Membrane recruitment of the cargo-selective retromer subcomplex is catalysed by the small GTPase Rab7 and inhibited by the Rab-GAP TBC1D5 - PubMed (original) (raw)

. 2009 Jul 15;122(Pt 14):2371-82.

doi: 10.1242/jcs.048686. Epub 2009 Jun 16.

Affiliations

Membrane recruitment of the cargo-selective retromer subcomplex is catalysed by the small GTPase Rab7 and inhibited by the Rab-GAP TBC1D5

Matthew N J Seaman et al. J Cell Sci. 2009.

Abstract

Retromer is a membrane-associated heteropentameric coat complex that functions in the endosome-to-Golgi retrieval of the cation-independent mannose-6-phosphate receptor, the Wntless protein and other membrane proteins of physiological significance. Retromer comprises two functional subcomplexes: the cargo-selective subcomplex is a trimer of the VPS35, VPS29, VPS26 proteins, whereas the sorting nexin proteins, Snx1 and Snx2 function to tubulate the endosomal membrane. Unlike the sorting nexins, which contain PtdIns3P-binding PX domains, the cargo-selective VPS35/29/26 complex has no lipid-binding domains and its recruitment to the endosomal membrane remains mechanistically uncharacterised. In this study we show that the VPS35/29/26 complex interacts with the small GTPase Rab7 and requires Rab7 for its recruitment to the endosome. We show that the Rab7K157N mutant that causes the peripheral neuropathy, Charcot-Marie-Tooth disease, does not interact with the VPS35/29/26 complex, resulting in a weakened association with the membrane. We have also identified a novel retromer-interacting protein, TBC1D5, which is a member of the Rab GAP family of proteins that negatively regulates VPS35/29/26 recruitment and causes Rab7 to dissociate from the membrane. We therefore propose that recruitment of the cargo-selective VPS35/29/26 complex is catalysed by Rab7 and inhibited by the Rab-GAP protein, TBC1D5.

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Figures

Fig. 1.

Fig. 1.

VPS35/29/26 interacts with Rab7. (A) Cells stably expressing GFP-Rab7 were treated with nocodazole before fixation and labelling with antisera against VPS26. There is some colocalisation between VPS35/29/26 and GFP-Rab7 (indicated by arrows), which is enhanced by treatment with nocodazole. Scale bar: 20 μm. (B) Cells expressing GFP-tagged Rab5, Rab7, Rab9 or VPS29 were treated with nocodazole, lysed and then incubated with anti-GFP. After washing, the immunoprecipitated proteins were subjected to SDS-PAGE and western blotting with anti-VPS35 and anti-VPS26. VPS35/29/26 interacts substoichiometrically with GFP-Rab7 but not GFP-Rab5 or GFP-Rab9. (C) Cells expressing GFP-Rab5, GFP-Rab7 or GFP-Rab9 were treated with nocodazole, lysed and incubated with anti-GFP. The immunoprecipitated proteins were analysed by SDS-PAGE, silver staining and mass spectrometry. VPS35 and VPS26 were detected in the Rab7 lane along with Rab escort protein and GDI-2. (D) Cells expressing GFP-Rab7, GFP-Rab7Q67L or GFPRab7T22N were treated as above except that there was no incubation with nocodazole before lysis and immunoprecipitation with anti-GFP. VPS35 and VPS26 were detected in the GFP-Rab7 and GFP-Rab7Q67L lanes.

Fig. 2.

Fig. 2.

VPS35/29/26 and Rab7 colocalise on endosomal membranes. Cells expressing either GFP-Rab7 (A-C) or GFP-Rab7Q67L (D-F) were grown in 60 mm dishes, washed and then snap frozen to permeabilise them. After fixation, the cells were labelled with anti-VPS26 and anti-GFP antibodies followed by 5 nm anti-rabbit IgG colloidal gold and 10 nm anti-mouse IgG colloidal gold. The cells were then fixed again, embedded in resin and sectioned for EM. The gold particles have been false colour coded to aid the distinction between the 5 nm and 10 nm colloidal gold. The 5 nm particles are coloured blue and the 10 nm particles are yellow. VPS26 and GFP-Rab7 or GFP-Rab7Q67L colocalise extensively. Scale bar: 250 nm.

Fig. 3.

Fig. 3.

The GDP-locked Rab7T22N mutant inhibits VPS26 membrane association. (A) Cells stably expressing GFP-Rab7T22N were seeded with untransfected HeLa cells onto cover slips before fixation and labelling with anti-VPS26 or anti-Snx1 antibodies. Cells expressing the GFP-Rab7T22N mutant (*) display reduced VPS26 localisation but Snx1 is unaffected. Scale bar: 20 μm. (B) The fluorescence intensity of the VPS26 labelling was quantified within a perinuclear region of interest (ROI). Cells expressing the GFP-Rab7T22N mutant had ∼40% less intense VPS26 labelling. (C) Cells expressing GFP-Rab5, GFP-Rab7, GFP-Rab7T22N or GFP-Rab7Q67L were snap frozen to permeabilise them, washed and scraped into buffer before centrifugation to pellet the membranes. Pellet (P) and supernatant (S) samples were subjected to SDS-PAGE and analysed by western blotting. (D) The amount of VPS35 in the pellet (P) and supernatant (S) fractions from two western blots was quantified and the average is shown graphically expressed as a percentage of the total in the cell-line assayed. VPS35 is ∼70% membrane associated except in cells expressing the Rab7T22N mutant where VPS35 is <50% membrane associated. Error bars represent s.e.m.

Fig. 4.

Fig. 4.

The Rab7K157N CMT mutant does not interact with VPS35/29/26. (A) Cells stably expressing GFP-Rab5, GFP-Rab7, GFP-Rab7L129F, GFP-Rab7K157N, GFP-Rab7N161T and GFP-Rab7V162M were treated with nocodazole before lysis and incubation with anti-GFP antibody. After washing, the immunoprecipitated proteins were subjected to SDS-PAGE and analysed by western blotting. VPS35 did not co-immunoprecipitate with GFP-Rab5 but did co-immunoprecipitate with GFP-Rab7, GFP-Rab7L129F and GFP-Rab7N161T. VPS35 only weakly interacted with GFP-Rab7K157N. (B) The data from two experiments were quantified by densitometry (normalising to the respective GFP-Rab7 signal) and the average is shown graphically. Error bars represent s.e.m.

Fig. 5.

Fig. 5.

Loss of Rab7 expression results in VPS26 redistribution to the cytoplasm. (A) Lysates from cells treated with siRNA to knock down (KD) expression of Rab7, Rab9, VPS26 or VPS35 were analysed by western blotting. The siRNA abolished expression of the respective target gene. (B) Cells treated with siRNA in A were labelled with antibodies against VPS26 and Snx1. Loss of Rab7 expression results in VPS26 becoming cytosolic whereas Snx1 remains membrane associated. Loss of Rab9 expression did not produce a similar effect. Scale bar: 20 μm. (C) The amount of membrane associated VPS35 and VPS26 was determined quantitatively using the same assay in Fig. 3B. Loss of Rab7 expression results in a notable shift of VPS35 and VPS26 into the cytosolic fraction. The effect is rescued by expression of GFP-Rab7. (D) The amount of VPS35 in the pellet (P) and supernatant (S) fractions from two western blots was quantified and the average is shown graphically expressed as a percentage of the total in the cell line assayed. 70-80% of VPS35 is membrane associated, except in cells treated with siRNA to silence Rab7 expression, where <50% of VPS35 is membrane associated. Error bars represent s.e.m.

Fig. 6.

Fig. 6.

GFP-Rab7 can rescue the loss of endogenous Rab7. (A) Cells expressing GFP-Rab7 or the endosomal membrane protein Slc11a2-GFP were treated with siRNA to knock down Rab7. Expression of GFP-Rab7 was able to rescue VPS26 localisation but expression of Slc11a2-GFP does not rescue VPS26 redistribution to the cytoplasm. Scale bar: 20 μm. (B) Control (con) or knockdown (KD) lysates from untransfected HeLa cells or cells expressing GFP-Rab7, Slc11a2-GFP, GFP-Rab7L129F, GFP-Rab7K157N or GFP-Rab7N161T, which had been treated with siRNA to knock down Rab7 expression were western blotted for Rab7 or the respective GFP-Rab7 protein. (C) The localisation of VPS26 in cells expressing GFP-Rab7L129F, GFP-Rab7K157N or GFP-Rab7N161T, which had been treated with siRNA to knock down Rab7 (see B) was investigated by immunofluorescence. Scale bar: 20 μm. (D) The Rab7K157N mutant results in less membrane-associated VPS35 and VPS26. Cells expressing GFP-Rab7 or GFP-Rab7K157N were treated with siRNA to knock down endogenous Rab7 expression and were then analysed using a centrifugation assay to measure membrane-associated VPS35 and VPS26. (E) Results from the western blot (D) were quantified and are shown graphically. Error bars represent s.e.m. (F-I) Rab7 expression was ablated in cells expressing GFP-Rab7 (F,G) or GFP-Rab7K157N (H,I) and the cells were then prepared for pre-embedding labelling EM. VPS26 labelling (5 nm gold, coloured blue) colocalises with anti-GFP (10 nm gold, coloured yellow) in GFP-Rab7 cells but there is little VPS26 labelling observed in GFP-Rab7K157N cells. Scale bar: 250 nm. (J) Gold particles from six micrographs each of GFP-Rab7 and GFP-Rab7K157N expressing cells were counted confirming that there is a marked reduction in VPS26 labelling.

Fig. 7.

Fig. 7.

Loss of Rab7 expression causes an endosome-to-Golgi retrieval defect. (A) Cells expressing the CD8-CIMPR reporter protein were treated with siRNA to knock down Rab7, Rab9 or VPS35. The cells were incubated with anti-CD8 at 4°C and then warmed to 32°C for 24 minutes before fixation and labelling with anti-TGN46 followed by appropriate fluorescent secondary antibodies. Loss of Rab7 expression results in a partial block in endosome-to-Golgi retrieval. Scale bar: 20 μm. (B) The microscopy-based endosome-to-Golgi retrieval assay was quantified by determining the extent of colocalisation of between endocytosed anti-CD8 and TGN46 relative to the total anti-CD8 fluorescence. The efficiency of endosome-to-Golgi retrieval in the knockdown cells is expressed relative to the control cells.

Fig. 8.

Fig. 8.

Identification of a novel retromer-interacting protein, TBC1D5. (A) Cells expressing either Hrs-1-GFP or VPS29-GFP were washed, lysed and then incubated with anti-GFP and protein-A-Sepharose to immunoprecipitate the respective GFP-tagged protein. After washing, the proteins were eluted at low pH, acetone precipitated, and subjected to SDS-PAGE and mass spectrometry. (B) GFP-tagged TBC1D5 was transiently transfected into HeLa cells along with empty vector and the truncation of TBC1D5 that lacks the TBC domain. 24 hours after transfection, the cells were lysed and immunoprecipitated with anti-GFP and analysed by western blotting. VPS35 and VPS26 co-immunoprecipitated with GFP-TBC1D5 but not GFP alone or the ΔTBC domain construct.

Fig. 9.

Fig. 9.

Expression of TBC1D5 negatively regulates VPS26 recruitment. (A) Cells transiently transfected with GFP-TBC1D5 were fixed and labelled with anti-VPS26 antibodies. GFP-TBC1D5 is predominately cytosolic but colocalisation with VPS26 can be observed occasionally (see inset box). Scale bar: 20 μm. (B) Cells were transiently transfected with GFP-TBC1D5, the ΔTBC domain construct or GFP alone. After fixation, cells were labelled with antibodies against VPS26 or Snx1. Overexpression of TBC1D5 results in reduced VPS26 labelling but has no effect on Snx1 localisation. The ΔTBC domain or GFP alone had no effect. Scale bar: 20 μm. (C) Cells expressing GFP-Rab5, GFP-Rab7, GFP-Rab9 or GFP-Rab7Q67L were transiently transfected with red fluorescent protein (RFP)-TBC1D5, fixed and then labelled with anti-VPS26 antisera. Overexpression of TBC1D5 can redistribute GFP-Rab7 into the cytoplasm but does not affect GFP-Rab5, GFP-Rab9 or the Rab7Q67L mutant. Scale bar: 20 μm.

References

    1. Arighi, C. N., Hartnell, L. M., Aguilar, R. C., Haft, C. R. and Bonifacino, J. S. (2004). Role of the mammalian retromer in sorting of the cation-independent mannose 6-phosphate receptor. J. Cell Biol. 165, 123-133. - PMC - PubMed
    1. Bache, K. G., Raiborg, C., Mehlum, A. and Stenmark, H. (2003). STAM and Hrs are subunits of a multivalent ubiquitin-binding complex on early endosomes. J. Biol. Chem. 278, 12513-12521. - PubMed
    1. Barlowe, C., Orci, L., Yeung, T., Hosobuchi, M., Hamamoto, S., Salama, N., Rexach, M. F., Ravazzola, M., Amherdt, M. and Schekman, R. (1994). COPII: a membrane coat formed by Sec proteins that drive vesicle budding from the endoplasmic reticulum. Cell 77, 895-907. - PubMed
    1. Béthune, J., Wieland, F. and Moelleken, J. (2006). COPI-mediated transport. J. Membr. Biol. 211, 65-79. - PubMed
    1. Bonifacino, J. S. and Traub, L. M. (2003). Signals for sorting of transmembrane proteins to endosomes and lysosomes. Annu. Rev. Biochem. 72, 395-447. - PubMed

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