Correspondence to: Lisa A. Dunbar, Department of Cellular and Molecular Biology, 333 Cedar St., Sterling Hall of Medicine, BE-30, New Haven, CT 06510. Tel:(203) 785-6833 Fax:(203) 785-4951 E-mail:ldunbar{at}biomed.med.yale.edu.
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Abstract |
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The H,K-adenosine triphosphatase (ATPase) of gastric parietal cells is targeted to a regulated membrane compartment that fuses with the apical plasma membrane in response to secretagogue stimulation. Previous work has demonstrated that the subunit of the H,K-ATPase encodes localization information responsible for this pump's apical distribution, whereas the ß subunit carries the signal responsible for the cessation of acid secretion through the retrieval of the pump from the surface to the regulated intracellular compartment. By analyzing the sorting behaviors of a number of chimeric pumps composed of complementary portions of the H,K-ATPase
subunit and the highly homologous Na,K-ATPase
subunit, we have identified a portion of the gastric H,K-ATPase, which is sufficient to redirect the normally basolateral Na,K-ATPase to the apical surface in transfected epithelial cells. This motif resides within the fourth of the H,K-ATPase
subunit's ten predicted transmembrane domains. Although interactions with glycosphingolipid-rich membrane domains have been proposed to play an important role in the targeting of several apical membrane proteins, the apically located chimeras are not found in detergent-insoluble complexes, which are typically enriched in glycosphingolipids. Furthermore, a chimera incorporating the Na,K-ATPase
subunit fourth transmembrane domain is apically targeted when both of its flanking sequences derive from H,K-ATPase sequence. These results provide the identification of a defined apical localization signal in a polytopic membrane transport protein, and suggest that this signal functions through conformational interactions between the fourth transmembrane spanning segment and its surrounding sequence domains.
Key Words: sorting, polarity, epithelia, Na,K-, ATPase, H,K-ATPase
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Introduction |
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Polarized epithelial cells localize distinct classes of transport proteins to different membrane surfaces in order to carry out their secretory and absorptive functions. The generation of apical and basolateral plasma membrane domains, which differ substantially in both their lipid and protein contents, requires polarized epithelial cells to target newly synthesized membrane components to their appropriate destinations and to retain them there preferentially after delivery. To become substrates for polarized sorting and retention, membrane proteins must be endowed with sorting signals that specify the proteins' destinations for the cellular sorting machinery. Currently, the nature of these sorting signals has been resolved for only a few membrane proteins. Tyrosine-based signals and dileucine motifs have been shown to direct proteins to the basolateral surface in many epithelial cell types (
The Na,K-ATPase and gastric H,K-ATPase are highly homologous proteins that are located at opposite membrane domains. These members of the P-type ATPase family are both composed of a 110-kD subunit that spans the membrane 10 times, and a heavily glycosylated 5560-kD ß subunit that spans the membrane once. Catalytic activity has been attributed to the
subunit, although the ß subunit may play a role in modulating ion affinity (
subunits of these pumps are 63% identical, whereas the ß subunits share 35% identity (
Through the generation of chimeric proteins incorporating complementary portions of these two ATPases, it has been possible to identify regions of the proteins that confer their distinctive plasma membrane distributions and catalytic properties ( subunit chimeras expressed in LLC-PK1 cells, it was found that the first 519 amino acids (roughly the NH2-terminal half) of the H,K-ATPase
subunit assures an apical distribution of a construct (H519N) in which this sequence is fused to the COOH-terminal half of the Na,K-ATPase polypeptide (
subunit is paired with the COOH-terminal half of the H,K-ATPase protein (N519H), accumulates at the basolateral membrane in association with the ß subunit of the H,K-ATPase (
subunits contain sorting information that is dominant over any that exists in the ß subunits, since the apical chimera H519N assembled with and redistributed the normally basolateral Na,K-ATPase ß subunit. Similarly, the N519H chimera redirected the normally apical H,K-ATPase ß subunit to the basolateral cell surface. To narrowly define the sorting information that exists within the first 519 amino acids of the gastric H,K-ATPase, we have constructed several new chimeras from complementary portions of the Na,K- and H,K-ATPase
subunits. Here, we report the identification of a transmembrane domain in the gastric H,K-ATPase, which is sufficient for apical localization. Unlike previously identified localization signals in the transmembrane domains of single spanning membrane proteins, this transmembrane domain does not appear to function through glycolipid interactions. Rather, it acts through long range interactions with its flanking loop domains.
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Materials and Methods |
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Construction of Chimeras
Chimeras were constructed between the rat Na,K-ATPase 1 subunit (cDNA provided by E. Benz, Johns Hopkins University, Baltimore, MD) and the rat gastric H,K-ATPase
subunit (cDNA provided by G. Shull, University of Cincinnati, Cincinnati, OH). All manipulations were performed on
subunit cDNAs subcloned into the Bluescript plasmid chimeras (Promega Corp.) at the ClaI and XbaI sites unless noted. The restriction sites ApaI, corresponding to H,K-ATPase amino acid number 85, AccI at amino acid 326, HpaI at amino acid 356, and NarI at amino acid 519, were used to construct the chimeras. Where necessary, sites were introduced at corresponding positions of both
subunit cDNAs through silent site-directed mutagenesis using the Kunkel method. Chimera I was made by subcloning the small ApaI fragment of the H,K-ATPase into the complementary portion of the Na,K-ATPase. To generate chimeras IIV, portions of the H,K-ATPase were excised at the appropriate restriction sites and ligated into the corresponding sites within chimera I. Chimeras VI and VII were made by ligating annealed oligos, encoding the appropriate amino acids into chimera I at the AccI and HpaI sites. Chimera VIII was made by ligating the ClaI/Hpa1 fragment of chimera VI into the corresponding sites within chimera IV. The constructs were sequenced through the ligation points. All chimeras were subcloned into the mammalian expression vector pCB6 (kindly provided by M. Roth, University of Texas Southwestern Medical Center, Dallas, TX) at the ClaI, XbaI sites before being transfected into LLC-PK1 cells.
Cell Culture and Transfection
LLC-PK1 cells were grown in -MEM (GIBCO BRL) supplemented with 10% FBS, 2 mM L-glutamine, 50 U/ml penicillin, and 50 µg/ml streptomycin. Subconfluent LLC-PK1 cells were transfected by the calcium phosphate method (
Immunofluorescence
Immunofluorescence was performed as described ( subunit (1:50). The endogenous Na,K-ATPase, a basolateral marker, was stained with mAb 6H (1:100), which is directed against the NH2 terminus of the Na,K-ATPase
subunit. Secondary goat antimouse or antirabbit antibodies (1:200) were conjugated to either rhodamine or fluorescein (Sigma Chemical Co.). All antibody incubations took place in goat serum dilution buffer for 1 h at room temperature. Between primary and secondary antibody incubations, the cells were subjected to three 5-min washes in the PBS-based wash buffer. After incubation with the secondary antibody, cells were washed in PBS three times for 5 min, and finally in 10 mM NaPi for 10 min before being mounted on coverslips with Vectashield (Vector Laboratories). Confocal sections were taken using a Zeiss LSM 410 laser scanning confocal microscope. Images are the product of eightfold line averaging. xz cross sections were generated with a 0.2-µm motor step. Contrast and brightness were set so that all pixels were in the linear range.
Detergent Solubility Assay
The detergent solubility assay was performed as described previously ( subunit, respectively, followed by either goat antimouse or goat antirabbit antibodies (1:1,000) conjugated to HRP (Sigma Chemical Co.). The resultant product was detected by ECL (Amersham Pharmacia Biotech) and quantified using an IS-1000 Digital Imaging System (Alpha Innotech Corp.) densitometer.
Ouabain Survival Assay
LLC-PK1 cells were plated in 6-well culture tissue dishes and allowed to attach overnight before media containing ouabain (Sigma Chemical Co.) at a concentration of 10 uM or 5 mM was added. The media were changed every 2 d during the assay. Cell survival was scored by light microscopy as the presence or absence of attached proliferating cells at the end of 5 d.
Acidification Assay
LLC-PK1 cells stably expressing chimera III and untransfected LLC-PK1 cells were grown to confluence on Transwell porous cell culture inserts (Corning Costar Corp.). The cells were rinsed in PBS++ and the media were replaced with weakly buffered DME containing 0.2 mM Hepes, pH 7.4. The cells were placed in a 37°C incubator with atmospheric CO2 levels. At the time points indicated, 100-ul samples were removed from the apical and basolateral chambers, placed under oil, and the pH was measured on a Corning blood gas pH analyzer. All measurements were performed using three separate filters for each time point.
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Results |
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A comparison of the amino acid sequences of the Na,K- and H,K-ATPase subunits reveals a site of striking nonhomology at the extreme NH2 terminus. Of the first 46 NH2-terminal residues, only 9 are identical. Furthermore, the NH2 terminus of the H,K-ATPase
subunit is 13 amino acids longer than that of the Na,K-ATPase
subunit. To examine the potential sorting function of this region, we constructed a chimera that consists of the first 85 amino acids of the H,K-ATPase fused to the complementary sequence of the Na,K-ATPase (Fig 1, chimera I). When transfected into LLC-PK1 cells, this chimera was found exclusively at the basolateral membrane as shown by indirect immunofluorescence (Fig 1A and Fig C). The endogenous Na,K-ATPase was also found at the basolateral membrane, as would be expected (Fig 1B and Fig D). It is clear from this result that the first 85 amino acids of the H,K-ATPase are not responsible for the apical distribution seen with the first chimera, H519N.
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Although the first 85 amino acids of the H,K-ATPase do not appear to mediate localization, this sequence does contain an epitope that enables us to discriminate between chimeric subunits and the endogenous Na,K-ATPase with our panel of antibodies. The HK9 antibody, raised against a synthetic peptide whose sequence was derived from rat gastric H,K-ATPase, recognizes an epitope at the extreme NH2 terminus between amino acids 3 and 23. Our antibody directed against the Na,K-ATPase
subunit also recognizes an epitope within the NH2 terminus between amino acids 1 and 21. Both of these antibodies have been well-characterized and do not show any cross-reactivity to other ATPases. By retaining the first 85 amino acids of the H,K-ATPase
subunit sequence as an epitope tag on the chimeras that were subsequently constructed, we are able to distinguish the chimeric
subunit proteins consisting largely of Na,K-ATPase
subunit sequence from the endogenous Na,K-ATPase. Western blots using the HK9 antibody show that chimeras prepared for this study migrated with the expected molecular weights in SDS-PAGE gels (data not shown).
To define the specific sequences within the remaining 434 amino acids that manifest sorting information, a set of overlapping chimeras was generated by taking advantage of two engineered restriction sites, AccI and HpaI, at positions corresponding to H,K-ATPase subunit amino acids 324 and 356, respectively. The chimera containing the NH2-terminal 324 amino acids of the H,K-ATPase (Fig 1, chimera II) does not appear to embody apical sorting information, since it is found at the basolateral membrane in transfected cells (Fig 1E and Fig G). However, the chimera in which H,K-ATPase sequence constitutes the second ectodomain loop fourth transmembrane domain (TM4), and part of the large cytoplasmic loop (Fig 1, chimera III) is localized predominantly to the apical membrane in LLC-PK1 cells (Fig 1I and Fig K), indicating that the sorting information that leads to the apical distribution of the chimera lies between amino acids 324 and 519 of the H,K-ATPase
subunit.
We further dissected the region between amino acids 324 and 519 by examining two chimeras consisting of the 85amino acid epitope tag and H,K-ATPase sequence between either amino acids 356 and 519 (Fig 2, chimera IV) or amino acids 324 through 356 (Fig 2, chimera V). The chimera containing H,K-ATPase sequence between amino acids 356 and 519, which corresponds to part of the large cytoplasmic loop, resides at the basolateral membrane in transfected LLC-PK1 cells (Fig 2A and Fig C). The second chimera (Fig 2, chimera V) on the other hand, is predominantly localized to the apical membrane when it is expressed in LLC-PK1 cells (Fig 2E and Fig G) as seen from the microvillar staining. Therefore, the second ectodomain loop and TM4 of the gastric proton pump appear to contain information that is sufficient to allow the chimeric ATPase to reach the apical membrane.
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It has been shown that both the Na,K-ATPase and H,K-ATPase subunits must associate with their respective ß subunits in order to leave the ER and be transported to the plasma membrane. The
subunit chimeras presented here contain the COOH-terminal half of the Na,K-ATPase, which has been shown to determine specificity in ß subunit assembly (
subunit, we would expect that the Na,K-ATPase ß subunit would assemble in the ER with both the Na,K-ATPase
subunit and chimeric
subunits, and thus be transported to both the apical and basolateral surfaces. Immunofluorescence localization performed on the cell line expressing chimera V shows that the chimeric protein is localized to only the apical membrane (Fig 3A and Fig C), whereas the endogenous Na,K-ATPase ß subunit protein is found at both the basolateral membrane and at the apical membrane in those cells expressing the chimera (Fig 3B and Fig D). Identical results were found with all of the apical chimeras depicted in Fig 1, Fig 2, and Fig 6 (data not shown). We conclude that these chimeras assemble with the endogenous Na,K-ATPase ß subunit protein, and like the first chimera H519N, redirect this normally basolateral protein to the apical surface.
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To discern whether it is the ectodomain or the transmembrane domain that mediates the apical localization of the protein, another set of chimeras was designed to contain only the second ectodomain loop or TM4 of the H,K-ATPase. When transfected into LLC-PK1 cells, the chimeric protein, which includes the ectodomain of the H,K-ATPase subunit (Fig 2, chimera VI), is restricted to the basolateral membrane (Fig 2I and Fig K). In contrast, the majority of the chimera incorporating the H,K-ATPase
subunit TM4 (designated as VII in Fig 2) is localized to the apical membrane (Fig 2M and Fig O). From these results, it is clear that the TM4 of the gastric H,K-ATPase
subunit is sufficient for the apical localization of these chimeric ion pumps.
Comparison of the sequences of the TM4s of the two ion pump subunits shows surprisingly little nonhomology, considering that this region can mediate the strikingly different membrane distributions exhibited by these proteins (Fig 4 A). Of the 28 amino acids that comprise the putative transmembrane domain, only 8 are nonidentical. Seven of the nonconserved amino acids in this segment are found in the portion of the transmembrane domain that is predicted to pass through the outer leaflet of the lipid bilayer (Fig 4 B). The outer leaflet of the apical plasma membrane of many epithelial cell types is enriched in glycosphingolipids (GSLs). Furthermore, there is evidence that GPI-linked proteins, as well as other apical polypeptides, are incorporated into GSL-rich domains during their biosynthetic passage through the Golgi complex (
GPI-linked proteins that have become associated with GSL-rich membrane domains are insoluble in 1% Triton X-100 at 4°C. When a cell lysate prepared in this fashion is fractionated on a sucrose floatation gradient, insoluble proteins are found near the top of the gradient, whereas soluble proteins remain in the heavier fractions (
During the course of our dissection of the sorting information residing between residues 324 and 519 of the H,K-ATPase subunit sequence, we constructed an eighth chimera to determine whether the TM4 of the H,K-ATPase was required for apical localization. This chimera contains the TM4 of the Na,K-ATPase flanked by the TM3-TM4 ectodomain and a portion of the large cytoplasmic loop of the H,K-ATPase. Since this chimera lacks the H,K-ATPase TM4, we anticipated that it would be expressed at the basolateral plasma membrane. Surprisingly, we found that it is predominantly located at the apical membrane when expressed in LLC-PK1 cells (Fig 6A and Fig C). The apical polarity of this chimera is dependent upon the presence of both segments of H,K-ATPase sequences flanking the TM4, since the presence of either domain alone results in expression at the basolateral membrane (chimeras IV and VI). This result demonstrates that whereas TM4 of the gastric H,K-ATPase is sufficient to achieve apical localization of the chimeric ion pump, it is clearly not necessary. It also indicates that noncontiguous pump sequence domains predicted to lie on opposite sides of the bilayer can collaborate to create a signal for the pump's polarized distribution.
We wondered if the steady-state localization of the chimeras correlated with their enzymatic activities. To determine whether the chimeras can function as sodium pumps, we assayed the ability of the cells expressing chimeras to survive under conditions that block the endogenous Na,K-ATPase. Ouabain is a specific inhibitor of the Na,K-ATPase. The endogenously expressed pig Na,K-ATPase in LLC-PK1 cells has a Ki for ouabain of 10-7 µM, whereas the Ki for the rat Na,K-ATPase, which was used in the construction of the chimeras, is 10-4 µM. Taking advantage of this disparity in ouabain sensitivity, cells expressing the chimeras were tested for their ability to survive in the presence of 10 µM ouabain over the course of 5 d. This concentration is lethal to untransfected LLC-PK1 cells. As seen in Fig 7 A, the cells expressing chimeras I, II, IV, VI, and VIII survived 10 µM ouabain. Presumably, the chimeras expressed in these cells are enzymatically active and can mediate K+ influx and Na+ efflux, since they were able to compensate for the ouabain-inhibited activity of the endogenous Na,K-ATPase. Extensive characterization of the activities catalyzed by chimeras I, IV, and VI is presented elsewhere (
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Chimeras III, V, and VII were not able to confer ouabain resistance, suggesting that they may be inactive. However, it is also possible that these chimeras possess activity that resembles that of the gastric H,K-ATPase. The H+ efflux activity catalyzed by the gastric H,K-ATPase can not substitute for the essential Na,K-ATPasedriven sodium efflux. Hence, expression of the gastric H,K-ATPase does not confer ouabain resistance to transfected cells (Gottardi, C.J., and M.J. Caplan, unpublished observations). Qualitative evidence that at least one of these apical chimeras that did not confer ouabain resistance is indeed enzymatically active is provided by the observation that cells expressing chimera III acidify their apical media compartment when grown on permeable filter supports. Typical measurements of this acidification are presented in Fig 7 B, which shows that over the course of 4 h there is a small but significant fall in the pH of the apical media bathing cells expressing chimera III. This phenomenon appears to be due to the expression of chimera III, since it is not seen in untransfected cells and the effect is abolished by high concentrations of ouabain added to the apical media.
The ability of chimeras I, II, IV, VI, and VIII to confer ouabain resistance strongly suggests that these chimeras are capable of mediating Na+ efflux, since cells expressing gastric H,K-ATPase are not rendered ouabain-resistant. It is interesting to note that chimeras I, II, IV, and VI, which all appear to exhibit sodium pump function, are located at the basolateral plasma membrane, whereas the chimeras H519N and III, which exhibit H,K-ATPaselike function, are apical proteins (
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Discussion |
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The evidence presented here demonstrates that the TM4 of the gastric H,K-ATPase contains information that is sufficient for this protein's apical localization in LLC-PK1 cells. Chimeras generated from complementary portions of the apical H,K-ATPase and the basolateral Na,K-ATPase are found at the apical membrane of LLC-PK1 cells if their TM4s are derived from the H,K-ATPase. Pulse-chase metabolic labeling experiments show that the apical chimera proteins appear to be at least as stable as the endogenous Na,K-ATPase, supporting the conclusion that the localization seen by immunofluorescence most likely reflects the true steady-state distributions of these proteins (data not shown).
Hydropathy plots predict that amino acids 329356 of the gastric H,K-ATPase comprise the TM4. Although this type of analysis is not definitive proof that these amino acids pass through the lipid bilayer, in this case it is supported by biochemical evidence ( subunit TM4 region and/or its flanking sequences.
The steady-state localization of the chimeras does not appear to based on the ion selectivity of the pumps. Both apical and basolaterally located chimeras are capable of pumping sodium, as measured by their ability to confer ouabain resistance. Several of these chimeras (I, IV, VI, and VIII) have also been found to possess Na+-stimulated ATPase activity, further demonstrating that they are functional as sodium pumps (
It must be noted, that although the apical sorting behavior of chimeras containing the H,K-ATPase TM4 (III, V, and VII) is consistent with our interpretation that the H,K-ATPase TM4 encodes apical localization information, it is also possible that the behavior of these chimeras is instead attributable primarily to the disruption of a basolateral localization signal in the TM4 of the Na,K-ATPase. According to this interpretation, these chimeras accumulate at the apical membrane by a default mechanism, as has been documented for other basolateral membrane proteins whose basolateral sorting signals have been perturbed (
There is mounting evidence that transmembrane domains can play important roles in protein trafficking. The apical sorting of two influenza virus proteins, neuraminidase and hemagglutinin, appears to be encoded in their transmembrane domains (
The fact that the amino acid residues of a transmembrane domain may be in direct contact with lipid molecules prompted us to examine whether the detergent solubility of the apical chimeras resembled that of GPI-linked proteins, which associate with GSLs in detergent-resistant domains (
In the absence of evidence for specific lipid associations, it is tempting to propose that proteinprotein interactions are involved in the recognition and interpretation of this novel sorting signal. In one line of MDCK cells, cytoskeletal interactions appear to play an important role in establishing the basolateral distribution of the Na,K-ATPase. These MDCK cells, which missort glycolipids, target newly synthesized Na,K-ATPase to both the apical and basolateral membrane domains ( subunits have been shown to bind ankyrin and share two conserved putative ankyrin-binding sites located in the cytoplasmic loops between transmembrane domains 2 and 3 and between transmembrane domains 4 and 5 (
The TM4 of the gastric H,K-ATPase could function as a localization signal by interacting with other proteins within the plane of the membrane. Evidence for such intra-membranous protein associations has been obtained in studies of the assembly of major histocompatibility complex class II molecules. The transmembrane helices of the major histocompatibility complex class II and ß subunits appear to be necessary and sufficient to ensure the interaction of these two polypeptides (
chain of the T cell receptor with the CD3
chain (
The gastric H,K-ATPase TM4 sequence shows no significant homology to any other apically located proteins. The gastric H,K-ATPase's closest molecular relatives are the nongastric H,K-ATPases. These pumps are expressed in colonic and renal epithelial cells and appear to reside at the apical plasma membrane ( subunits are each 63% identical to one another at the amino acid level (
subunits are almost identical to that of the Na,K-ATPase. There is mounting evidence that the similarity of the TM4 of the nongastric H,K-ATPases to that of the Na,K-ATPase may confer a shared ability to transport Na+ ions (
subunit sequence to generate similar apical localization signals through modification of the conformations of their TM4s.
In light of the apical localization of the nongastric H,K-ATPases, the behavior of chimera VIII, which contains the TM4 of the Na,K-ATPase, is especially interesting. This chimera resides at the apical plasma membrane of LLC-PK1 cells, demonstrating that TM4 of the H,K-ATPase is sufficient but not necessary for apical localization. Although it is possible that this chimera contains an apical localization signal that is completely distinct from that present in TM4 of the H,K-ATPase, it must be noted that the apical polarity of this chimera is dependent upon the simultaneous presence of two stretches of H,K-ATPase amino acids flanking TM4. Alone, each of these sequences is unable to direct the chimera to the apical membrane. The predicted ectodomain loop between TM3 and TM4 is only six amino acids in length and differs at only three positions between the Na,K and gastric H,K-ATPases. As only two of the nonidentical amino acids were exchanged in chimeras incorporating the H,K-ATPase TM3-TM4 ectodomain, one or both of these amino acids must account for the change in localization seen between chimera IV and chimera VIII. This ectodomain region could play a role in creating an apical localization signal by cooperating with the large cytoplasmic loop to cause TM4 to adopt a particular conformation or orientation. The H,K-ATPase TM4 segment alone may independently achieve this same conformation. Consistent with this interpretation, recent structural analysis of the P-type Ca-ATPase suggests that cytosolic segments of the enzyme may anchor the transmembrane helices in specific positions ( subunit does not itself carry a specific localization signal. Instead, it may exert its effects on localization by imposing certain conformations on other parts of the
subunit, such as the ectodomain or cytoplasmic domain adjacent to TM4. These conformational motifs could then be recognized by components of the cellular sorting machinery. Future studies will determine the specific residues of both the TM4 and its flanking regions, which are responsible for apical localization.
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Footnotes |
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1 Abbreviations used in this paper: GPI, glycophosphatidyl inositol; GSL, glycosphingolipid; TM4, fourth transmembrane domain.
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Acknowledgements |
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We thank G. Shull and E. Benz for the gifts of the subunit cDNAs, and Deborah Brown for helpful suggestions. We also thank Vanathy Rajendran and Jeff Possick for valuable assistance with experiments, and the entire Caplan Lab for comments and discussion.
This work was supported by National Institutes of Health (NIH) grant GM-42136 (M.J. Caplan), a National Science Foundation National Young Investigator Award (M.J. Caplan), and a National Research Service award from NIH (L.A. Dunbar).
Submitted: 15 July 1999
Revised: 10 December 1999
Accepted: 4 January 2000
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References |
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