The Intracellular Pool of Exocyst Subunits Is Associated in Part with EEA1-, Tf-, and Rab11a-positive Endosomes But Not the TGN of Polarized MDCK Cells
Initial studies of exocyst subunit distribution in MDCK cells showed that Sec6 and Sec8 were localized at or near the tight junctions of cells after initiation of cell-to-cell contact or tubulogenesis (Grindstaff et al., 1998
; Lipschutz et al., 2000
; Rogers et al., 2003
). Other studies showed that the exocyst was associated with intracellular compartments including the Golgi, the TGN of MDCK cells expressing a kinase-inactive mutant of protein kinase D, and the recycling endosomes of wild-type MDCK cells (Yeaman et al., 2001
; Ang et al., 2003
; Prigent et al., 2003
). The discrepancy in exocyst localization may reflect, in part, the degree of cellular polarization, the growth conditions of the cells, the observation that some monoclonal antibodies differentially recognize pools of junctional versus intracellular populations of exocyst subunits (Yeaman et al., 2001
), and the possibility that the exocyst complex may exist in different conformational states (extended or closed) depending on its localization in the cell (Munson and Novick, 2006
Using a number of fixation conditions and monoclonal antibodies to Sec6 (rSec6), Sec8 (rSec8, 10C2, 5C3, 2E12), and Exo70 (13F3), we found that permeabilizing cells with saponin (a treatment that removes the cytoplasmic pool of exocyst), just before fixation, revealed a large intracellular pool of exocyst subunits in polarized MDCK cells (see Supplementary Figures 1 and 2). This intracellular pool was also observed in preextracted, semipolarized MDCK cells grown on glass coverslips (data not shown). By removing soluble proteins, the extraction procedure may cause exocyst-interacting proteins to dissociate from the complex, thus allowing antibody binding. Alternatively, the extraction procedure may alter the conformation of the exocyst, revealing the intracellular pool of subunits. Importantly, the intracellular pool of Exo70 (and of Sec8 using the 10C2, 5C3, and 2E12 antibodies) was observed even in cells not permeabilized with saponin before fixation (see for example, and ), confirming that the intracellular pool of exocyst is not simply an artifact of the saponin pretreatment.
Figure 1. Localization of exocyst subunits in polarized MDCK cells. (A and B) Cells were treated with saponin and then fixed using a pH-shift protocol. (A) Distribution of Sec8 (green), furin (red), and ZO-1 (blue). (B) Distribution of Sec8 (green), EEA1 (red), (more ...)
Figure 3. Localization of exocyst subunits to Rab11a- and IgA-positive recycling endosomes. (A–D) Distribution of Sec8/Exo70 (green) and Rab11a (A and C) or IgA (B and D). The distribution of ZO-1 was also examined but is not apparent in all of the panels. (more ...)
We initially assessed whether there was colocalization between Sec8 (using mAb 10C2) or Exo70 (using mAb 13F3) and the TGN marker furin. The tight junction protein ZO-1 was labeled to mark the position of the apicolateral junction. Furin was localized to a ribbon-like structure that resided in a supranuclear position in the cell (, A and C). We observed occasional regions where the furin-labeled TGN and Sec8 were in close proximity, but generally there was little colocalization between furin and Sec8 (A). This lack of colocalization was also apparent for two other Sec8 antibodies (5C3 or 2E12) and Sec6 (data not shown). Exo70 was occasionally found associated with the ends of the TGN ribbons (see boxed region, C), but there was generally not much overlap between Exo70 and furin (C). Next, we analyzed if any of the exocyst-associated tubulovesicular elements were associated with EEA1-positive apical and basolateral early endosomes (AEE and BEE, respectively) (Leung et al., 2000
). Although there was no colocalization between EEA1 and Sec8 (B), or EEA1 and Sec6 (data not shown), there appeared to be some localization of Exo70 to EEA1-positive endosomes (D). In this case, Exo70 appeared to concentrate at the periphery of the EEA1-positive endosomal elements (D, inset).
We next examined whether Sec6, Sec8, or Exo70 were associated with Tf-positive BEE or the common recycling endosomes (CRE) of polarized MDCK cells. BEE are found closely apposed to the basal and lateral surfaces of the cell, whereas CRE are found in a peri- and supranuclear distribution (Sheff et al., 1999
; Wang et al., 2000a
). After basolateral uptake of Tf for 45 min, the cells were fixed and double-labeled with antibodies specific for canine Tf, Sec8, or Exo70. We observed some colocalization between the peripherally localized Tf-labeled BEE and the exocyst subunits (bottom panels, , A and B). However, colocalization was more apparent for the Exo70 subunit. Colocalization was also readily observed in the supranuclear CRE (top panels, , A and B), and Sec6 showed a similar degree of colocalization with Tf as that observed for Sec8 (data not shown). As further confirmation of our localization studies, we immunoisolated Sec8-positive endosome-enriched compartments and observed that the Tf receptor was associated with these membranes (C). As a control we used nonspecific mouse IgGs, which failed to capture the Tf receptor.
Figure 2. Association of exocyst subunits with Tf-positive endosomes. (A) Distribution of Sec8 (green), Tf (red), and ZO-1 (blue). (B) Distribution of Exo70 (green), Tf (red), and ZO-1 (blue). Top, an XZ section; middle, a 3D reconstruction of optical sections (more ...)
The recent reports that Sec15 interacts with Rab11 (Zhang et al., 2004
; Wu et al., 2005
) prompted us to explore whether the exocyst was associated with the Rab11-positive ARE of polarized MDCK cells. The ARE, is morphologically distinct from the AEE, BEE, and CRE, is located at the apical pole of polarized MDCK cells, and is a site of regulation of apical recycling and basolateral-to-apical transcytosis of the pIgR (Apodaca et al., 1994
; Casanova et al., 1999
; Brown et al., 2000
; Wang et al., 2000a
). The pIgR normally transports basolaterally internalized IgA from BEE, to CRE, to the ARE, and then to the apical surface where the receptor is cleaved, releasing it along with bound IgA into secretions (Apodaca et al., 1994
; Brown et al., 2000
). However, a significant fraction of pIgR escapes proteolysis and is then endocytosed and recycled through the ARE en route to the apical cell surface. In our experiments, IgA was either internalized basolaterally for 10 min and chased for 20 min or internalized from the apical pole of the cell for 10 min, to accumulate ligand in the ARE. After apical uptake surface-bound ligand was removed by proteolysis, the cells were then fixed and labeled with antibodies specific for exocyst subunits, Rab11a, and IgA. We observed colocalization between Sec8 or Exo70 and Rab11a (, A and C) or Sec8 or Exo70 with basolaterally internalized IgA (, B and D). Triple label experiments confirmed that Sec8/Exo70, Rab11a and apically internalized IgA were codistributed in a subset of ARE (, E and F).
Additional experiments confirmed the association of exocyst subunits with Rab11-positive endosomes and directly with dominant active Rab11aSV. An endosomal-enriched fraction was incubated with Dynabeads coated with an antibody that recognizes both a and b isoforms of Rab11 (the Rab11a specific antibodies we used for IF were not functional in this protocol). The immunoisolated fractions were resolved by SDS-PAGE and Western blots were probed with pIgR-, Rab11-, and Sec8-specific antibodies (G). Consistent with the IF analysis, a fraction of Sec8 and the pIgR was associated with Rab11-positive endosomes. Little pIgR, Rab11, or Sec8 association was observed in control reactions incubated with nonspecific rabbit IgGs (G). The Golgi marker GM130 was not observed in the Rab11a-positive endosome fraction (data not shown), confirming that the immunoisolated sample represents an endosome-enriched fraction and does not pulldown other non–Rab11a-associated membrane-bound compartments.
Likely reflecting the transient nature of the Rab11a/exocyst interaction, we were unable to coimmunoprecipitate endogenous Rab11a with antibodies to Sec8, Sec15A, or Exo70. However, when we infected MDCK cells with an adenovirus encoding a GTPase-deficient mutant of Rab11a fused to GFP and containing an HA tag (GFP/HA-Rab11aSV), we observed that Exo70 was associated with GFP-Rab11aSV–positive endosomes (A). Furthermore, when we performed coimmunoprecipitation using Sec8 antibodies, we observed that multiple exocyst subunits (Sec6/Sec8/Sec15A/Exo70) as well as GFP/HA-Rab11aSV were found in a complex (B). Exo70 did not colocalize with a dominant negative mutant of Rab11a (GFP/HA-Rab11aSN), which appeared to be primarily cytosolic (data not shown).
Figure 4. Association of exocyst subunits with GFP/HA-Rab11aSV. (A) MDCK cells were infected with virus encoding GFP/HA-Rab11aSV and then fixed and processed for IF. The distribution of GFP/HA-Rab11aSV (green) and Exo70 (red) are shown. A merged image is shown (more ...)
Taken together, the above data indicate that exocyst subunits are localized to multiple endocytic compartments including early endosomes, Tf-positive recycling endosomes, and the Rab11a-positive ARE, but not to a significant degree with the TGN of polarized MDCK cells.
Basolateral Recycling, Apical Recycling, and Basolateral-to-Apical Transcytosis are Exocyst-dependent Trafficking Pathways
Next, we examined whether there was a functional role for the exocyst in postendocytic trafficking pathways. For this analysis we used SLO-permeabilized cell assays that we previously developed to measure transcytosis and recycling (Apodaca et al., 1996
; Leung et al., 1998
). Important advantages of this technique include the ability to examine exocyst function after the cells have already polarized, the ability to test the acute effects of inhibiting exocyst function on defined trafficking events and the ability to uniformly permeabilize the entire monolayer, ensuring equal delivery of the reagents to each cell.
We first examined whether Tf recycling in polarized MDCK cells was dependent on the exocyst. 125
I-Tf was internalized from the basolateral surface of filter-grown MDCK cells, the cells were permeabilized with SLO, and after cytosol washout, vesicle trafficking was reconstituted at 37°C in the presence of exogenous cytosol and an ATP-regenerating system. A pool of function-blocking Sec8 mAbs (10C2, 5C3, 2E12; Grindstaff et al., 1998
) was included in the washout step, and the reconstitution reaction. As a control we substituted the Myc 9E10 mAb for the Sec8 antibodies in the reaction. At the end of the reconstitution reaction, the percentage of ligand that was recycled was calculated. The ATP-dependent values were normalized to control reactions that contained an ATP-regenerating system and cytosol, but no antibody. The addition of Sec8 antibodies significantly inhibited basolateral recycling of Tf by ~45%; however, no effect was observed upon addition of the nonspecific Myc antibody (A).
Figure 5. Exocyst requirement for endocytic traffic in SLO-permeabilized MDCK cells. (A) Basolateral recycling of 125I-Tf in SLO-permeabilized MDCK cells incubated in the presence of an ATP-regenerating system (ATP), cytosol, and either Myc antibodies (Myc) or (more ...)
In the next experiment, we examined whether transit of 125I-IgA from the ARE and release from the apical pole of the cell was exocyst-dependent. 125I-IgA was internalized for 10 min at 37°C, washed, and then chased for 20 min to accumulate IgA in the Rab11a-positive elements of the ARE. IF confirmed that under these internalization conditions, IgA was present at the apical pole of the cell in tubulovesicular structures that colocalized with Rab11a, but not with Tf receptor (B). The cells were then permeabilized with SLO and 125I-IgA release from the ARE was measured in the presence or absence of exocyst antibodies. Blocking antibodies significantly inhibited IgA trafficking from the ARE by ~40% (C), whereas Myc antibodies had no effect.
As further confirmation that the exocyst modulated apically directed traffic, we also explored apical recycling of IgA in SLO-permeabilized cells. Filter-grown cells were pulsed with 125I-IgA from the apical domain for 10 min, the cells were washed and chased in the absence of ligand for a total of 5 min, and then membrane bound IgA was stripped from the surface with trypsin at 4°C. The cells were permeabilized with SLO, and apical IgA release was reconstituted in the presence of Sec8 or Myc antibodies as described above. In the presence of the Sec8 antibodies, the pool of IgA that recycled apically and was dependent upon ATP and cytosol was significantly inhibited by ~80% relative to control (D). It is worth noting that we observed a relatively large ATP independent pool of recycling IgA in these assays (~30%), indicating that either reconstitution was inefficient or that apical recycling had little requirement for ATP. The ATP-independent pool of recycling was insensitive to the addition of Sec8 antibodies (data not shown).
It was previously shown that apical delivery of newly synthesized p75 neurotrophin receptor was independent of exocyst function (Grindstaff et al., 1998
). Consistent with this previous analysis, we found that addition of function blocking Sec8 antibodies to SLO permeabilized MDCK cells had no significant effect on apical delivery of this protein (data not shown). This latter observation confirms that only a subset of trafficking events are exocyst dependent in SLO-permeabilized cells.
Although the exocyst is generally thought to be involved in promoting transit between intracellular compartments and the plasma membrane, some studies indicate that it may also play a role in modulating cargo exit from the TGN or endosomes (Yeaman et al., 2001
; Beronja et al., 2005
; Langevin et al., 2005
). To explore this possibility, we reconstituted vesicle budding from ARE in mechanically perforated cells. 125
I-IgA was internalized basolaterally for 20 min at 18°C and then chased for 20 min to accumulate IgA in the ARE. The apical membrane was then mechanically perforated with nitrocellulose (Bomsel and Mostov, 1993
) and release of 125
I-IgA in transport vesicles was measured in the presence of Sec8 or Myc antibodies, cytosol, and an ATP-regenerating system. Addition of antibodies against Sec8, but not Myc, resulted in a significant inhibition of 125
I-IgA release from labeled ARE (E).
Taken together the above results indicate that the exocyst modulates a broad spectrum of endocytic trafficking events in polarized cells, including those directed toward the apical and basolateral pole of the cell. Furthermore, the exocyst may modulate the exit of IgA-pIgR cargo from the ARE.
The C-terminus of Sec15A Binds to Rab11a
The potential requirement for the exocyst in basolateral-to-apical transcytosis and apical recycling prompted us to further explore the molecular requirements for this dependence. We initially focused on the previously described interaction between Sec15 and Rab11 (Zhang et al., 2004
; Wu et al., 2005
). Although mapping of Sec15-Rab11 interactions was recently described using Drosophila
proteins (Wu et al., 2005
), we confirmed these interactions with their mammalian orthologues (). Using a two-hybrid approach and a quantitative β-galactosidase assay, we observed that full-length rat Sec15A interacted with wild-type Rab11a as well as with GTPase-deficient Rab11a-SV. However, no interaction was observed with the dominant negative mutant Rab11aS25N (Rab11a-SN), Lamin C, or empty vector (B). Next, we broadly examined the region of Sec15A that was involved in these interactions. The Sec15A N-terminus (Sec15NT; amino acids 1–390) showed no interactions with Rab11a (C). However, the Sec15A C-terminus (Sec15CT; amino acids 391–822) interacted, like the intact protein, with wild-type Rab11a and Rab11a-SV, but not with Rab11a-SN (D). It was previously reported that a point mutation that converted Asn659
to an alanine residue in Drosophila
Sec15CT blocked its interaction with Rab11 (Wu et al., 2005
). We observed that the analogous mutation in mammalian Sec15CT, in which Asn709
was converted to an alanine residue (Sec15CT(NA)), prevented the interaction of Rab11a with the C-terminus of Sec15A (E).
Figure 6. Interaction between Rab11a and the C-terminus of Sec15A. (A) Sec15A constructs used to identify the Sec15A-Rab11a interaction domain. (B–E) Results of CPRG assay between the Sec15A constructs shown in panel A and either wild-type Rab11a (Rab11a), (more ...)
To confirm that Sec15CT can interact with Rab11a in vivo, we generated stable cell lines expressing GFP-Sec15CT or GFP-Sec15CT(NA), which were subsequently infected with GFP/HA-Rab11aSV adenovirus. The cells were cross-linked with the reversible cross-linker DSP, lysed, and GFP/HA-Rab11aSV was immunoprecipitated using an anti-HA antibody. Western blots of the immunoprecipitates were probed with the 15S2G6 antibody (which recognizes the C-terminus of Sec15A) or with antibodies against Rab11a. Consistent with our two-hybrid analysis, anti-HA antibodies coimmunoprecipitated a complex between GFP/HA-Rab11aSV and GFP-Sec15CT, but little interaction was observed between GFP/HA-Rab11aSV and GFP-Sec15(NA) (F).
Expression of Sec15CT or Down-Regulation of Sec15A Impairs Basolateral-to-Apical Transcytosis of pIgR-IgA Complexes
We next examined whether Sec15CT expression affected the distribution of Rab11a, potentially by impairing interactions between endogenous Sec15 and Rab11a. We transiently transfected polarized filter-grown MDCK cells with GFP-tagged Sec15CT (GFP-Sec15CT), and ~24 h after transfection the cells were fixed and labeled with Rab11a-specific antibodies (A). We estimate that ~20–30% of the cells expressed GFP-Sec15CT after transfection. We observed that GFP-Sec15CT was localized to small vesicular structures at the apical pole of the cell as well as very large “vesicular” structures in the medial cytoplasm. Rab11a colocalized with both pools of GFP-Sec15CT (B), as did the pIgR (see B). However, the large medial GFP-Sec15CT–positive elements did not colocalize with or alter the distribution of Sec8 nor did they colocalize with basolaterally internalized IgA (data not shown). When examined by live-cell imaging, GFP-positive vesicular elements were observed to enter and exit the large vesicular structures (data not shown), demonstrating that these structures are dynamic and unlikely to be cytoplasmic accumulations of GFP-Sec15CT in aggresomes. The mutant version of GFP-tagged Sec15CT (GFP-Sec15CT(NA)) showed some puncta at the apical pole of the cells that were positive for Rab11a (C), indicating that the mutant may have bound to this compartment in a Rab11a-independent manner. However the mutant did not induce the formation of large vesicular structures in the medial cytoplasm and appeared to be predominantly cytoplasmic.
Figure 7. Expression of GFP-Sec15CT and GFP-Sec15CT(NA) in polarized MDCK cells. (A) Filter transfection protocol. (B) Distribution of GFP-Sec15 (green), Rab11a (red), and the nucleus (blue). (C) Distribution of GFP-Sec15(NA) (green), Rab11a (red), and the nucleus (more ...)
Figure 8. IgA transcytosis in polarized MDCK cells expressing GFP-Sec15CT or GFP-Sec15CT(NA). (A) Protocol for detecting basolaterally internalized IgA at the apical cell surface. (B) Distribution of GFP-Sec15CT or GFP-Sec15CT(NA) (green), the pIgR (red), and IgA (more ...)
Next, we examined whether expression of GFP-Sec15CT altered basolateral-to-apical transcytosis of IgA using a morphological assay that scored the delivery of basolaterally internalized IgA to the apical surface of polarized MDCK cells (A). IgA was internalized from the basolateral surface of the cells for 20 min at 18°C to trap a cohort of IgA in BEE (Song et al., 1994
). After this pulse, we confirmed that basolaterally internalized IgA was found in BEE subjacent to the basolateral surface of the cells expressing either GFP-Sec15CT or GFP-Sec15CT(NA) (see medial projections, B). The cells were also labeled with an anti-pIgR mAb (SC166) to confirm pIgR expression. To initiate transcytosis, the basolateral surfaces of the cell were washed, and the cells were incubated in the absence of ligand for 20 min at 37°C. A Cy3-labeled secondary antibody specific for IgA was included in the apical medium during the 37°C chase to label IgA-bound pIgR complexes as they appeared at the apical cell surface (A). As described above, a significant fraction of the pIgR escapes cleavage and recycles at the apical pole of the cell. After the chase in the presence of anti-IgA antibody, the cells were washed, fixed, and stained. We observed that in cells expressing GFP-Sec15CT, there was little uptake of Cy3-labeled secondary antibody (C, blue), whereas in untransfected cells or those transfected with GFP-Sec15CT(NA), significant uptake of the anti-IgA antibody was detected at the apical pole of the cells (C, blue), which colocalized with the pIgR. These results indicate that the expression of GFP-Sec15CT inhibited IgA delivery to the apical surface of the cell, whereas Sec15CT(NA), which binds inefficiently to Rab11a, had less of an effect. We also examined the effect of expressing GFP fused to full-length Sec15A; however, our analysis was thwarted by preliminary studies that showed expression of this construct resulted in the rapid formation of apoptotic cells, which had very low pIgR expression.
As further evidence of the effects of Sec15CT on transcytotic traffic, we used the stable MDCK cells lines expressing GFP-Sec15CT or GFP-Sec15CT(NA) described above. The level of expression of these two constructs was approximately equivalent to that of the endogenous protein (A), which is likely to be an underestimate as only ~ 40–50% of the cells expressed these constructs, and was less than that observed using the transient transfection protocol described above. The medial vesicular structures were present in these cells, but were somewhat smaller in dimension, likely reflecting the lower levels of GFP-Sec15CT expression in these cell lines. To measure transcytosis, the cells were first infected with an adenovirus encoding the pIgR. After 24 h to allow for receptor expression, 125I-IgA was internalized from the basolateral surface of the cell for 10 min at 37°C, the cells were washed, and the percentage of internalized 125I-IgA released into the basolateral medium (recycled) or apical medium (transcytosed) was measured during a 2-h incubation at 37°C. Consistent with the morphological assay, we observed that IgA transcytosis was significantly impaired by expression of GFP-Sec15CT. The effect was kinetic with an ~50% inhibition observed at 15 min and ~20% at the 2-h time point (B). There was little effect on basolateral recycling or degradation, but there was a compensatory increase in the amount of cell-associated ligand after the 2-h chase (data not shown). In contrast, there was no effect on apical recycling of IgA (D) or basolateral recycling of Tf (F). Expression of GFP-Sec15CT(NA) resulted in a small but significant stimulation of basolateral-to-apical transcytosis of IgA (C), but had no effect on apical recycling of 125I-IgA (E), or basolateral recycling of 125I-Tf (G).
Figure 9. Effect of expressing GFP-Sec15CT and GFP-Sec15CT(NA) on the postendocytic fate of IgA and Tf in polarized MDCK cells infected with adenovirus encoding the pIgR. (A) Lysates of cells expressing GFP-Sec15CT or GFP-Sec15CT(NA) were resolved by SDS-PAGE, (more ...)
As a final experiment we down-regulated expression of Sec15A by transiently expressing a plasmid (pSuper-Sec15A) that expresses a Sec15A specific shRNA. Compared with cells expressing vector alone (not shown) or a control construct (pSuper-control), expression of pSuper-Sec15A resulted in a decrease in both Sec15A mRNA and protein expression as assessed by RT-PCR analysis or by Western blotting (A). The decrease in protein and mRNA expression was somewhat variable (see triplicate samples in A). By comparing the average amount of mRNA/protein found in cells expressing pSuper-Sec15A to cells expressing pSuper-control, we estimate that expression of pSuper-Sec15A decreased Sec15A mRNA expression by ~80% and Sec15A protein expression by ~50%. We used RT-PCR to confirm that pSuper-Sec15A had little effect on mRNA expression for Sec15B (A; decrease of ~20%); however, the lack of isoform specific antibodies prevented us from examining the protein levels of Sec15B in the cells. There was no effect of silencing Sec15A expression on levels of Sec8 (A). Expression of pSuper-Sec15A shRNA, but not pSuper-control, had a similar phenotype to expression of GFP-Sec15CT: basolateral-to-apical transcytosis was significantly inhibited at all time points (B), but there was no effect on apical or basolateral recycling (, C and D). Taken together, the above results indicate that Sec15A, possibly acting through Rab11a, modulates basolateral-to-apical transcytosis in polarized MDCK cells.
Figure 10. Dependence of basolateral-to-apical transcytosis on expression of Sec15A. (A) The upper panel shows Sec15A or Sec15B mRNA expression in three cell samples expressing pSuper-Sec15A (lanes 1–3) or pSuper-control (lanes 4–6). The lower panel (more ...)