To study the intracellular trafficking of the Na pump, we have used a new method that permits the direct observation of temporally defined cohorts of proteins via the combination of fluorescence microscopy with pulse–chase labeling protocols. The 20-kD SNAP tag is a modified version of the DNA repair protein 06
-alkylguanine-DNA alkyltransferase, which cleaves para-substituted benzylguanines (BGs) by covalently and irreversibly transferring the substituted benzyl group to its active thiol. Fluorescent BG derivatives allow for the labeling and detection of SNAP-tagged fusion proteins in either live or fixed cells (Keppler et al., 2004
). We engineered a Na pump construct harboring a SNAP tag connected at the α subunit N terminus via a short linker region containing an HA tag (). MDCK cells stably transfected with the SNAP-tagged Na pump and labeled with tetramethylrhodamine-conjugated BG (TMR-STAR) yield a robust signal that colocalizes with gp58. gp58 detects the endogenous β subunit of the Na,K-ATPase (Füllekrug et al., 2006
), indicating that PM localization is not affected by addition of the SNAP tag (). In addition, several lines of evidence indicate that the fusion of the SNAP tag with Na,K-ATPase does not perturb the trafficking behavior or enzymatic activity of the pump (Fig. S1
Figure 1. Detection of Na pump localization via the SNAP tag. (A) Diagram of the SNAP tag labeling reaction for the N-terminally tagged Na,K-ATPase α subunit. TMR, tetramethylrhodamine. (B) Stably transfected MDCK cells expressing both SNAP–Na,K-ATPase (more ...)
Newly synthesized Na,K-ATPase is labeled by TMR-STAR and rapidly traffics to the PM
To follow the postsynthetic fate of the Na,K-ATPase, we used a block–chase strategy using our MDCK cell line expressing the SNAP-tagged Na,K-ATPase α subunit (). Preincubation of live cultures with the nonfluorescent BG-Block substrate was found to saturate the SNAP tags on all previously synthesized pumps and effectively blocked this pool of pump from labeling with TMR-STAR. BG-Block treatment did not achieve this effect by inducing pump degradation, as the distribution of the total cellular pool of the SNAP-tagged Na pump was unaltered when detected by HA staining (). In addition, we monitored the rate of protein turnover in metabolically labeled cultures after treatment with BG-Block and did not observe any effects relative to mock-treated samples (unpublished data).
Figure 2. Newly synthesized Na pump trafficking imaged via the SNAP tag. (A) Live SNAP cells were preincubated with nonfluorescent BG (BG-Block) for 30 min, fixed, stained with TMR-STAR (red), and processed for immunofluorescence with the indicated antibodies (green). (more ...)
We next investigated the recovery of TMR-STAR labeling in BG-blocked cultures to determine how soon after cessation of the block newly synthesized SNAP-tagged Na,K-ATPase could be detected. Within 30 min after the block, a perinuclear reticular staining pattern reminiscent of the ER was visible. The magnitude of signal increased over time, and within 1 h, we could detect significant colocalization of the TMR-STAR label with the Golgi marker protein GM130 (). To analyze the reappearance of the TMR-STAR–labeled pump at the membrane, we used the HA epitope as a marker for Na pump resident at the PM. When an area in a plane of focus below that of the Golgi was imaged, we could detect TMR-STAR signal colocalizing with the basolateral PM as early as 1 h after block, which increased dramatically by 90 min ().
Golgi-accumulated Na pump is rapidly trafficked to the lateral membrane in polarized MDCK cells
To accumulate a cohort of newly synthesized Na pump into a single organelle, we used a Golgi block step by incubating BG-blocked cells at 19°C for 2 h after a short post–BG-Block recovery period at 37°C. Using this protocol, we were able to synchronize the pump specifically in the Golgi complex, where it colocalized with the Golgi markers GS15 and Vti1a (), whereas no significant signal was detected in the ER or at the PM (). In this and subsequent experiments, all post-Golgi trafficking incubations were performed at 31°C to allow direct comparison with previous studies that used a temperature-sensitive mutant of the VSV-G protein (Ts 045; Ang et al., 2003
; Schuck et al., 2007
). Ts 045 trafficking is more efficient when assayed below 37°C because of misfolding of the mutant protein at higher temperatures (Ang et al., 2004
). When samples were warmed to 31°C for 5 min, very little delivery to the PM was observed (). However, by 10 min, Na pump was readily detected at the lateral PM, and the majority of the Golgi signal was depleted by 20 min after release (). It is interesting to note that the HA staining corresponding to the pool of old Na pump was clustered into beadlike plaques strung along the length of the lateral PM, whereas the newly delivered pump exhibited a uniform distribution (10 min) and only later localized with HA into these clusters (20 min). It has been suggested that the Na pump is localized into this beaded arrangement in association with contacts between adjacent cells (Vagin et al., 2006
). Our data indicate that the pump is first delivered randomly over the entire surface of the lateral PM and subsequently becomes incorporated into the preexisting aggregates at the sites of these contacts.
Figure 3. TGN-accumulated Na pump is rapidly trafficked to the lateral membrane. (A) SNAP cells were subjected to BG-Block, incubated at 37°C for 30 min to begin synthesis of new Na pump, and placed at 19°C for 2 h to accumulate newly synthesized (more ...)
To analyze quantitatively the trafficking of SNAP-tagged Na pump to the PM, z stacks were generated for each time point, and the percentage of newly synthesized Na pump colocalizing with the PM was determined (Fig. S2 A
). In samples maintained at 19°C, ~6 ± 3% of the TMR-STAR–labeled Na pump signal colocalized with the PM, indicating that there may be a small degree of leakage of Na pump out of the Golgi during incubation at 19°C. When the temperature was raised to 31°C, the TMR-STAR Na pump signal at the PM increased to 12 ± 8%, 27 ± 9%, 53 ± 11%, 84 ± 2%, 90 ± 4%, and 88 ± 3% at 5 min, 10 min, 15 min, 20 min, 40 min, and 60 min after Golgi block release, respectively.
A previous study of VSV-G trafficking in polarized MDCK cells demonstrated that upon release from Golgi block, only 40% of the Golgi-accumulated VSV-G signal reached the PM after 30 min (Hua et al., 2006
). Because this suggests that the Na pump and VSV-G traffic to the PM at quite different rates, we compared VSV-G trafficking with that of the Na pump in our assay system (Fig. S2 A). For this experiment, we used VSV-G Ts 045, which is blocked from exiting the ER when host cells are incubated at 40°C. Next, cells were shifted from 40 to 19°C to allow the VSV-G protein to depart the ER and accumulate in the Golgi. Under Golgi block conditions, we detected slightly more signal for VSV-G at the lateral PM (7 ± 4%) than was observed for Na pump. When the temperature was shifted to 31°C, lateral PM accumulation increased to 11 ± 6%, 21 ± 4%, 28 ± 9%, 35 ± 4%, 52 ± 4%, 66 ± 12%, and 76 ± 9% for 5 min, 10 min, 15 min, 20 min, 30 min, 40 min, and 60 min, respectively, demonstrating that these two proteins traffic from the Golgi to the PM at quite different rates. It is possible that the relatively slow accumulation of VSV-G at the PM in comparison with the Na,K-ATPase is the result of comparatively rapid internalization of newly delivered VSV-G back into REs. To test this possibility, we monitored the uptake of newly delivered VSV-G from the PM by carrying out the temperature shift Golgi release protocol in the presence of an antibody directed against the extracellular domain of VSV-G (Fig. S2 B). Only a small fraction of the externally labeled VSV-G is endocytosed in samples released from the Golgi block for 20–40 min. Furthermore, much of the intracellular pool of VSV-G that is detected after release from the Golgi block does not colocalize with transferrin (Tfn) receptor (Tfn-R), indicating that it is not associated with endosomes and is instead most probably still resident in the TGN. Collectively, these findings suggest that rapid internalization does not account for the slow accumulation of VSV-G protein at the PM after release from the Golgi block.
The Na pump traffics directly to and uniformly across the entire length of the lateral membrane
Previous studies analyzing the delivery of the Na,K-ATPase to the PM in the Heidelberg strain of MDCK cells demonstrated that newly synthesized pumps are delivered directly to the lateral PM without significant appearance at the apical surface (Caplan et al., 1986
; Gottardi and Caplan, 1993
; Mays et al., 1995
). As biochemically based membrane delivery assays often involve complex procedures to label a single membrane surface, we investigated whether we could detect similar vectorial delivery directly using the SNAP system. As expected from the data depicted in , under the 19°C Golgi block, we detected a globular staining pattern concentrated beneath the apical PM () that colocalizes with Golgi markers (not depicted). When samples were warmed for 5–20 min at 31°C, we were unable to detect colocalization of the Na pump with the apical marker gp135 at any time point, whereas a rapid accumulation of this Na pump population at the lateral PM was observed. Our data are consistent with those of previous biochemical studies (Caplan et al., 1986
; Gottardi and Caplan, 1993
; Mays et al., 1995
), suggesting that the SNAP system can be applied successfully to follow the postsynthetic trafficking of membrane proteins to the PM.
Figure 4. TGN-accumulated Na pump is trafficked directly to and randomly throughout the lateral membrane. SNAP cells were BG blocked, and newly synthesized Na,K-ATPase was accumulated in the TGN for 2 h at 19°C. After the Golgi block, samples were fixed (more ...)
Basolateral vesicle delivery to the lateral PM is often mediated through the specific tethering of these vesicles to the membrane via the exocyst complex (Grindstaff et al., 1998
; Yeaman et al., 2001
). This multicomponent protein complex is localized, in polarized epithelial cells, at the apical–lateral junction (Grindstaff et al., 1998
) and in association with REs (Oztan et al., 2007
). The localization of the exocyst at the site of tight junctions suggests that basolaterally targeted proteins must first be delivered to this region followed by redistribution across the entire surface of the lateral PM (Rodriguez-Boulan et al., 2005
). However, conflicting evidence exists supporting both junctional and random delivery to the lateral PM. Live cell imaging experiments with GFP-tagged low density lipoprotein receptor (LDLR), an AP-1B–dependent basolateral cargo, showed that vesicles fuse with the lateral PM primarily in a region between 6–12 µm above the basal surface (Kreitzer et al., 2003
). In addition, a GFP-tagged version of the VSV-G protein was found to accumulate in the region of the junction when cells were treated with tannic acid to block membrane fusion (Polishchuk et al., 2004
). However, when VSV-G–YFP trafficking was monitored in live MDCK cells, specific delivery to the region of the junction was not observed (Hua et al., 2006
To analyze the initial delivery of newly synthesized Na,K-ATPase to the PM, we monitored the sites of first appearance of the Na pump relative to the localization of the tight junctional marker ZO-1 (). As expected from the data depicted in , there was a lag period over which little pump was delivered to the PM after 5 min. Interestingly, at both 10 and 20 min after release from the 19°C Golgi block, we were unable to detect any specific accumulation at the region of the junction. When we monitored delivery of VSV-G, a protein known to use the exocyst in its trafficking to the PM (Yeaman et al., 2001
; Moskalenko et al., 2002
), random delivery was also observed (unpublished data). At early time points, we noted the appearance of concentrated zones of comparatively intense TMR-STAR–labeled Na pump signal that were distributed at random over the entire length of the lateral PM (, arrows). Because the Na pump is known to interact with the spectrin–ankyrin cytoskeleton (Woroniecki et al., 2003
), it is possible that these local concentrations are attributable to the selective stabilization of the pump in subdomains of the PM rather than corresponding to sites of initial vesicle delivery. Of course, it remains possible that carrier vesicle fusion is occurring for both proteins at the site of the junction, and subsequent rapid diffusion in the plane of the PM accounts for their initial detection near the basal surface. However, the data presented in this study are not consistent with an obvious requirement for delivery of basolaterally directed cargo vesicles to spatially discrete fusion hot spots localized to specific subdomains of the lateral PM.
Unlike the VSV-G protein, the Na pump does not pass through REs en route to the PM
Previous studies have shown that REs are primary sorting stations for AP-1B–dependent cargoes en route to the PM (Ang et al., 2004
; Cancino et al., 2007
). However, little is known about the pathway taken by AP-1B–independent cargoes, as they traffic to the basolateral surface. To analyze whether the Na pump traverses REs before its arrival at the PM, we assessed whether newly synthesized Na pump colocalized with internalized fluorescently labeled Tfn, a marker for the RE, after release from the Golgi block. Under Golgi block conditions, the Alexa Fluor 488 Tfn signal corresponding to REs was detected adjacent to but not colocalized with elements of the Golgi complex (). Interestingly, when the temperature was raised to 31°C for periods ranging from 2 to 10 min, no colocalization between the Na,K-ATPase and Tfn was detected ( and not depicted). Using a similar strategy, Ang et al. (2004)
demonstrated marked colocalization of VSV-G with Tfn after a 10-min release from a 19°C Golgi block in nonpolarized MDCK cells. We confirmed that VSV-G protein behaves in this fashion when it is expressed in our SNAP tag–expressing MDCK cells. We also found that VSV-G colocalizes with Tfn in REs when cells have been treated with tannic acid, a membrane impermeant fixative which blocks vesicle delivery to the PM. Thus, the localization of VSV-G to REs after release from Golgi block must occur as the VSV-G protein is en route to the cell surface and is not caused by endocytosis after surface delivery (Fig. S3
Figure 5. The Na,K-ATPase does not pass through REs en route to the lateral membrane. (A) Cells were infected with adenovirus expressing the human Tfn-R, after which they were BG blocked, and newly synthesized Na pump was accumulated in the TGN (red) for 2 h at (more ...)
To further analyze whether delivery of the Na pump to the PM depends on the participation of functional REs, we used a modified version of an RE inactivation strategy (Ang et al., 2004
). HRP is capable of reacting with DAB in the presence of H2
to form an insoluble precipitate, and using HRP that is conjugated to Tfn (Tfn-HRP), it has been shown that VSV-G is blocked from reaching the PM in both nonpolarized (Ang et al., 2004
) and polarized MDCK cells (Cresawn et al., 2007
) subjected to HRP-mediated RE ablation.
In this experiment, cells were infected with adenoviruses expressing Tfn-R to aid in Tfn uptake and VSV-G Ts 045 to verify whether the ablation manipulation was successful. Cells were treated as described in Materials and methods to accumulate both newly synthesized Na pump and VSV-G intracellularly and to accumulate Tfn-HRP in REs. Cells were subjected to RE ablation and either fixed immediately or allowed to recover for 1.5 h at 31°C in the presence of cycloheximide (CHX). Under these conditions, we detected a predominantly intracellular pool of both VSV-G and Na,K-ATPase at time 0 for both ablated and control cells (). In the absence of ablation, both VSV-G and the Na pump readily trafficked to the PM, with 74% of the Na pump and 64% of VSV-G resident at the membrane after incubation at 31°C (). Furthermore, in control experiments in which cells were either subjected to the DAB reaction without prior exposure to Tfn-HRP or in which DAB was omitted, both VSV-G and the Na,K-ATPase reached the membrane (unpublished data). In samples that were subjected to HRP- and DAB-mediated ablation, VSV-G was retained intracellularly after release from the Golgi block, with only 33% of the protein reaching the membrane (). In these ablated cells, the bulk of VSV-G had trafficked out of the TGN and was retained within the RE compartment, as revealed by costaining with an antibody directed against the Tfn-R (). In marked contrast to the behavior of the VSV-G protein, 79% of the Na pump reached the PM unimpeded in cells that had been subjected to the ablation protocol (). Our results demonstrate that the ablation reaction does not globally affect the TGN, as the Na pump is able to depart the Golgi complex after ablation and to traffic to the PM. Furthermore, these experiments show that the newly synthesized Na,K-ATPase bypasses the RE en route from the Golgi complex to the PM.
Trafficking of the Na pump is not governed by the same set of small GTPase regulators that are involved in the trafficking of AP-1B–dependent membrane proteins
We next analyzed the effects of mutant forms of selected Rab and Rho family GTPases on Na pump trafficking. Small GTPases are important regulators of many membrane trafficking events (Grosshans et al., 2006
). In polarized MDCK cells, expression of constitutively active forms of Rab8 and Rab10 or dominant-negative CDC42 has been shown to induce the missorting of AP-1B–dependent basolateral cargo proteins to the apical membrane (Kroschewski et al., 1999
; Ang et al., 2003
; Schuck et al., 2007
). Rab11, which is resident in apical REs, appears to have no effect on AP-1B–dependent basolateral trafficking (Ang et al., 2003
); however, it has been shown to disrupt sorting of E-cadherin, an AP-1B–independent cargo (Desclozeaux et al., 2008
To test the effects of these proteins on Na pump trafficking, cells were microinjected with constructs encoding fusion proteins in which GFP is appended to constitutively active forms of Rab8 (Q67L), Rab10 (Q68L), Rab11 (Q70L), or dominant-negative CDC42 (T17N). As a positive control to demonstrate that these mutated proteins disrupt AP-1B–dependent trafficking, each Rab construct was simultaneously injected with a plasmid encoding LDLR. To ensure that the mutant Rab proteins were expressed at significant levels before analyzing pump trafficking, cells were allowed to synthesize the mutant Rab proteins for 45 min before the BG-Block reaction was initiated, and then labeling proceeded as described in Materials and methods. When cells were injected with a plasmid expressing GFP alone, both LDLR and the Na pump were detected at the lateral PM (). Expression of either Rab8 Q67L or CDC42 T17N routed LDLR to the apical surface (), whereas they had no visible effect on the trafficking of the Na pump, which is consistent with the apparent independence of Na,K-ATPase sorting from AP-1B–dependent mechanisms. Rab10 is known to participate in the sorting of at least two AP-1B–dependent cargoes; however, its effects on AP-1B–independent proteins have not been tested. Our assay demonstrated that expression of activated Rab10 led to apical accumulation of the LDLR, whereas Na pump trafficking was completely unaffected. In cells expressing levels of Rab11 Q70L equivalent to those obtained with Rab8 Q67L, there was no apparent effect on either LDLR or Na pump distribution, which is in agreement with previous observations (; Ang et al., 2003
Figure 6. Na pump trafficking is not regulated by the same small GTPases as AP-1B–dependent cargo and does not require E-cadherin. (A) SNAP cells were grown on clear polyester filters for 4 d to form a fully polarized monolayer and microinjected with plasmids (more ...)
E-cadherin is not required for the polarized delivery of the Na pump
E-cadherin is a Ca-dependent cell adhesion molecule that links the lateral membranes of adjacent cells together through homotypic interactions (Takeichi, 1990
). The introduction of E-cadherin into mouse fibroblasts induces the formation of cell–cell contacts and results in the localization of the Na,K-ATPase into these junctions (McNeill et al., 1990
). Suppression of E-cadherin expression in MDCK cells by RNAi demonstrated that E-cadherin is required for the establishment of epithelial polarity, but it does not appear to be needed for its maintenance once polarity is established (Capaldo and Macara, 2007
). In these experiments, even though E-cadherin depletion approached 100% in transfected cells, tight junctions remained intact, and both apical and basolateral markers (including the Na pump) were properly localized. However, in at least one strain of MDCK cells, Na pump polarity is achieved through selective stabilization of the pump in the basolateral PM after random delivery to both surfaces (Mays et al., 1995
). Thus, the fact that the steady-state basolateral localization of the Na,K-ATPase is maintained in the E-cadherin knockdown MDCK cells does not provide any insight into the question of whether E-cadherin is required for the direct targeting of newly synthesized Na,K-ATPase to the basolateral surface.
To determine whether newly synthesized Na pumps are vectorially targeted to the lateral membrane in E-cadherin–depleted cells, we transfected MDCK cells expressing the SNAP-tagged Na pump with a plasmid expressing a short hairpin RNA (shRNA) directed against either E-cadherin or against the luciferase gene, which served as a negative control. Transfected cells were then plated onto trans-well filters at a high enough density to immediately form a monolayer and allow cell–cell contacts to form before E-cadherin knockdown (Capaldo and Macara, 2007
). Using this method, E-cadherin was significantly depleted in transfected cells, whereas steady-state Na pump distribution and tight junctions were unaffected (). In addition, we cotransfected cells with shRNAs directed against both E-cadherin and Cadherin-6, as codepletion of these two proteins is required for the loss of β-catenin localization at cell–cell contacts in MDCK cells (Capaldo and Macara, 2007
). As expected, no effect was observed for ZO-1 staining in double knockdown cells relative to control cells (). The Na pump also remained localized to the lateral membrane, indicating that cadherin-mediated localization of β-catenin is not required to maintain the Na pump's distribution. To analyze whether cadherins are necessary for direct trafficking of the Na pump to the basolateral membrane, we accumulated newly synthesized Na pump into the Golgi in both control and knockdown cells, as described in the legend for , and followed its trafficking after release from the Golgi block (). Similar to the results of , the Na pump was rapidly trafficked from the Golgi to the lateral membrane in cells transfected with the luciferase RNAi construct. Similar results were observed in the cadherin-depleted cells, indicating that neither E-cadherin nor Cadherin-6 is required for the vectorial delivery of the Na pump once polarity has been established.
The Na pump and VSV-G are segregated into distinct PGTIs en route to the PM
It has been shown that some apical and basolateral cargoes segregate from one another in the Golgi complex and are delivered to the PM in separate PGTIs (Wandinger-Ness et al., 1990
; Keller et al., 2001
). However, the presence of separate PGTIs for different basolateral cargoes in the same cell has not been demonstrated. Our data suggest that VSV-G, and possibly all AP-1B–dependent cargo proteins, uses a pathway that is distinct from that pursued by the Na pump during its biosynthetic trafficking to the PM. This behavior suggests that during at least one presumably late step in their transport to the PM, the VSV-G protein and the Na,K-ATPase must occupy separate transport vesicles.
To test this possibility, we used a modified version of our Golgi block protocol on nonpolarized MDCK cells, as described in Materials and methods. Using this strategy, the vast majority of VSV-G and TMR-STAR–labeled Na pump colocalized in the TGN during the Golgi block (). Interestingly, even under Golgi block conditions, areas of local concentrations within the Golgi were observed for both Na pump and VSV-G, indicating that their initial distributions are partially segregated (, inset). Upon release from the block for 15 min, the VSV-G and Na pump signals diverged and appeared as populations of small puncta presumably corresponding to separate PGTIs (, arrows). Direct observation of 150 Na pump–containing carriers found that only 30 ± 6% of carriers costained for VSV-G. In some cases, individual puncta that were labeled exclusively for either the Na pump or the VSV-G protein appeared to reside in close proximity to one another. These observations support the surprising conclusion that at least some proteins destined for delivery to the same domain of the PM are segregated into distinct populations of vesicular carriers that pursue divergent courses before arriving at their common destination.
Figure 7. Na pump and VSV-G exit the TGN in separate PGTIs en route to the lateral membrane. SNAP cells were transfected with a plasmid encoding Ts 045 VSV-G–YFP and incubated for 24 h at 40°C to accumulate newly synthesized VSV-G in the ER. Next, (more ...)