A novel experimental system to measure STxB transport from EE/RE to the TGN
In the absence of a molecular understanding of membrane dynamics at the interface between the endocytic and the biosynthetic/secretory pathways, the existence of EE/RE-to-TGN transport has remained controversial. We have therefore reconstituted transport of STxB from EE/RE to the TGN in streptolysin O (SLO)-permeabilized HeLa cells. A previously constructed recombinant modified STxB with two COOH-terminal sulfation sites, termed STxB-Sulf2
, was chosen as a reporter molecule because its sulfation by TGN-localized sulfotransferase allows detection and quantification of arrival in the TGN (Mallard et al., 1998
was accumulated in EE/RE of intact HeLa cells by continuous incubation at low temperatures (Mallard et al., 1998
) ( A, main protocol). The cells were then subjected to SLO permeabilization, and transport to the TGN was assayed in the presence of [35
S]sulfate ( B). The SLO permeabilization technique was particularly adapted to our studies because only the plasma membrane is permeabilized following binding of SLO to cells on ice.
Figure 1. 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 (more ...)
In the absence of exogenous cytosol, transport was 19% (± 5.8%, n = 55) of that detected in the presence of 3 mg/ml of cytosol (, condition EE). The sulfation reaction per se was not dependent on exogenous cytosol because STxB-Sulf2, preaccumulated in the Golgi apparatus of intact cells ( D, Golgi), was sulfated, after permeabilization, in the same manner in the presence or absence of exogenous cytosol. In addition, the same dose-dependence on exogenous cytosol was observed when [35S]-labeled 3′-phosphoadenosine 5′-phosphosulfate (PAPS) instead of [35S]sulfate was used as a direct sulfuryl donor ( C).
To determine whether STxB transport to the TGN was energy dependent, we examined both complete and ATP-depleted cytosol ( E). These experiments were done with [35S]-labeled PAPS to render the sulfation reaction itself ATP independent. Under these conditions, TGN-localized STxB-Sulf2 was still efficiently sulfated, independent of the addition of an ATP regeneration system ( E Golgi, black bars). However, STxB transport to the TGN from the EE was strongly inhibited in the absence of ATP ( E, EE, white bars).
was transported to the TGN with comparable kinetics in permeabilized and intact cells. In fact, maximal sulfation was reached after 45 min in permeabilized cells ( F), as in intact cells (Mallard et al., 1998
). Furthermore, we found that the efficiency of transport in permeabilized cells was 25% of that in intact cells ( A, insert), comparable to other in vitro systems that reconstitute coupled budding and fusion reactions. Throughout this manuscript, this percentage was set to 100% for comparison purposes. Finally, electron microscopical studies established that in SLO-permeabilized cells, a significant part of internalized STxB (, G–H, 15 nm) gained access to structures labeled by the TGN markers TGN46 ( G, 10-nm gold particles, arrows) and galactosyl-transferase ( H, 10-nm particles, arrows), as previously described in intact cells (Johannes et al., 1997
; Mallard et al., 1998
). Morphologically identifiable Golgi stacks were also marked under these conditions ( H). In the absence of cytosol, no STxB transport to the Golgi could be detected (unpublished data).
Taken together, these results show that STxB transport from EE/RE to the TGN was efficiently reconstituted in SLO-permeabilized cells. The process exhibited the hallmarks characteristics of in vivo transport, and revealed canonical biochemical requirements observed for other in vitro reconstituted transport steps.
t-SNARE proteins in EE/RE-to-TGN transport
SNAREs are key regulators of vesicular membrane traffic. To test whether EE/RE-to-TGN transport was SNARE dependent, SNARE activity was inhibited using the dominant-negative α-SNAP mutant L294A that is unable to stimulate the ATPase activity of NSF (Barnard et al., 1997
). When added to permeabilized cells, recombinant α-SNAP(L294A) inhibited STxB transport in a dose-dependent manner ( A). Transport could also be slightly stimulated by the addition of low concentrations of wild-type α-SNAP ( A). These data strongly indicated a role for SNARE proteins in EE/RE-to-TGN transport.
Figure 2. Retrograde transport to the TGN is mediated by the t-SNAREs Syn6, Syn16, and Vti1a. An experimental protocol as shown in A was used. (A) STxB-Sulf2 transport to the TGN was assayed by sulfation analysis in the presence of the indicated concentrations (more ...)
We then set out to use the permeabilized cell system to identify the t-SNAREs that would function in the fusion process involving EE/RE-derived STxB-containing transport intermediates. Syn6, Syn10, Syn16, and Vti1a were chosen for our studies because of their localization in the Golgi apparatus (Bock et al., 1997
; Simonsen et al., 1998
; Tang et al., 1998a
; Xu et al., 1998
), and Syn7 as a negative control for its exclusive localization on endosomes (Nakamura et al., 2000
). Syn16 appeared of particular interest because of its extensive colocalization with the trans-Golgi marker TGN38 ( D, top panel), which persisted upon BFA treatment ( D, bottom panel). As shown in B, antibodies against Syn6, Syn16, and Vti1a potently inhibited transport, while anti-Syn7, anti-Syn10, or an irrelevant rabbit control IgG mixture had no significant effect. The use of anti-Syn16 at higher doses did not significantly increase inhibition ( C, Syn16), indicating that either the antibody itself was only partially inhibitory, or that Syn16 containing complexes are not the only ones controlling STxB transport to the TGN. Prebinding of anti-Syn16 to recombinant His-tagged Syn16 completely abolished the inhibitory effect of the antibody ( C). The anti-Syn16 effect was not due to cross-linking of Syn16 on target membranes, since monovalent Fab fragments generated from the antibody also potently inhibited transport ( C). Prebinding of Fab fragments to His-tagged Syn16 resulted in the loss of inhibition ( C). Taken together, these results suggest that Syn16, Syn6, and Vti1a participate in STxB transport to the TGN.
Interestingly, we observed that the inhibitory effects of anti-Syn6 and anti-Syn16 antibodies were not additive ( E), indicating that their respective target molecules function in the same transport pathway, possibly within the same molecular complex. The latter indication was tested by coimmunoprecipitation. Syn16 was immunoprecipitated from Triton X-100 lysates of HeLa cells. The precipitate was then probed by immunoblot for the presence of a cis-Golgi SNARE, Syn5 (Dascher et al., 1994
; Rowe et al., 1998
), the Golgi/TGN and endosomal SNAREs Syn6, Vti1a, and Vti1b (Bock et al., 1997
; Advani et al., 1998
; Xu et al., 1998
), and the endosomal SNAREs Syn7 and Syn12 (Tang et al., 1998c
; Nakamura et al., 2000
). Syn6 was coimmunoprecipitated with Syn16 ( F), confirming their physical association. Vti1a was also found in the Syn16 immunoprecipitate ( F), an observation that is consistent with the inhibitory effect of the anti-Vti1a antibody on transport to the TGN ( B). In a separate experiment, both Vti1a and Syn16 were also coimmunoprecipitated with Syn6 (unpublished data).
We used an overexpression approach to further confirm the role of Syn6 and Syn16 in retrograde transport to the TGN in intact cells. Previous studies had indicated that the cellular TGN marker protein TGN38, like STxB, cycles from EE/RE to the TGN (Ghosh et al., 1998
; Mallard et al., 1998
). As syntaxins are tail-anchored proteins with a majority of the polypeptide exposed to the cytosol, the cytosolic domains (cyto) of these molecules are expected to function as dominant negative mutants. We therefore tested whether expression of Syn6-cyto and Syn16-cyto would have an effect on the trafficking of TGN38 in CHO cells stably expressing a Tac epitope–tagged version of the protein (Ghosh et al., 1998
). In most untransfected cells or cells expressing Syn7-cyto, anti-Tac antibody, added from the outside of the cell, was found to accumulate in the perinuclear Golgi area ( G). In contrast, >50% of cells expressing Syn16-cyto or Syn6-cyto did not display Golgi-accumulation ( G), indicating that the soluble cytosolic domains of Syn6 and Syn16 blocked TGN38 transport to the TGN. Similar results were obtained when STxB EE/RE-to-TGN transport was followed in cytosolic domain expressing cells (unpublished data). In conclusion, the same Golgi/TGN-localized t-SNAREs appeared to regulate the retrograde transport of two markers, STxB and TGN38, to the TGN.
Identification of v-SNAREs in EE-to-TGN transport
A SNARE-mediated EE/RE-to-TGN transport model would implicate an interaction between the TGN-localized Syn6/Syn16/Vti1a t-SNAREs and putative early endosomal v-SNAREs. Therefore, anti-Syn6 or anti-Syn16 immunoprecipitates were analyzed for the presence of four VAMP family proteins known to be localized to endosomes: VAMP3/cellubrevin (McMahon et al., 1993
; Galli et al., 1994
), VAMP4 (Steegmaier et al., 1999
), VAMP7/TI-VAMP (Galli et al., 1998
), and VAMP8/endobrevin (Wong et al., 1998
). Intact cells were treated before lysis either with NEM to accumulate cognate SNARE complexes, or with DTT-inactivated NEM as a control. An increase of SNARE complex recovery after NEM treatment indicates that the complexes did not form after lysis and dilution (Galli et al., 1998
). VAMP3/cellubrevin, VAMP4, and VAMP8/endobrevin coimmunoprecipitated with Syn6 ( A). Of these, only VAMP4, and to a lesser extent VAMP3/cellubrevin coimmunoprecipitated with Syn16 ( B). Association of VAMP3/cellubrevin and VAMP4 with Syn6 and Syn16 implied that they were candidate v-SNAREs for the regulation of EE-to-TGN transport.
Figure 3. 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 (more ...)
Antibodies to VAMP4 or VAMP3/cellubrevin also coimmunoprecipitated Syn6 and Vti1a (, C–D). Again, VAMP4 was more efficient than VAMP3/cellubrevin, suggesting that VAMP4 may be more important for retrograde transport at the EE/RE-TGN-interface than VAMP3/cellubrevin. Syn16 was clearly detected in the anti-VAMP4 immunoprecipitate ( C), whereas a smear from the heavy chain prohibited its detection in the anti-VAMP3/cellubrevin immunoprecipitate (unpublished data). A small amount of Vti1b could be found in the anti-VAMP4 immunoprecipitate ( C), much less than for Vti1a. Importantly, antibody to VAMP4 did not coimmunoprecipitate VAMP3/cellubrevin, and vice versa (, C–D). This observation suggests that both v-SNAREs, VAMP4 and VAMP3/cellubrevin, interacted independently with the same t-SNAREs.
To study the function of VAMP4, an antibody was made against a peptide from the NH2 terminus of the protein (residues 1–15) with maximal sequence divergence compared with other VAMPs. Anti-VAMP4 inhibited EE/RE-to-TGN transport in a dose dependent manner, and inhibition was lost when the antibody was prebound to its antigen ( A). We furthermore observed that purified VAMP4-cyto inhibited STxB transport to the TGN on permeabilized HeLa cells, whereas VAMP8-cyto had no effect ( A). As shown for Syn6 and Syn16 ( G), overexpression of VAMP4-cyto also inhibited TGN38 cycling to the TGN, whereas VAMP7-cyto was without effect ( B). Thus, VAMP4 function was necessary not only for retrograde transport of STxB, but also for that of TGN38.
Figure 4. Functional implication of VAMP4 in EE-to-TGN transport. An experimental protocol as shown in 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 (more ...)
VAMP4 and Syn16 appeared to interact physically and functionally. Indeed, antibodies against Syn16 and VAMP4, when combined, had no additive inhibitory effects ( C), suggesting that these two molecules functioned in the same molecular complex, as described above for Syn6 and Syn16 (, E–F).
Our anti-peptide antibody against the NH2
terminus of VAMP3/cellubrevin was without effect, even at high concentrations (unpublished data). Because it has been reported that VAMP3/cellubrevin is cleaved by tetanus neurotoxin (TeNT) (Galli et al., 1994
), we tested whether TeNT would modulate transport. As shown in A, TeNT cleaved VAMP3/cellubrevin (Galli et al., 1994
), but not VAMP7/TI-VAMP (Galli et al., 1998
), VAMP4, or VAMP8/endobrevin. Furthermore, an antibody that recognizes all synaptobrevin-like and TeNT-sensitive VAMPs (clone 10.1) (McMahon et al., 1993
) revealed that VAMP3/cellubrevin was the only member of this group to be expressed in HeLa cells ( B). At TeNT concentrations above 100 nM, a condition under which most VAMP3/cellubrevin was degraded ( A), EE/RE-to-TGN transport was inhibited by 25%, whereas an inactive mutant of TeNT, TeNT (E234Q), was without effect ( C). As a whole, these data suggest that the TeNT effect was due to modulation of VAMP3/cellubrevin activity.
Figure 5. 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 (more ...)
Importantly, when TeNT and anti-VAMP4 antibody were added together, transport inhibition was additive ( D). This observation is consistent with the above-mentioned hypothesis that both v-SNAREs interact independently with the Syn6/Syn16/Vti1a t-SNAREs (). The level of residual transport ( D) was comparable to that observed in the presence of anti-Syn6, anti-Syn16, or anti-Vti1a antibodies ( B), again confirming that these molecules act in the same transport step.
To further investigate the potential role of VAMP3/cellubrevin and VAMP4 in EE/RE-to-TGN transport, the distribution of both proteins was compared with that of STxB internalized at low temperatures into the EE (). Many of the STxB containing early endosomal structures were also labeled with VAMP3/cellubrevin. Using anti-VAMP4 (unpublished data) or GFP-coupled VAMP4 (), similar observations were made.
Figure 6. 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 (more ...)
The role of Rab6a′ in EE/RE-to-TGN transport
The addition of the nonhydrolyzable GTP-analogue GTPγS to the permeabilized cell assay resulted in a strong inhibition of EE/RE-to-TGN transport ( A), implicating a function for GTPases. We further showed the involvement of proteins of the Rab family of small GTPases (Zerial and McBride, 2001
), as their removal from membranes by preincubation of SLO-permeabilized cells with recombinant Rab-GDI (GDP-dissociation inhibitor) resulted in a strong inhibition of transport ( A). An antibody against Golgi-localized Rab6 potently inhibited EE/RE-to-TGN transport ( B). The inhibition obtained with the anti-Rab6 antibody was markedly stronger than that observed with an antibody to Rab11 ( C), which we had previously implicated in the regulation of membrane dynamics at the RE-TGN interface (Wilcke et al., 2000
). The combined use of both antibodies did not lead to a significant increase in inhibition ( C).
Figure 7. 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 (more ...)
Recently it has become clear that mammalian cells express two Rab6 isoforms, termed Rab6a and Rab6a′ (Echard et al., 2000
). Rab6a and Rab6a′ differ in only three amino-acids, and available anti-Rab6 antibodies recognize both proteins equally well (Echard et al., 2000
). To discriminate between Rab6a and Rab6a′ function, mutants were overexpressed in HeLa cells ( A). A dominant-negative Rab6a′ mutant (Rab6a′T27N) was found to be a potent inhibitor of EE/RE-to-TGN transport, as measured by the sulfation assay ( A) or immunofluorescence analysis in transfected cells ( C). In cells overexpressing Rab6a′T27N, STxB accumulated in transferrin receptor containing EE/RE ( D). In contrast, the GTPase deficient mutant Rab6a′Q72L only weakly affected transport ( A), an effect that probably coincided with a mild alteration of Golgi morphology ( C). These findings thus suggested that Rab6a′ regulates retrograde transport to the TGN.
Figure 8. 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 (more ...)
On the other hand, both Rab6aT27N and Rab6aQ72L inhibited transport by ~50% ( A). The block induced by Rab6aQ72L was actually expected, as this mutant causes redistribution of Golgi membranes (Martinez et al., 1997
) including the TGN-localized t-SNAREs Syn6, Syn16, and Vti1a (unpublished data). The inhibitory effect of Rab6aT27N, although less pronounced than that of Rab6a′T27N, was more surprising in the light of its function in Golgi-to-ER transport (Martinez et al., 1997
). However, it should be pointed out that all Rab6-binding proteins identified so far interact with both Rab6a and Rab6a′. The only noticeable exception is the Rab6a interacting protein Rabkinesin-6 (Echard et al., 2000
), a molecular motor likely to be involved in the long-range movement of Rab6a positive structures between the Golgi and the ER (Echard et al., 1998
; White et al., 1999
). Therefore, overexpression of Rab6aT27N might titrate molecules required for Rab6a′ function, resulting in an indirect inhibition of EE/RE-to-TGN transport.
All Rab6 mutants that were used in this study were expressed to similar levels ( B). The specificity of our observations is illustrated by the fact that overexpression under identical conditions as those described above of a dominant negative Rab4 mutant did not affect post-Golgi retrograde transport (unpublished data). Furthermore, recently published data suggests that dominant negative Rab5 does not interfere with STxB transport to the Golgi apparatus (Nichols et al., 2001
). In view of the potent and specific inhibition of retrograde transport by Rab6a′T27N, we therefore conclude that Rab6a′ function is required in this transport step.