doi: 10.1083/jcb.200110081. Epub 2002 Feb 11.
Early/recycling endosomes-to-TGN transport involves two SNARE complexes and a Rab6 isoform
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- PMID:11839770
- PMCID: PMC2174079
- DOI: 10.1083/jcb.200110081
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Early/recycling endosomes-to-TGN transport involves two SNARE complexes and a Rab6 isoform
Frédéric Mallard et al. J Cell Biol..
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Abstract
The molecular mechanisms underlying early/recycling endosomes-to-TGN transport are still not understood. We identified interactions between the TGN-localized putative t-SNAREs syntaxin 6, syntaxin 16, and Vti1a, and two early/recycling endosomal v-SNAREs, VAMP3/cellubrevin, and VAMP4. Using a novel permeabilized cell system, these proteins were functionally implicated in the post-Golgi retrograde transport step. The function of Rab6a' was also required, whereas its closely related isoform, Rab6a, has previously been implicated in Golgi-to-endoplasmic reticulum transport. Thus, our study shows that membrane exchange between the early endocytic and the biosynthetic/secretory pathways involves specific components of the Rab and SNARE machinery, and suggests that retrograde transport between early/recycling endosomes and the endoplasmic reticulum is critically dependent on the sequential action of two members of the Rab6 subfamily.
Figures
Characterization of the experimental permeabilized cell system. (A) Generic protocols used to reconstitute STxB transport from the EE to the TGN. (Perm.) Permeabilization in the absence of exogenous cytosol. (Inset) Variations 1 and 2 were used to compare transport efficiencies in intact or permeabilized HeLa cells (means of two experiments). (Detection) Incubation of permeabilized cells with [35S]sulfate and ATP-regenerating system (ATP reg. sys.) in the absence of cytosol. (B and C) STxB transport to the TGN depends on the presence of cytosol in a dose-dependent manner. A representative gel is shown in B, and the corresponding quantification in C. Means of two [35S]-PAPS (PAPS) or three [35S]sulfate (35SO42-) experiments (± SEM). Note that similar responses are observed when [35S]sulfate or [35S]-PAPS are used as sulfuryl donors (the total amounts of sulfated STxB-Sulf2 obtained in both conditions are comparable). (D) Sulfation as such is not cytosol dependent. STxB-Sulf2 was preaccumulated, either in the Golgi apparatus at 37°C, or in the EE at 19.5°C before permeabilization and incubated with or without cytosol. (E) STxB transport to the TGN is ATP dependent. As in D, STxB-Sulf2 was accumulated in the EE or the Golgi apparatus before permeabilization and incubation with complete or ATP-depleted cytosol in the presence of35S-PAPS to render the sulfation reaction as such ATP independent. (F) Kinetics of STxB transport to the TGN. Permeabilized cells were continuously incubated with [35S]sulfate for the indicated times. (G and H) STxB transport to the TGN in permeabilized cells detected by electron microscopy. STxB was internalized into the EE, and the cells were permeabilized and then incubated for 30 min at 37°C before fixation and cryosectioning. Cryosections were labeled for STxB (15-nm gold particles) and (G) TGN46 (10-nm gold particles) or (H) GalT (10-nm gold particles). Bars, 100 nm.
Retrograde transport to the TGN is mediated by the t-SNAREs Syn6, Syn16, and Vti1a. An experimental protocol as shown in Fig. 1 A was used. (A) STxB-Sulf2 transport to the TGN was assayed by sulfation analysis in the presence of the indicated concentrations of recombinant wild- type α-SNAP (wt) or a dominant negative α-SNAP mutant (L294A). As in the following parts of the figure, means (± SEM) of two to six experiments are shown. (B) 25–50 μg/ml of anti-Syn6, 7, 10, 16, or anti-Vti1a antibodies were continuously present from permeabilization on. Rb IgG, rabbit control IgG. The experiments with Syn6 were performed both with a monoclonal and a polyclonal antibody. (C) Anti-Syn16 antibody and Fab fragments generated from this antibody (Syn16[Fab]) had comparable inhibitory effects on STxB-Sulf2 transport to the TGN. Inhibition could be reversed by prebinding of the antibodies to recombinant His-tagged Syn16. Higher doses of anti-Syn16 (200 μg/ml; Syn16[200]) did not significantly increase the inhibitory effect. (D) Syn16 localization in the TGN. Note that upon BFA treatment, the perinuclear staining of TGN38 and Syn16 collapsed into a microtubule organizing center-like staining, a characteristic of TGN proteins. (E) Antibodies against Syn6 and Syn16 had no additive inhibitory effects on STxB-Sulf2 transport to the TGN, suggesting that both proteins function in the same molecular complex. (F) Antibody against Syn16 coimmunoprecipitated Syn6 and Vti1a, but not Vti1b, the cis-Golgi Syn5 or the endosomal Syn7 or Syn12. (G) Expression of Syn6-cyto and Syn16-cyto, but not of Syn7-cyto in Tac-TGN38–transfected CHO cells, specifically inhibited transport of internalized anti-Tac antibodies to the TGN.
Retrograde transport to the TGN is mediated by the t-SNAREs Syn6, Syn16, and Vti1a. An experimental protocol as shown in Fig. 1 A was used. (A) STxB-Sulf2 transport to the TGN was assayed by sulfation analysis in the presence of the indicated concentrations of recombinant wild- type α-SNAP (wt) or a dominant negative α-SNAP mutant (L294A). As in the following parts of the figure, means (± SEM) of two to six experiments are shown. (B) 25–50 μg/ml of anti-Syn6, 7, 10, 16, or anti-Vti1a antibodies were continuously present from permeabilization on. Rb IgG, rabbit control IgG. The experiments with Syn6 were performed both with a monoclonal and a polyclonal antibody. (C) Anti-Syn16 antibody and Fab fragments generated from this antibody (Syn16[Fab]) had comparable inhibitory effects on STxB-Sulf2 transport to the TGN. Inhibition could be reversed by prebinding of the antibodies to recombinant His-tagged Syn16. Higher doses of anti-Syn16 (200 μg/ml; Syn16[200]) did not significantly increase the inhibitory effect. (D) Syn16 localization in the TGN. Note that upon BFA treatment, the perinuclear staining of TGN38 and Syn16 collapsed into a microtubule organizing center-like staining, a characteristic of TGN proteins. (E) Antibodies against Syn6 and Syn16 had no additive inhibitory effects on STxB-Sulf2 transport to the TGN, suggesting that both proteins function in the same molecular complex. (F) Antibody against Syn16 coimmunoprecipitated Syn6 and Vti1a, but not Vti1b, the cis-Golgi Syn5 or the endosomal Syn7 or Syn12. (G) Expression of Syn6-cyto and Syn16-cyto, but not of Syn7-cyto in Tac-TGN38–transfected CHO cells, specifically inhibited transport of internalized anti-Tac antibodies to the TGN.
Identification of v-SNAREs in STxB transport from the EE to the TGN. Cells were treated either with NEM, or NEM quenched with DTT (NEM/DTT) before lysis in immunoprecipitation buffer. IP, immunoprecipitation. (A) VAMP3/cellubrevin, VAMP4, and VAMP8/endobrevin were coimmunoprecipitated with Syn6, but not VAMP7/TI-VAMP. (B) Among the VAMPs that interacted with Syn6, only VAMP3/ cellubrevin and VAMP4 were also coimmunoprecipitated with Syn16. (C) Antibodies to VAMP4 coimmunoprecipitated Syn6, Syn16, and Vti1a, and immunoprecipitated VAMP4 itself. Vti1b could also be detected, but to a much lesser extent than Vti1a. (D) Antibodies to VAMP3/cellubrevin immunoprecipitated VAMP3/cellubrevin itself, and coimmunoprecipitated Syn6 and Vti1a, but not Vti1b. The presence of Syn16 on blots could not be resolved due to its close proximity to the heavy chains of anti-VAMP3/cellubrevin antibody used for the immunoprecipitation. Note that anti-VAMP4 did not coimmunoprecipitate VAMP3/cellubrevin (C), and vice versa (D).
Functional implication of VAMP4 in EE-to-TGN transport. An experimental protocol as shown in Fig. 1 A was used. (A) Permeabilized HeLa cells were incubated in the continuous presence of 0.1 or 0.4 mg/ml of anti-VAMP4 antibody, 0.4 mg/ml of control Rb IgG, or 0.25 mg/ml recombinant cytosolic fragments of VAMP4 (VAMP4-cyto) or VAMP8 (VAMP8-cyto). STxB-Sulf2 transport from EE/RE to the TGN was sampled by sulfation analysis. The inhibitory anti-VAMP4 effect was reversed by prebinding of the antibody to its peptide antigen. Peptide antigen alone had no effect on transport (unpublished data). Means of three to seven experiments (± SEM) are shown. (B) Expression of VAMP4-cyto inhibited anti-Tac antibody transport to the TGN in CHO cells expressing Tac-TGN38, whereas expression of VAMP7-cyto had no effect. (C) Antibodies against VAMP4 and Syn16 had no additive inhibitory effects on STxB-Sulf2 transport from EE/RE to the TGN, suggesting that both molecules function within the same molecular complex. One experiment representative of two is shown.
Putative role of VAMP3/cellubrevin in EE/RE-to-TGN transport. (A) TeNT was added at the indicated doses to SLO-permeabilized HeLa cells. Lysates from these cells were blotted for the indicated v-SNAREs. Note that among the tested proteins, only VAMP3/cellubrevin was cleaved. (B) TeNT-treated and control HeLa cell extracts were probed with an antibody (10.1) which recognizes the synaptobrevin-like members of the VAMP family. (Arrow) Migration of VAMP3/cellubrevin on a parallel blot. (C) The incubation of permeabilized cells with TeNT led to a partial inhibition of STxB-Sulf2 transport to the TGN, whereas the TeNT mutant E234Q was without effect. (D) When anti-VAMP4 antibody and TeNT were used in the same reactions, additive inhibition of STxB-Sulf2 transport was observed, suggesting that VAMP4 and the TeNT targets function in different molecular complexes or in parallel pathways. In C and D, an experimental protocol as shown in Fig. 1 A was used. Means of three to nine experiments (± SEM) are shown.
VAMP3/cellubrevin and GFP-VAMP4 colocalized with STxB on membranes of EE/RE. Cy3-labeled STxB was internalized at low temperatures into EE/RE of untransfected (VAMP3) or GFP-VAMP4–transfected HeLa cells. The cells were then fixed and stained with anti-VAMP3, where indicated. A subset of STxB containing structures were also labeled for VAMP3/cellubrevin or VAMP4.
The Rab6 and Rab11 regulate EE/RE-to-TGN transport. (A) Permeabilized HeLa cells were incubated either continuously with GTPγS (1 mM), or pretreated with the indicated concentrations of recombinant Rab-GDI. (B) The indicated concentrations of anti-Rab6 antibody or control rabbit IgG (0.1 mg/ml) were continuously present from permeabilization on. (C) Anti-Rab6 (75 μg/ml) and anti-Rab11 (100 μg/ml) antibodies did not have additive inhibitory effects when added at the same time to the permeabilized cell assay.
Specific role of the Rab6a' isoform in EE/RE-to-TGN transport. (A) Sulfation analysis on intact cells overexpressing the indicated Rab6a and Rab6a′ mutants or a dominant negative Rab11 mutant. Note that dominant negative Rab6a′T27N strongly inhibited retrograde transport to the TGN. (B) Western blot analysis of the expression levels of the indicated Rab6 mutants. All mutants were equally well expressed. CTL, empty vector transfected cells. (C) Cy3-labeled STxB was internalized for 45 min at 37°C into cells expressing the indicated proteins. The cells were then fixed and stained with antibodies to the medial Golgi marker CTR433 or Rab6. (D) In Rab6a′T27N–expressing cells, STxB (red) accumulated in transferrin receptor (TfR, green) containing EE/RE.
Specific role of the Rab6a' isoform in EE/RE-to-TGN transport. (A) Sulfation analysis on intact cells overexpressing the indicated Rab6a and Rab6a′ mutants or a dominant negative Rab11 mutant. Note that dominant negative Rab6a′T27N strongly inhibited retrograde transport to the TGN. (B) Western blot analysis of the expression levels of the indicated Rab6 mutants. All mutants were equally well expressed. CTL, empty vector transfected cells. (C) Cy3-labeled STxB was internalized for 45 min at 37°C into cells expressing the indicated proteins. The cells were then fixed and stained with antibodies to the medial Golgi marker CTR433 or Rab6. (D) In Rab6a′T27N–expressing cells, STxB (red) accumulated in transferrin receptor (TfR, green) containing EE/RE.
References
- Advani, R.J., H.R. Bae, J.B. Bock, D.S. Chao, Y.C. Doung, R. Prekeris, J.S. Yoo, and R.H. Scheller. 1998. Seven novel mammalian SNARE proteins localize to distinct membrane compartments. J. Biol. Chem. 273:10317–10324. - PubMed
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