An accumulation of data in recent years has supported the existence of a distinct endosomal compartment in which membrane proteins and lipids are packaged for recycling to the cell surface. This compartment, termed the recycling endosome, has been characterized morphologically by its pericentriolar location, its tubulovesicular morphology, and its dependence on intact microtubules for its localization. It has also been characterized functionally by the presence of known recycling proteins (e.g., transferrin receptors), exclusion of fluid-phase markers, and the absence of markers of the sorting endosome such as Rab4 and Rab5 (Daro et al., 1996
). Recently, investigations have demonstrated that the small GTPase Rab11a is a reliable marker of the recycling endosome compartment in nonpolarized cells (Ullrich et al., 1996
; Green et al., 1997
). Ullrich et al. (1996)
also found that a mutant of Rab11a defective in GTP binding (Rab11aS25N) impaired recycling of internalized transferrin, apparently by inhibiting transport from sorting endosomes to the recycling endosome.
An equivalent of the recycling endosome has also been identified functionally and morphologically in epithelial cells (Apodaca et al., 1994
; Barroso and Sztul, 1994
; Odorizzi et al., 1996
). Like its counterpart in nonpolarized cells, this compartment is clustered around the centrosome, is tubulovesicular in morphology, and requires intact microtubules for its integrity; however, in addition the apical recycling endosomal compartment appears to serve a sorting function that is unique to polarized cells. Because it is accessed by endocytic pathways originating from both poles of the cell, apical and basolateral membrane components (including lipids) become mixed within the recycling endosome and must be resorted before transport back to the appropriate membrane domain. In addition, a subset of internalized proteins is transcytosed in epithelial cells. These transcytosing proteins must be separated from recycling proteins and packaged for transport to the opposite cell surface. Clearly, the function of the recycling endosome in polarized cells is more complex and is likely to require a specialized machinery that does not exist in nonpolarized cells.
In this study, we demonstrate the association of Rab11a with the apical recycling system in polarized (MDCK) cells. This association is based on our findings that 1) antibodies specific for Rab11a label a population of vesicles in close proximity to the centrosome; 2) this localization is dependent on intact microtubules; and 3) IgA internalized from either the apical or basolateral plasma membrane can enter Rab11a-immunoreactive membranes. We also demonstrate that Rab25, a close relative of Rab11a that is expressed exclusively in epithelia, colocalizes with Rab11a in MDCK cells, suggesting that it regulates an epithelial-specific function associated with this endosomal system. In support of this hypothesis, we found that overexpression of Rab25 in MDCK cells coexpressing the pIgR resulted in a decreased rate of receptor transcytosis and of apical, but not basolateral, recycling of internalized ligand. Moreover, Rab25 expression had no detectable effect on basolateral recycling of internalized transferrin, further suggesting a selective role for this GTPase in apically directed postendocytic trafficking.
Although nocodazole caused dispersion of the apical recycling endosomes, taxol, a compound that stabilizes microtubules, induced a distinct concentration of both Rab11a and Rab25 immunostaining in the region of the tight junctions. The reasons for this distribution are not yet clear; however, an association between at least one Rab protein (Rab6) and a novel isoform of kinesin (rabkinesin-6) has been reported recently (Echard et al., 1998
) and was found to play an important role in Golgi membrane dynamics. This raises the intriguing possibility that Rab11a and/or Rab25 may also interact with microtubule motors to regulate the movement of vesicles or endosomes along microtubules.
As described above, mammalian Rab11a and Rab25 are closely related and in fact comprise a subfamily of Rab proteins along with mammalian Rab11b (Lai et al., 1994
), the Ypt3 protein of Schizosaccharomyces pombe
(Drivas et al., 1991
), and Ypt31p and Ypt32p of Saccharomyces cerevisiae
(Benli et al., 1996
; Jedd et al., 1997
). The putative effector domains of Rab11a and Rab25 are very similar in sequence (90% conserved, versus 53% between Rab25 and Rab5, and 50% with Rab17), suggesting that they interact with similar or identical targets to achieve their function. Because Rab11a appears to regulate transport of transferrin from sorting endosomes to the recycling endosome, it is tempting to speculate that Rab25 may play a similar role in the transport of apically internalized membrane from apical sorting (Rab5-positive) endosomes to the common recycling endosome in epithelia. Certainly, the selective inhibition of apical but not basolateral recycling of internalized IgA (or basolateral recycling of transferrin) is consistent with such a model, but how could the effects of Rab25 overexpression on basolateral-to-apical transcytosis be explained? Both transferrin receptor and the pIgR are transported from the basolateral cell surface to the apical recycling compartment with similar kinetics (Apodaca et al., 1994
); however, transferrin receptors are recycled to the basolateral plasma membrane with high efficiency, whereas the pIgR is thought to enter the apical recycling pathway to reach the apical surface (Apodaca et al., 1994
). If this is indeed the case, a decrease in the rate of membrane flow through the apical recycling pathway would be expected to have similar effects on both apical recycling and basolateral-to-apical transcytosis, precisely what we have observed in Rab25-expressing MDCK cells.
Recent investigations have demonstrated the association of another epithelial-specific Rab protein, Rab17, with the apical recycling system in both a mouse mammary epithelial cell line, Eph4 (Zacchi et al., 1998
), and MDCK cells (Hunziker and Peters, 1998
); however, the functional consequences of Rab17 overexpression in these two studies are contradictory and therefore difficult to interpret. In Eph4 cells, expression of wild-type Rab17 had no effect on transcytosis; however, both a constitutively active (Q77L) and dominant-negative (N132I) mutant stimulated transcytosis as well as apical recycling (Zacchi et al., 1998
). In contrast, in MDCK cells, expression of wild-type Rab17 had a significant inhibitory effect on transcytosis of IgA and increased basolateral recycling (Hunziker and Peters, 1998
). Neither activating nor inhibitory mutants were tested in this latter study. The inhibition of transcytosis by Rab17 in MDCK cells is similar to that observed here for Rab25, except that we did not detect a stimulation of basolateral recycling. Rather, overexpression of Rab25 led to an intracellular accumulation of internalized ligand.
We also found that expression of a Rab25 mutant predicted to be deficient in GTP binding (Rab25T26N) had no effect on either transcytosis or apical recycling. This was somewhat surprising, given that a similar mutation in Rab11a (S25N) significantly impaired transferrin receptor recycling in baby hamster kidney cells (Ullrich et al., 1996
); however, the apparent lack of a Rab25T26N phenotype can be interpreted in several ways. The simplest explanation is that the mutant protein is aberrantly folded and therefore completely nonfunctional; however, the level of expression of Rab25T26N was similar to that of the wild-type protein as determined by immunoblotting (Figure E, inset), suggesting that it was not subject to an increased rate of turnover. Alternatively, it is possible that the mutant protein is poorly prenylated. A nonprenylated mutant of Rab17 was similarly without effect when expressed in MDCK cells (Hunziker and Peters, 1998
). Still, perhaps the most likely explanation is that because the effects of overexpressed Rab25 are inhibitory, the T26N mutation yields a GTPase that is no longer functionally inhibitory. Clearly further work will be required to distinguish among these possibilities.
The sequence of the GTP-binding site of Rab25 is unique among Rab proteins, in that the P-3 domain consensus sequence WDTAGQE contains a leucine residue in place of the conserved glutamine. An equivalent substitution in Ras creates a dominant-active GTPase (Haubruck and McCormick, 1991
). Although the effects of this mutation on Ras GTPase activity have been well characterized, the phenotype of Rab proteins with similar mutations is not as well established. For example, although the Q to L substitution in Rab5 results in a dominant-active Rab–GTP state (Hoffenberg et al., 1995
), the same mutation in SEC4 has no effect on GTPase activity (Walworth et al., 1989
). The results presented here demonstrate that despite the presence of a leucine residue at position 71, Rab25 is not a constitutively active GTPase. Indeed, recombinant Rab25 exhibited higher intrinsic GTPase activity than recombinant Rab11a in vitro. Furthermore, we found that Rab11aQ70L is also an active GTPase, with cytosol (GAP)-stimulated activity that is indistinguishable from that of wild-type Rab11a. These findings demonstrate that in members of the Rab11 subfamily, substitution of the conserved glutamine in the P-3 GTP-binding domain with leucine does not significantly alter GTPase activity and may explain the unexpectedly minor effects of Rab11aQ70L expression on trafficking through recycling endosomes in nonpolarized cells (Ullrich et al., 1996
). We have found that overexpressed wild-type Rab11a and Rab11aQ70L both are localized in a centrosomally clustered distribution that is similar to that observed for endogenous Rab11a in nontransfected MDCK cells (Goldenring and Casanova, our unpublished results). All of these results emphasize the need to document the GTPase activities of P-3 mutations among Rab proteins.
Rab11a was initially characterized as a 24-kDa GTP-binding protein from brain membranes (Kikuchi et al., 1988
) and was subsequently cloned from a number of sources, including MDCK (Chavrier et al., 1990
), mouse kidney (Chavrier et al., 1992
), and rabbit gastric parietal cells (Goldenring et al., 1994
). In epithelial tissues, prominent immunostaining for Rab11a was observed in the subapical region of a number of cell types, including surface mucous cells of the gastric mucosa, intestinal enterocytes and colonocytes, kidney collecting duct, pancreatic acinar cells and pancreatic ducts, and hepatocytes and hepatic ducts, as well as the medial layers of the squamous mucosa of the skin and esophagus (Goldenring et al., 1996
). Although our initial investigations showed the highest expression in epithelial cells, recent studies have documented that Rab11a is also present in nonpolarized cells, including fibroblasts (Ullrich et al., 1996
) and erythroleukemia cells (Green et al., 1997
). In brain, Rab11a is associated with cell bodies and dendrites in various nerve cells (Sheehan et al., 1996
). Interestingly, Rab11a is highly enriched in rabbit gastric parietal cells, where it is associated with intracellular tubulovesicles containing the H/K-ATPase (Goldenring et al., 1994
; Calhoun and Goldenring, 1997
). These tubulovesicles comprise a specialized regulated recycling organelle that sequesters the H/K-ATPase intracellularly and releases it to the apical plasma membrane in response to acid secretagogues. We have also observed that Rab25 colocalizes with Rab11a on parietal cell tubulovesicles (Calhoun and Goldenring, 1997
) as well as in other gastrointestinal cells (Nwokeji et al., 1998
), suggesting that the two proteins act together to regulate membrane recycling in various epithelial cell types.
In summary, we have demonstrated that Rab11a and Rab25 associate with the apical recycling system in polarized MDCK cells. The inhibition of transit through the apical recycling system by overexpression of Rab25 indicates that members of the Rab11 family may coregulate the rate and efficiency of processing through plasma membrane recycling systems. The presence of at least three different Rabs (Rab11a, Rab25, and Rab17) on recycling endosomes in polarized cells is indicative of the complexity of the recycling system, and it will be of interest to determine the precise role of each in the recycling process.