Rab22a Localizes on Nonclathrin-derived Endosomes
We examined whether any of the Rab proteins known to have a role in endosomal trafficking colocalized with the nonclathrin-derived endosomal pathway. Due to the lack of good immunological reagents suited to detect endogenous Rab proteins, we transiently transfected HeLa cells with various GFP-tagged Rabs (Rab4, Rab5, Rab7, Rab11a, Rab11b, Rab14, Rab15, Rab21, Rab22a, and Rab25) because, in most instances, the GFP tag does not affect either the localization or the function of the Rab proteins (Sonnichsen et al., 2000
). To label the clathrin-independent and the clathrin-dependent endosomal systems, respectively, Rab-transfected cells were allowed to internalize an antibody directed against MHCI and Alexa-633–conjugated Tfn (633-Tfn) for 30 min. In control cells (either untransfected or transfected with the empty GFP vector), MHCI was localized 1) in vesicular endosomes located in the perinuclear area that contained Tfn (, top, inset), hereafter referred to as the merge compartment (Naslavsky et al., 2003
); 2) in tubular recycling endosomes devoid of Tfn emanating from the perinuclear area and occasionally observed originating from endosomal structures which contained Tfn (, top, arrowheads); and 3) in vesicular structures clustered at the cell periphery and devoid of Tfn (, top, arrows). In 40–60% of the cells, the tubular endosomes extended for several micrometers from the juxtanuclear region, whereas in the other cells tubules were much shorter (our unpublished observations). These long tubular structures were not an artifact of fixation or specimen processing because they also could be visualized by time-lapse imaging of living cells (see below). The reason why long tubules were not seen in all cells is not clear.
Figure 1. MHCI colocalizes with GFP-Rab22a on tubular endosomes. HeLa cells were transiently transfected with either GFP (top), GFP-Rab5 (middle), or GFP-Rab22a (bottom) for 20 h and then incubated for 30 min at 37°C in the presence of an antibody against (more ...)
When overexpressed at a low level, most of the Rabs did not affect the morphology of either the clathrin-independent or the clathrin-dependent pathway. Rab5 (, middle) and to a minor extent Rab4, Rab14, and Rab21 (our unpublished observations) were localized on the merge compartment (, middle, inset), but were not present on the MHCI-containing endosomes devoid of Tfn (tubules and peripheral vesicles; , middle, arrows and arrowheads). Rab7, Rab15, and Rab25 did not colocalize with MHCI under these experimental conditions (our unpublished observations). Rab11a and Rab11b occasionally localized to the MHCI-containing tubular endosomes and only partially overlapped with the merge compartment (our unpublished observations). Strikingly, Rab22a showed a distinct, overlapping pattern. At low level of expression Rab22a colocalized with MHCI in the tubules and in the peripheral vesicles that were both devoid of Tfn (, bottom, arrows and arrowheads) and occasionally localized with MHCI and Tfn in the merge compartment (, bottom, inset). Similar results were obtained overexpressing Rab22a in other cell types (e.g., Cos7 and A431; our unpublished data) or overexpressing Rab22a tagged with either a myc or a FLAG tag (our unpublished observations).
Next, we examined whether endogenous Rab22a also localized on the nonclathrin-derived endosomes and to this aim, we generated a polyclonal antibody directed against the carboxy terminus of human Rab22a (see MATERIALS AND METHODS). The Rab22a antiserum was first tested by immunoblotting lysates from either FLAG-Rab22a–transfected or nontransfected HeLa cells. In untransfected cells, the antiserum recognized a single band of the predicted molecular size for Rab22a (, lane 1). The band was not detected by Rab22a antiserum that was preincubated with the immunizing peptide (, lane 3) or by preimmune serum (, lane 5). The Rab22a antibody detected the overexpressed protein, which ran at a slightly higher molecular weight due to the FLAG tag (, lane 2, asterisk), but did not recognize any of the other Rabs overexpressed as controls (our unpublished observations). Similar results were obtained with lysates derived from other cell types and tissues (Cos7, A431, and bovine brain; our unpublished observations).
Figure 2. Detection of endogenous Rab22a with a specific antibody. (A) Lysates from nontransfected (lanes 1, 3, and 5) and FLAG-Rab22a-transfected (lanes 2, 4, and 6) HeLa cells were probed with a Rab22a-antiserum in the absence (lanes 1 and 2) or in the presence (more ...)
Next, we examined whether the Rab22a antibody could specifically label Rab22a in cells. Importantly, the Rab22 antiserum detected the overexpressed Rab22a, but did not recognize any of the other Rab proteins that were overexpressed as controls (our unpublished observations). Nontransfected HeLa cells that had internalized MHCI and 633-Tfn for 30 min were probed with the Rab22a antiserum. The endogenous Rab22a localized on both the tubular endosomes and the vesicles at the cell periphery, which contained MHCI but did not contain Tfn (, inset), and only occasionally did we observe labeling of the merge compartment. The labeling of the endosomal structures was neither detected by preimmune serum nor by the Rab22a antiserum that was preincubated with the immunizing peptide ().
Because Rab11a and Rab11b also were occasionally localized on the MHCI-containing tubules, we checked whether the endogenous Rab11 also was present on these structures. MHCI-containing tubules but not other MHCI-containing endosomes were indeed occasionally labeled by an affinity-purified antibody directed against Rab11a (Volpicelli et al., 2002
). However, the extent and frequency of the labeling was much lower than by Rab22a antibody (our unpublished observations).
Rab22a Is Associated with the Recycling Endosome and Not the Incoming Clathrin-independent Endosome
The tubular endosomes containing MHCI also have Arf6 associated with them and have been implicated in the recycling of MHCI back to the PM (Radhakrishna and Donaldson, 1997
; Caplan et al., 2002
; Naslavsky et al., 2003
). Because Arf6 is associated with both the incoming and outgoing transport carriers of this clathrin-independent endosomal system, we examined where Rab22a was localized with respect to Arf6 and its mutants. We used expression of GFP-Rab22a to visualize Rab22a because it localized similarly to endogenous Rab22a with the exception that GFP-Rab22a also localized to the merge compartment (see DISCUSSION). Additionally, GFP-Rab22 had the advantages of a strong fluorescent signal and better resolution. In cells expressing wild-type Arf6, GFP-Rab22a colocalized with Arf6 on the tubular endosomal membranes and on peripheral vesicles that accumulated at the periphery of the cells (, top), but it did not colocalize with Arf6 on the PM, where Arf6 is believed to be in its active, GTP-bound form (, top, arrowheads). Expression of the constitutively active mutant of Arf6 (Arf6-Q67L) blocks the clathrin-independent endosomal pathway shortly after internalization; it causes the Arf6 early endosomal structures to undergo fusion, leading to the accumulation of large vacuolar structures, enriched in phosphatidylinositol bisphosphate and actin, that trap Arf6 and cargo molecules (Brown et al., 2001
; Naslavsky et al., 2003
). Under these conditions, both the convergence of the nonclathrin-derived endosomes with the early endosomes and the recycling to the plasma membrane are inhibited (Naslavsky et al., 2003
; our unpublished data). Remarkably, GFP-Rab22a did not associate with the vacuoles formed in cells expressing Arf6-Q67L (, middle). Furthermore, GFP-Rab22a–labeled tubules were no longer observed and the number of Rab22a-labeled peripheral vesicles was significantly decreased. Conversely, in cells expressing the inactive, dominant negative mutant of Arf6 (Arf6-T27N), GFP-Rab22a was localized predominantly on the Arf6-labeled tubules, and fewer peripheral vesicles also were observed (, bottom). In all of the cells, the prominent juxtanuclear distribution of GFP-Rab22a represents the merge compartment that also contained the transferrin receptor (our unpublished observations), and as mentioned above, this localization is not observed for the endogenous protein. Endogenous Rab22a localized on the Arf6-labeled tubules in both Arf6 wt- and Arf6 T27N-expressing cells, whereas it was not localized onto the vacuoles in Arf6-Q67L–expressing cells (our unpublished observations). These data suggest that Rab22a is not associated with Arf6 at the PM or on early endosomal structures, but rather on the outgoing recycling arm of the pathway.
Figure 3. GFP-Rab22a is not localized on the vacuoles accumulated in Arf6-Q67L–expressing cells. HeLa cells were cotransfected with GFP-Rab22a and either Arf6-wt (top), Arf6-Q67L (middle), or Arf6-T27N (bottom). Cells were probed with a polyclonal antiserum (more ...)
To confirm that Rab22a is associated with MHCI on outgoing recycling structures and not on incoming early endosomes, we compared internalization at early and late times. MHCI and 633-Tfn were internalized for 5 min in HeLa cells that were transiently transfected with GFP-Rab22a. As described previously, after 5 min of uptake, MHCI and Tfn were found on distinct endosomes, mostly vesicular in nature (Naslavsky et al., 2003
) that were not labeled by GFP-Rab22a (, inset). Tubules and peripheral vesicles that were labeled with GFP-Rab22a were mostly devoid of both MHCI and Tfn (, arrowheads). A similar experiment was performed using time-lapse imaging. Movie-1 (see Supplementary Information) and show a cell transfected with GFP-Rab22a after the addition of an antibody against MHCI that was conjugated to Cy3 (Cy3-MHCI). At early time points, MHCI-containing endosomes occurred close to the PM and at the bottom of the cell (, 1′ and inset). These pleiomorphic structures were not labeled with GFP-Rab22a, and none of the GFP-Rab22a-labeled tubules and vesicles contained any MHCI. After 5 min, some Rab22a-labeled tubules seemed to contain MHCI and also some vesicular structures in the perinuclear area (, 5′, 10′, and 20′, and insets). The amount of MHCI in GFP-Rab22a tubules increased over time. The overexpression of GFP-Rab22a did not alter either the kinetics of internalization or the morphology of the MHCI tubules compared with the adjacent nontransfected cell. In Movie 2 (see Supplementary Information), the movement of GFP-Rab22a–labeled tubules from the perinuclear area to the cell periphery is shown along with the disappearance of the tubules in the cluster of peripheral vesicles. In some cases, tubules are seen breaking down into small vesicles before fusion.
Figure 4. Rab22a is associated with the recycling endosomes and not with the incoming, nonclathrin-derived endosomes. (A) HeLa cells overexpressing GFP-Rab22a were allowed to internalize an antibody against MHCI and 633-Tfn for 5 min. Cells were fixed and processed (more ...)
Together, these data suggest that Rab22a associates with the nonclathrin-derived cargo at a later stage of the endocytic processes, after the convergence of nonclathrin derived endosomes with the early endosomes, and during recycling.
Expression of Rab22a-S19N Inhibits the Formation of Tubular Recycling Endosomes Containing MHCI
Because Rab22a is localized on the recycling endosomes, we wondered whether its activity might regulate recycling. To address this question, we used two mutants of Rab22a that are impaired in the GTP cycle: Rab22a-Q64L, defective in GTP hydrolysis, and Rab22a-S19N, defective in GTP binding (Mesa et al., 2001
; Kauppi et al., 2002
). HeLa cells were transiently transfected with either GFP-Rab22a-Q64L or GFP-Rab22a-S19N and allowed to internalize MHCI antibody and 633-Tfn for 30 min. Rab22a-Q64L localized to tubular structures and peripheral vesicles that contained MHCI but not Tfn (). The phenotype was almost identical to that of the wild-type protein, although larger MHCI-containing structures could be observed at the periphery, and the number of cells showing MHCI- and Rab22a-positive tubules was significantly increased ().
Figure 5. Expression of Rab22a mutants alters the morphology of the MHCI-containing endosomes and inhibits recycling of MHCI, but not of Tfn, back to the plasma membrane. (A) HeLa cells transfected with either GFP-Rab22a-Q64L or GFP-Rab22a-S19N were loaded with (more ...)
Strikingly, in cell expressing Rab22a-S19N, tubular structures and vesicles at the cell periphery were no longer observed (), and MHCI and Tfn were clustered in the perinuclear area in a compartment (, arrowheads) that was labeled by an antibody against Rab11a but not by an antibody against Rab4 (our unpublished observations). Rab22a-S19N was localized to the cytoplasm and to a structure in the perinuclear area () that colocalized with GM130, a marker for the Golgi apparatus (our unpublished observations and previously reported by Kauppi et al., 2002
). At low levels of expression of either the wild type or mutants of Rab22a, we did not observe any effect on Golgi morphology (our unpublished observations). However, the Golgi became fragmented upon high expression of wild-type or Q64L mutant of Rab22a (our unpublished observations) as had been observed previously (Kauppi et al., 2002
Expression of Rab22a and Its Mutants Inhibits the Recycling of MHCI but Not Tfn to the PM
Because Rab22a is localized on MHCI tubules that were suggested to mediate the recycling of MHCI to the PM (Radhakrishna and Donaldson, 1997
; Caplan et al., 2002
) and the overexpression of GFP-Rab22a-S19N significantly reduced the number of tubules in HeLa cells, we examined whether Rab22a was required for recycling of MHCI back to the PM. Recycling of MHCI was estimated by measuring the amount of MHCI reappearing at the PM after its accumulation in the recycling compartment (Radhakrishna and Donaldson, 1997
; see MATERIALS AND METHODS). We previously showed that treatment of cells with cytochalasin D, an inhibitor of actin polymerization, reversibly inhibits MHCI recycling causing MHCI to accumulate in the tubular endosomes (Radhakrishna and Donaldson, 1997
). In these experiments, we used LatA, another actin inhibitor, to accumulate MHCI in the tubular endosomes because LatA was less toxic to the cell. (white circles) shows the time course of the reappearance of MHCI to the surface with the maximal amount of recycling occurring at ≈30 min. Strikingly, the continuous presence of LatA in the medium significantly inhibited recycling of MHCI (, black circles), whereas it had no effect on the recycling of Tfn ().
To study the effect of Rab22a and its mutants on the recycling of MHCI, HeLa cells were transiently transfected with GFP-Rab22a wt, GFP-Rab22a-Q64L, or GFP-Rab22a-S19N, and the amount of recycled MHCI was estimated as described above. Expression of the dominant negative mutant of Rab22a, S19N, resulted in a significant inhibition in recycling of MHCI compared with nontransfected or GFP-transfected controls (, solid bars). Expression of the constitutively active mutant Q64L also inhibited recycling as potently as S19N mutant (, solid bars), suggesting that both activation and inactivation of Rab22a was required for recycling of MHCI. Some inhibition of MHCI recycling also was observed in cells expressing wild-type Rab22a (, solid bars). Notably, neither Rab22a nor its mutants affected the recycling of Tfn to the PM (, open bars), as had been reported previously (Kauppi et al., 2002
Rab22a seems to specifically regulate the recycling of MHCI to the plasma membrane. The inhibition of recycling caused by overexpression of Rab22a and its mutants was not due to an effect on the internalization of MHCI (our unpublished observations) or an inhibition of fusion between MHCI-containing and clathrin-derived endosomes. We did not observe any qualitative () or quantitative (our unpublished observations) difference in the fusion between the MHCI-containing and the transferrin-containing endosomes. The lack of effect of Rab22a constructs on the internalization of both MHCI and Tfn (our unpublished observations) is consistent with the fact that neither the endogenous nor the overexpressed Rab22a were localized on the incoming endosomes at early times of internalization (Figures and and Movie 1).
siRNA-mediated Depletion of Rab22a Delays MHCI Recycling to the PM
Overexpression of Rab22a and its mutants affects recycling of MHCI. Surprisingly, both the “active” (wt and Q64L) and the “inactive” (S19N) forms of Rab22a impaired recycling. The impairment could be due to the perturbation of the GTP cycle of Rab22a, cellular levels of Rab22a, or to the sequestration of some GEF or GAP that Rab22a might have in common with other Rab proteins. To determine whether endogenous Rab22a regulates recycling, HeLa cells were treated with a siRNA directed against Rab22a to reduce the endogenous levels of Rab22a, as described in MATERIALS AND METHODS. The efficiency of Rab22a depletion was first assessed by immunoblotting. In siRNA-treated cells, Rab22a was depleted below the detection level, whereas other proteins (namely, Rab4, Rab5, Rab11, Arf6, TfnR, and actin) were not affected (). The siRNA-treated cells had normal morphology and could be grown for several passages in the presence of the siRNA. In siRNA-treated cells, labeling with the Rab22a-antiserum was greatly diminished () and when Rab22a-depleted cells were allowed to internalize cy3-MHCI and 633-Tfn, the percentage of cells showing MHCI-containing tubular structures and peripheral vesicles was significantly reduced, as previously seen when Rab22a-S19N was expressed (compare Figures and ). The trafficking of Tfn was not impaired, and the morphology of other organelles in both the secretory (Golgi and endoplasmic reticulum) and the endocytic pathway (early endosomes and lysosomes) was not affected (our unpublished observations). Recycling of MHCI back to the PM also was measured in siRNA-treated cells. The recycling of MHCI to the PM was substantially inhibited, whereas the recycling of Tfn was slightly inhibited (). The internalization of MHCI and Tfn was not altered in siRNA-treated cells (our unpublished observations).
Figure 6. siRNA-mediated depletion of Rab22a inhibits MHCI recycling to the PM. (A) Lysates from either mock- or siRNA-treated cells were subjected to electrophoresis and probed with antibodies directed against Rab22a, Rab4, Rab5, Rab11, Arf6, TfnR, and actin. (more ...)
Expression of Rab11a-S25N Inhibits Recycling of MHCI to the Plasma Membrane
Rab11 has been implicated in the recycling of membranes from the juxtanuclear endocytic recycling compartment (ERC) (Ullrich et al., 1996
; Volpicelli et al., 2002
), and recently a dominant negative mutant of Rab11a has been shown to inhibit the recycling to the plasma membrane of Integrin β1 (Powelka et al., 2004
), a molecule known to traffic with MHCI (Radhakrishna and Donaldson, 1997
). Because both exogenous Rab11a and endogenous Rab11a were localized on the tubules (our unpublished observations), we investigated whether Rab11a and Rab22a regulate the same steps in the recycling of MHCI to the PM. HeLa cells overexpressing the dominant negative mutant of Rab11a (GFP-Rab11a-S25N) were allowed to internalize antibody to MHCI and 633-Tfn for 30 min. In cells expressing GFP-Rab11a-S25N, internalized MHCI was localized in tubular structures and in the perinuclear area, but it was no longer present in peripheral vesicles in cells (). Fusion of MHCI-containing endosomes with Tfn-containing endosomes was not affected (our unpublished observations). Strikingly, expression of GFP-Rab11a-S25N did not affect the percentage of cell showing MHCI-containing tubules, whereas the percentage of cells showing MHCI in peripheral vesicles was reduced (). The overexpression of GFP-Rab11a-S25N impaired the recycling of both MHCI and Tfn () as reported previously (Ullrich et al., 1996
; Schlierf et al., 2000
; Powelka et al., 2004
). GFP-Rab11a-S25N localized to the juxtanuclear region and partially overlapped with TfnR and Golgi markers, but no alteration in Golgi structure was observed (our unpublished observations). Expression of the dominant negative mutant of Rab11b (GFP-Rab11b-S25N), a related isoform of Rab11a, gave similar results (our unpublished observations).
Figure 7. Dominant negative mutant of Rab11a inhibits the recycling to the PM of both MHCI and Tfn. (A) HeLa cells were transfected with GFP-Rab11a-S25N and processed as described in . Bars, 10 μm. (B) HeLa cells were transfected as indicated, processed (more ...)