyclonal antibody to Vps26 followed by Alexa Fluor 594–conjugated donkey anti–rabbit IgG. Images were captured by epifluorescence microscopy. Bar, 10 μm.
Figure 7.
Dominant-negative forms of Rab5 or Rab7 alter the association of retromer with endosomes. (A–I) HeLa cells were transfected with plasmids encoding GFP-Rab4a–S22N (A–C), GFP-Rab5a–S34N (D–F), GFP-Rab7–T22N (G–I), or GFP-Rab7–Q67L. At 8 h after transfection, the cellular distribution of Vps26 (B, E, and H) and SNX1 (C, F, and I) was analyzed by indirect immunofluorescent staining using rabbit polyclonal antibody to Vps26 and mouse monoclonal antibodies to SNX1 followed by Alexa Fluor 594–conjugated donkey anti–rabbit IgG and Alexa Fluor 647–conjugated donkey anti–mouse IgG. (J–L) Cells were treated with 200 nM wortmannin in DMSO for 30 min at 37°C and the distribution of Vps26 and SNX1 was analyzed by indirect immunofluorescent staining using the respective primary antibodies and fluorescently labelled secondar)y antibodies. Images were captured by epifluorescence microscopy. Arrows point to cells expressing the GFP-Rabs. Bar, 10 μm.
Figure 8.
Depletion of Rab7 blocks the transport of CI-MPR from endosomes to the TGN. HeLa cells were treated twice at 24-h intervals with an inactive siRNA (control; A, C, E, and G) or siRNA to Rab7 (B, D, F, and H–N). At 48 h after treatment, the steady-state distribution of CI-MPR (A–D) was assessed by indirect immunofluorescent staining of fixed cells using rabbit polyclonal antibody to cytosolic tail of CI-MPR followed by Alexa Fluor 488–conjugated donkey anti–rabbit IgG. C and D correspond to high magnification views of CI-MPR–positive structures from control or Rab7-depleted cells, respectively. Live control cells (E and G) or Rab7-depleted cells (F and H–N) were incubated with an antibody to the luminal domain of CI-MPR for 2 h at 37°C. Cells were washed, fixed, permeabilized, and stained with Alexa Fluor 488–conjugated donkey anti–mouse IgG to detect internalized antibody to CI-MPR (E–H, J, and M). G and H show high magnification views from control (E) or Rab7-depleted cells (F), respectively. Cells in I–N were additionally stained with rabbit polyclonal antibody to TfR (I–K) or giantin (L–N) followed by Alexa Fluor 594–conjugated donkey anti–rabbit IgG. Images in A–H were captured using an epifluorescence microscope, and images in I–N were captured with a confocal microscope. (K and N) For merged images, yellow indicates colocalization. Arrows in I–K indicate examples of foci where proteins colocalize. (O) Extracts of HeLa cells treated with siRNAs to the proteins indicated on top were analyzed by 4–20% acrylamide gradient SDS-PAGE and immunoblotting (IB) with antibodies to the proteins indicated on the right. Equal amounts of total protein were loaded. Bars: (A, B, E, and F) 15 μm; (C, D, G, and H) 1.5 μm; (I–N) 10 μm.
Figure 9.
Rab7 depletion impairs processing of cathepsin D. HeLa cells were treated twice at 24-h intervals with inactive siRNA (control; lane 1) or siRNAs to the proteins indicated on top. At 24 h after the second round of siRNA treatment, cells were rinsed with PBS and incubated in serum-free culture medium for 24 h. The medium was collected and precipitated with trichloroacetic acid, and the resulting pellets were dissolved in Laemmli sample buffer. Cell extracts and media samples were analyzed by 4–20% acrylamide gradient SDS-PAGE and immunoblotting with rabbit polyclonal antibody to cathepsin D. Equal amounts of total protein were loaded on each lane. Blots were also probed with antibody to actin as a loading control. The positions of molecular mass markers (in kilodaltons) and of the precursor (pCatD), intermediate (iCatD), and mature (mCatD) forms of cathepsin D are indicated.
Figure 10.
Schematic representation of the regulation of retromer by Rab5 and Rab7. The scheme depicts a section of an endosome with its vacuolar and tubular aspects. The sequence of reactions is represented in the context of the progression from Rab5- to Rab7-positive endosomes and from the vacuolar to the tubular aspect. Details of this model are described in the Discussion section. BAR, Bin–Amphiphysin–Rvs; HOPS, homotypic fusion and vacuole protein sorting; PX, phox homology.
References
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