Two approaches were taken to identify the SNARE proteins responsible for MPR transport to the Golgi. First, we tested the ability of antibodies to block the transport of cation-dependent (CD) MPR to the TGN in vitro. SNARE identification was initially hindered by the lack of cross-reactivity of anti-SNARE antibodies with the CHO cell components of our original in vitro transport assays (Itin et al., 1997
). To circumvent this, we reestablished our in vitro transport assay using an HEK293 human cell line that stably expresses a tagged version of the CD-MPR containing an N-terminal His tag for purification, a myc-tag for localization, and a consensus tyrosine sulfation site that can be modified by tyrosine sulfotransferase upon its arrival at the TGN (Itin et al., 1997
As shown in , anti-STX16 and -Vti1a antibodies inhibited in vitro transport in a concentration-dependent manner, but anti-STX6 antibodies did not. STX6 is required for the transport of Shiga toxin and cholera toxin from early endosomes to the Golgi complex (Mallard et al., 2002
). Three monoclonal and one polyclonal anti-Rab6 antibody were tried without effect (Fig. S1 A, available at http://www.jcb.org/cgi/content/full/jcb.200707136/DC1
). This observation provided an important molecular distinction between these two transport pathways, which have already been shown to differ in their requirements for the Rab9 GTPase (Iversen et al., 2001
) and the putative tether, Golgin-97 (Lu et al., 2004
; Reddy et al., 2006
Figure 1. Retrograde transport of MPRs to the TGN is mediated by STX10, STX16, Vti1a, and VAMP3. (A) In vitro transport of CD-MPRs in reactions containing 100 μg/ml (light gray bars) or 200 μg/ml (dark GRAY bars) anti-SNARE IgG. Cytosol-dependent (more ...)
To determine whether these SNARE antibodies inhibited a Rab9-dependent step, we tested whether the addition of both anti-Rab9 IgG and anti-STX16 IgG led to an even greater level of inhibition. As shown in , anti-STX16 and anti-Rab9 antibodies each inhibited in vitro transport ~40–50%. When added together, no further inhibition was observed. This suggests that Rab9 and STX16 antibodies are inhibiting the same transport process in vitro.
As a second independent test for SNARE involvement, we expressed and purified soluble forms of 12 TGN and endosome-localized SNARE proteins () and tested their ability to inhibit in vitro transport reactions by presumably forming nonfunctional complexes with endogenous SNARE proteins. In agreement with the antibody inhibition data (), soluble GST-tagged STX16 and Vti1a proteins inhibited transport ~50–60% (). Removal of GST by thrombin cleavage of GST-STX16 yielded the same level of transport inhibition (41 ± 0.1% MPR transport relative to control reactions). In addition, STX10 and soluble VAMP3 and 4 inhibited transport (). In contrast, soluble STX5, 6, 11, 13, Vti1b, and VAMP7 and 8 were without significant effect ().
Analysis of the concentration dependence of the inhibition observed showed that transport could be inhibited as much as 70% with ~2 μM STX10, STX16, and Vti1a (). VAMP3 and 4 were less potent but also showed concentration-dependent inhibition. When evaluated on a concentration basis (), VAMP3 was significantly more potent than VAMP4 and is likely to be the more relevant vesicle SNARE for this reaction. The control SNARE protein STX13 failed to inhibit transport when added to similar concentrations. Together, these experiments implicate a SNARE complex comprised of STX10, STX16, Vti1a, and VAMP3 in the transport of MPRs from late endosomes to the Golgi complex. This set of SNAREs includes one member each from the categories Qa, Qb, Qc, and R SNAREs that are required to form a functional SNARE complex (Jahn and Scheller, 2006
). Further evidence suggesting that these SNAREs function in a common step (and thus are members of the same complex) is shown in ; the inhibitory effects of STX10 and 16 were not additive. This specific SNARE complex (STX10–STX16–Vti1a–VAMP3) has been detected in living cells (Wang et al., 2005
), but its precise function was unknown until now.
To verify the physiological significance of our in vitro experiments, we tested the consequences of soluble SNARE protein overexpression in cultured cells. A block in MPR recycling caused by an inhibitory SNARE protein should decrease the level of MPRs available within the Golgi to transport newly synthesized lysosomal hydrolases efficiently. Under these conditions, hydrolases would be missorted and secreted. Thus, hexosaminidase secretion can be used to monitor a block in MPR trafficking. We have shown this to be true in cells expressing a dominant inhibitory Rab9 protein (Riederer et al., 1994
) or depleted of the putative tether, GCC185 (Reddy et al., 2006
Expression of soluble versions of STX10, STX16, and Vti1a but not STX6, STX13, or VAMP4 led to roughly a doubling in the amount of hexosaminidase secreted (). Comparison of the levels of exogenous SNARE protein expression () shows that STX10 was the most potent inhibitor. VAMP4 was poorly expressed; therefore, the results for this SNARE are inconclusive. Only a slight increase in hexosaminidase secretion was seen in cells overexpressing VAMP3. Together, these data support the in vitro findings () and the conclusion that a STX10–STX16–Vti1a-containing complex is needed for MPR recycling in living cells. In addition, soluble v-SNAREs were less potent inhibitors than soluble t-SNAREs both in vitro () and upon expression in cultured cells ().
Figure 2. Hexosaminidase secretion is increased in cells when STX10, STX16, or Vti1a function is compromised. (A) HeLa cells grown in 6-cm dishes were either mock transfected (control) or transfected with the indicated myc-tagged cytosolic SNARE and incubated in (more ...)
We confirmed the importance of the STX10 t-SNARE in MPR trafficking by siRNA depletion (, inset). In cells depleted of ~90% of their STX10 content, hexosaminidase secretion was stimulated ~2.5-fold (). Thus, by two independent criteria, STX10 is required for proper lysosomal enzyme sorting in living cells.
Loss of STX10 enhances MPR degradation
Steady-state levels of MPRs were also significantly decreased in cells depleted of STX10 and, to a lesser extent, in cells depleted of STX16 () relative to a control protein, p115. This is reminiscent of the increased turnover of MPRs observed when cells are depleted of TIP47, Rab9, or GCC185 (Diaz and Pfeffer, 1998
; Ganley et al., 2004
; Reddy et al., 2006
) or seen upon the loss of Retromer and PIKfyve proteins (Arighi et al., 2004
; Seaman, 2004
; Rutherford et al., 2006
Figure 3. MPRs are destabilized in cells depleted of STX10. CI-MPR and SNARE protein levels in HEK293 cells transfected with the indicated siRNAs. (A) Immunoblot. (B) Quantitation of the data shown in A. Values are from two independent experiments, normalized to (more ...)
Interestingly, siRNA depletion of STX16 led to a concomitant 80% loss of STX6 with little change in STX10; the depletion of STX6 led to a 30% loss of STX16. This suggests that the majority of STX6 is complexed to a significant fraction of STX16 in the cell and that STX16 is an important stabilizer of STX6. Cells depleted of STX10 showed increased levels of STX6 (60%) and STX16 (40%; ). In contrast, the depletion of STX6 led to a 60% increase in STX10 levels. It is possible that the depletion of one SNARE frees up STX16 to provide additional stabilization for another partner SNARE protein.
The loss of MPRs in STX10-depleted cells was caused by an increase in their degradation rate as measured in a pulse-chase labeling experiment. Cells were transfected with STX10 siRNA at time zero, and the loss of STX10 protein was monitored. At 45 h of siRNA treatment, STX10 protein was decreased by 36%; by 54 h, it was decreased by 83% (). Cells were pulse labeled with [35S]methionine and cysteine, and the chase was begun at a time when STX10 began to be significantly depleted. MPR levels were then determined at various times by immunoprecipitation. As shown in , cells depleted of STX10 showed significantly increased MPR degradation compared with control-treated cells. The half-life of MPRs was estimated to be ~13 h in the absence of STX10, which was much shorter than that of control cells (>36 h) under these conditions. This confirms the importance of STX10 for MPR stability and, by inference, MPR trafficking.
MPR missorting in cells lacking STX10
The missorting of MPRs in cells depleted of STX10 was confirmed by light microscopy. As shown in , cells containing STX10 showed a typical perinuclear concentration of cation-independent (CI) MPRs (, top; cell at bottom left). In contrast, cells depleted of STX10 (, arrows) showed highly dispersed staining for CI-MPRs (, top right; arrows). Under these conditions, the TGN was intact as determined by the localization of the TGN protein Golgin-97 (, bottom). Therefore, in cells lacking STX10, CI-MPRs are mislocalized to vesicles that are dispersed throughout the cytoplasm. Quantitation of the peripheral vesicles showed that their number increased more than twofold in cells depleted of STX10 ().
Figure 4. Depletion of STX10 leads to CI-MPR dispersal to sorting nexin-2–positive structures but does not disrupt Golgin 97 localization. (A) Summation of confocal z sections of HeLa cells treated with STX10 siRNA and double labeled with rabbit anti-STX10 (more ...)
Characterization of the accumulated vesicles revealed that they contain sorting nexin-1 (not depicted) and sorting nexin-2 proteins (), which are constituents of the retromer complex that is important for MPR recycling to the trans-Golgi complex (Arighi et al., 2004
; Seaman, 2004
; Rojas et al., 2007
). Indeed, 85% (766/904) of peripheral MPR-positive vesicles were sorting nexin-2 positive in STX10-depleted cells (). A significant proportion of sorting nexin-2 protein colocalizes with early endosome markers such as early endosome antigen 1 (EEA1; Arighi et al., 2004
; Seaman, 2004
; Carlton et al., 2005
; Rojas et al., 2007
). In the current study, EEA1 was present on 44% (304/695) of sorting nexin-2–positive structures in control cells; only 123/942 (13%) of the peripheral sorting nexin-2 structures contained CI-MPRs, and few peripheral MPR structures were observed (159; ). Upon STX10 depletion, 43% (301/708) of sorting nexin-2–positive vesicles contained EEA1, and 84% (766/916) contained CI-MPRs. As before, we saw a significant increase in peripheral MPR-positive structures (904/159). These peripheral MPRs showed a similar degree of overlap with EEA1 as sorting nexin-2 in STX10-depleted cells (~45%; unpublished data). In summary, upon the loss of STX10, the newly peripheral MPRs are tightly associated with sorting nexin-2 in small peripheral structures; at least some of these represent early endosomes because of their content of EEA1.
Are the peripheral MPR-containing structures that accumulate in STX10-depleted cells transport vesicle intermediates? If so, they would be expected to contain Rab9, which resides on the surface of vesicles traversing from late endosomes to the trans-Golgi (Barbero et al., 2002
). HeLa cells depleted of the putative tether GCC185 also accumulate MPRs in peripheral vesicles, the majority of which contain Rab9 protein (Reddy et al., 2006
). In contrast, in STX10-depleted cells, only 18% (15/84) of MPR-containing, peripheral vesicles also contained Rab9 (unpublished data). This suggests that MPRs are blocked in a prelate endosome compartment before a Rab9-dependent vesicle transfer process. Wang et al. (2005)
showed that cells depleted of STX10 display an alteration in transferrin receptor localization. If STX10 participates in more than one transport event, its depletion could lead to the accumulation of MPRs in a peripheral Rab9-negative prelate endosome compartment. Importantly, because our in vitro transport assay measures transport from a late endosome compartment (Goda and Pfeffer, 1988
), we are able to discern a specific role for STX10 in transport from that compartment to the Golgi complex.
STX10 functions in the late endosome to Golgi pathway
To verify that STX10 is required at the TGN for the receipt of late endosome but not early endosome-derived cargoes, we tested whether cells depleted of this t-SNARE were competent to transport cholera toxin B from early endosomes to the Golgi complex. For these experiments, fluorescent cholera toxin B fragment was internalized by receptor-mediated endocytosis for 30 min, and its arrival to the TGN was monitored microscopically after an additional 30 min. As shown in , the depletion of STX10 did not compromise the ability of HeLa cells to transport fluorescent cholera toxin B fragment from the early endocytic pathway to the TGN. In contrast, consistent with previous papers studying Shiga toxin (Mallard et al., 2002
; Wang et al., 2005
), the disruption of STX6 blocked this transport step (). As was true for STX10 depletion, the loss of STX6 did not alter the TGN, as monitored by immunostaining for Golgin-97 (not depicted) or GCC185 (), suggesting that cholera toxin mislocalization in STX6-depleted cells is not simply caused by Golgi complex disruption. Quantitation of these experiments revealed a block in cholera toxin transport in >50% of cells analyzed (). This morphological determination provides a minimum estimate of the extent of a block in transport, as only severely impaired cells are scored.
Figure 5. Cholera toxin B fragment transport in cells depleted of STX10 or 6. (A) HeLa cells were treated with control siRNA (left) or STX10 siRNA (right) and were allowed to internalize AlexaFluor488-conjugated cholera toxin B (CTxB) for 30 min followed by a 30-min (more ...)
Figure 6. TGN46 transport in cells depleted of STX10 or S6. HeLa cells were treated with STX10 siRNA (top) or STX6 siRNA (middle) and were stained for the indicated proteins; TGN46 was detected with sheep antibody and AlexaFluor594 anti–sheep antibodies. (more ...)
As an independent test of the specificity of STX10, we also examined the localization of TGN46, a protein that shuttles between endosomes and the Golgi complex (). Cells depleted of STX10 showed perfect Golgi localization of TGN46 (, top; arrows). In contrast, cells depleted of STX6 showed a vesicular pattern for TGN46 in >80% of cells scored (, middle; arrows). This was not caused by disruption of the TGN, at least by the criteria of GCC185 staining (, bottom; arrows).
These data directly show that the transport of proteins from early and late endosomes to the Golgi complex are mediated by distinct SNARE protein complexes: a SNARE complex containing STX6, STX16, and Vti1a mediates the receipt of cargo from early endosomes, whereas a complex containing STX10, STX16, and Vti1a mediates the receipt of cargo from late endosomes. Moreover, they reveal that the steady-state localization of TGN46 is sensitive to the loss of STX6, a finding that suggests that this protein follows a transport route that is shared with cholera toxin but is distinct from MPRs.
A STX10 SNARE complex component binds the GCC185 tether
Essentially every putative tether studied to date interacts with cognate SNARE proteins (for review see Lupashin and Sztul, 2005
). Thus, we were interested to test whether components of the STX10 complex interact with GCC185. GCC185 is a long coiled-coil protein that is required for MPR transport to the Golgi complex in vitro and in vivo (Reddy et al., 2006
). In addition, GCC185 binds directly to the Rab9 GTPase, which is also needed for this transport process (Reddy et al., 2006
We incubated purified, soluble SNARE proteins with recombinant GCC185 protein and analyzed bound proteins by immunoblotting. For these experiments, a GCC185 fragment comprised of the 110 C-terminal–most amino acids (C-110) was used. Importantly, the GCC185 C terminus bound directly to GST-STX16 but not to GST-STX10, -STX13, or Vti1a (). These data demonstrate that STX16 has the capacity to bind directly to GCC185. Although the binding is of low efficiency, it is specific.
Figure 7. STX16 interacts directly and specifically with GCC185. (A) GST-tagged cytosolic SNAREs were incubated with purified GCC185–C-110. Tagged SNAREs were recovered by glutathione–Sepharose beads, and the bound proteins were analyzed by SDS-PAGE (more ...)
We next tested whether full-length cytosolic GCC185 bound to immobilized SNARE proteins. In this case, GST-tagged STXs were incubated with cytosol; bound proteins were collected on glutathione–Sepharose beads and analyzed by immunoblotting. As shown in (top), full-length GCC185 bound to immobilized STX16 but did not show significant interaction with STX6, STX13, or the GST control (). In contrast, the structurally related Golgin-97 failed to bind to any of these SNARE proteins (, middle). This was somewhat surprising, as Golgin-97 is implicated in early endosome to TGN transport (Lu et al., 2004
), and it might have shown binding to STX6 or 16, which participate in that transport step. In summary, these data confirm the ability of STX16 to bind specifically to full-length GCC185.
STX16 could interact with GCC185 alone or as part of an intact SNARE complex. As shown in , incubation of cell extracts with anti-GCC185 antiserum led to the coimmunoprecipitation of small amounts of coexpressed, myc-tagged, soluble STX10, STX16, and Vti1a but not STX13, VAMP3, or VAMP4. This protocol would yield an underestimate of protein interactions because the initial GCC185 immunoprecipitation was of low efficiency (, top). Together, these data suggest that GST-STX10 and Vti1a can interact with GCC185 indirectly as part of an STX16-containing complex of t-SNARE proteins ().
If this conclusion is correct, STX16 might also mediate the interaction of GCC185 with the STX6–STX16–Vti1a complex. As shown at the bottom of , myc-tagged soluble STX6 was also coimmunoprecipitated with GCC185 antibodies. Again, this interaction was indirect, as full-length GCC185 failed to bind to immobilized STX6 protein (). However, we found no evidence that GCC185 interacts functionally with this complex in vivo, as the depletion of GCC185 did not prevent cholera toxin transport to the Golgi (Fig. S1 B). It should be noted that Derby et al. (2007)
reported a requirement for GCC185 in Shiga toxin transport, but the Golgi complex was much more disrupted in those siRNA depletion experiments than seen in our studies, and Golgin-97 may have been codepleted upon the efficient loss of GCC185 (Reddy et al., 2006
The ability of STX16 to bind to the GCC185 C terminus led us to test whether Rab GTPase binding might compete for this interaction because we have shown that the GCC185 C terminus also contains a Rab-binding site (Reddy et al., 2006
). The C-110 fragment binds to both Rab6 and Rab9 GTPases but not Rab1 or Rab5 GTPases (Reddy et al., 2006
; Schweizer Burguete et al., 2008
), and Rab binding might regulate SNARE interaction. As shown in , STX16 binding to GCC185 was decreased when reactions contained Rab6-GTP. At the same concentrations, Rab9 had less of an effect (unpublished data). Competition was specific for the active Rab protein in that Rab6-GDP failed to compete with STX16 for GCC185 binding. A mutant GCC185 protein construct (I1588A/L1595A) that fails to bind Rab GTPases (Schweizer Burguete et al., 2008
) also failed to bind STX16 (), which is consistent with these proteins sharing interaction sites on GCC185. Failure to bind was not the result of protein misfolding, as the mutant protein is entirely dimeric and soluble (unpublished data).
Figure 8. Rab6 competes with STX16 for GCC185 binding. (A) Purified, GCC185–C-110 and GST-soluble STX16 were incubated together, either alone or with Rab6 preloaded with GDP or GTPγS. GST-STX16 was recoveredwith glutathione–Sepharose beads; (more ...)
In summary, these data demonstrate a role for STX10–STX16–Vti1a and VAMP3 in the transport of MPRs (but not TGN46 or cholera toxin) from late endosomes to the TGN. In addition, the putative tether for this transport reaction, GCC185, binds to the t-SNARE complex that mediates vesicle fusion. Finally, the ability of a Rab GTPase that would be present at the target membrane to regulate SNARE–tether interactions implies that Rabs may regulate such interactions during or after vesicle docking and fusion.