Lisa A. Dunbar, Paul Aronson, Michael J. Caplan
Review by Michael J. Osgood
Mechanisms for cellular localization of membrane proteins in polarized epithelial cells following expression have been determined for only a few proteins. For example, proteins have been shown to localize to cellular basolateral membranes through tyrosine-based signals and dileucine motifs in epithelial cells. Localization to the opposite luminal-facing apical membrane has been demonstrated to occur through a glycophosphatidyl inositol (GPI) anchor.
Gastric parietal cell H,K-ATPase and Na,K-ATPase are highly homologous integral membrane peptides that localize to opposite cellular membranes. These proteins consist of an ? subunit (110-kD) that is highly homologous (63% amino acid similarity), as well as a ? subunit (55-60-kD) that is also similar (35% amino acid similarity). Furthermore, the proteins have the same number of transmembrane domains and ectodomains, and the corresponding domains are very similar in size, the greatest difference being in the amino-termini. Despite the similarities in shape and sequence of H,K-ATPase and Na,K-ATPase, they localize to different regions of gastric epithelial cells. The gastric H,K-ATPase is localized to either subapical tubulovesicular elements or, more commonly, the apical membrane itself, which exposes the protein to the stomach lumen. The Na,K-ATPase, however, resides solely in basolateral membrane regions of gastric parietal cells. It is known that the H,K-ATPase does not localize to the apical membrane through a GPI linked mechanism. In this paper, the authors seek to determine the mechanism through which this non-GPI linked H,K-ATPase protein localizes to the apical membrane of gastric epithelial cells. They successfully demonstrate that a unique transmembrane domain of the H,K-ATPase is sufficient for localization to the apical membrane of gastric parietal cells.
The authors began their search with the knowledge that the amino-terminus half of H,K-ATPase (519 amino acids) is responsible for localization of the protein to the apical membrane. This was previously shown through the construction of two chimeras of the ? subunit, one of which consisted of the amino-terminus half of H,K-ATPase and the carboxyl-terminus half of Na,K-ATPase, and the other consisting of the amino-terminus half of Na,K-ATPase and the carboxyl-terminus half of H,K-ATPase. Both chimeras were paired with the ? subunit of Na,K-ATPase. The chimera with the H,K-ATPase amino-terminus subunit localized to the apical membrane, while the chimera with the Na,K-ATPase carboxyl-terminus subunit localized to the basolateral membrane. Two conclusions were reached from these observations. First, the region specifying cellular localization of H,K-ATPase to the apical membrane resides in the amino-terminus half of H,K-ATPase, since both the amino- and carboxyl-termini of Na,K-ATPase directed localization to the basolateral membrane. Second, the ? subunits direct sorting independent of the ? subunit because the chimera with the H,K-ATPase amino-terminus half localized to the apical membrane despite the presence of a Na,K-ATPase ? subunit on the chimera. Relying on these findings, the authors designed experiments to determine which domain of the 519 amino acid H,K-ATPase amino-terminus is responsible for apical localization.
The authors designed chimeras with several amino-terminus substitutions in order to locate a more specific domain within the first 519 amino acids responsible for apical membrane localization. Chimeras were first assembled and subcloned into Bluescript plasmid and then subcloned and expressed within expression vector pCB6. The recombinant pCB6 vector was introduced into LLC-PK1 cells, where the adenosine triphosphatase pumps were expressed and tested for membrane localization.
In order to determine the effect on localization of the large variation in amino acid sequence between H,K-ATPase and Na,K-ATPase at the extreme amino-terminus, the authors tested the potential sorting function of this region by forming a chimera consisting of amino acids 1-85 of the H,K-ATPase fused to the remaining complementary ? subunit of Na,K-ATPase using restriction site ApaI. Labeled as chimera I in Figure 1, this protein localized to the basolateral membrane (Fig. 1, A and C) of the LLC-PK1 cells as determined by immunofluorescence using secondary antibody that recognizes polyclonal antibody HK9 raised against amino acids 3-23 of the amino-terminus H,K-ATPase portion of the chimera. Figure 1, B and D, shows immunofluorescence using secondary antibody that recognizes mAb 6H raised against amino acids 1-21 of the Na,K-ATPase. This data serves as a positive control to establish the fluorescence pattern of endogenous Na,K-ATPase in the basolateral membrane of LLC-PK1 cells. The immunofluorescent patterns of the chimera and the endogenous Na,K-ATPase correspond, indicating that the apical localization signal does not reside within the first 85 amino acids of H,K-ATPase. The confocal images show an en face perspective and a xz cross section. The en face perspective from above the cell layer shows clear basolateral membrane affiliation, in which the proteins are congregated mostly around the lateral margins of the cell membranes. The xz cross section shows a view from the side of the cells, indicating clearly that the chimeric pumps and the endogenous Na,K-ATPase are localized to all membrane regions except the luminal. A separate western blot was conducted which verified the expected size of this chimera and all the others that were produced, so we know that the full-size chimeras are immunofluorescing (data not shown). This region of amino acids 1-85 was retained throughout all of the chimeras as an epitope tag for immunofluorescent differentiation of the chimera pumps from endogenous Na,K-ATPase.
So far, the authors have narrowed the possible location of the apical localization signal to amino acids 85-519 of the H,K-ATPase ? subunit amino-terminus. In order to further differentiate the areas of this region responsible for apical localization, two engineered restriction sites – AccI and HpaI – were produced by site-directed mutagenesis at amino acids 324 and 356, while retaining the amino acid sequence of the protein in these regions. Chimera II, which consisted of the first 324 amino acids of H,K-ATPase and the complementary remainder of Na,K-ATPase, localized to the basolateral membrane, indicating that the membrane localization signal does not reside within this region of H,K-ATPase. Again, basolateral localization was confirmed by immunofluorescence of endogenous Na,K-ATPase as a positive control, with images similar to chimera I. The remaining amino acid segment from amino acids 324-519 should therefore contain the information directing cellular localization. Chimera III contained the H,K-ATPase segment consisting of these amino acids, as well as amino acids 1-85 as an epitope. Indeed, immunofluorescence verified that this chimera localized to the apical membrane, confirming that this region is responsible for sorting of the H,K-ATPase to the apical membrane. The en face and xz confocal images show localization of chimeric pumps across the luminal membranes of the cell lines. Endogenous Na,K-ATPase control images are localized to basolateral membrane regions.
This region was further differentiated in chimeras IV and V by implementing the engineered restriction sites. Chimera IV, which contained the H,K-ATPase sequence between amino acids 356-519, localized to the basolateral membrane (Figure 2, A and C). Chimera V contained the H,K-ATPase sequence between amino acids 324-356, and this chimera localized to the apical membrane (Figure 2, E and G). This sequence includes the second ectodomain region as well as the fourth transmembrane domain (TM4) of H,K-ATPase. Confocal images of the chimeric localization show the expected pattern for both basolateral localization of chimera IV and apical localization of chimera V. To differentiate the roles of the second ectodomain region and TM4 of H,K-ATPase, chimeras VI and VII were even more specific. Chimera VI contained the ectodomain region from H,K-ATPase and localized to the basolateral membrane, while chimera VII contained TM4 and localized to the apical membrane. Confocal en face and xz images show the expected patterns, though the image of chimera VII is not as intense as previous images. The authors concluded from these results that TM4 is sufficient for sorting and localization of H,K-ATPase to the apical membrane.
In an effort to determine the pattern of assembly of the of chimeric and Na,K-ATPase ? subunits with the Na,K-ATPase ? subunit, the authors chose to introduce ? subunit to cells expressing chimera V. En face and ez section confocal images show that binding of ? subunit did not affect the apical localization of chimera V (Fig. 3, A and C), nor did it affect the basolateral localization of endogenous Na,K-ATPase (Fig. 3, B and D). This is an important test because it confirms that the amino acid sequence containing ectodomain 2 and TM4 of H,K-ATPase is still sufficient for localization of the ? subunit to the apical membrane when bound to the ? subunit, as in the wild type conformation.
After suggesting TM4 as a region responsible for cellular localization of H,K-ATPase to the apical membrane, the authors note that there is surprisingly little difference between the TM4 sequences of H,K-ATPase and Na,K-ATPase. Of the 28 amino acids comprising the transmembrane domain, only 8 are nonidentical, and these are restricted mostly to the first half of the transmembrane domain (Figure 4A). This portion of noncomplementary amino acids is predicted to pass through the outer leaflet of the apical plasma leaflet (Figure 4B), which is often rich with glycosphingolipids (GSLs). In search of a mechanism by which TM4 targets H,K-ATPase to the apical membrane, the authors investigate the possibility that TM4 associates with GSLs during its route through the Golgi complex, which could mediate the targeting of the protein-GSL complex to the apical membrane due to the typical GSL polarity of epithelial cells. Restriction of Na,K-ATPase from this GSL pathway would therefore result in targeting of Na,K-ATPase to the basolateral membranes. This theory relies on the observation that MDCK cell lines that lack GSL polarity misdirects Na,K-ATPase, targeting it equally to the apical and basolateral regions. Moreover, MDCK cells that are normally GSL polar direct Na,K-ATPase equally to apical and basolateral membranes when treated with fumonisin, a drug that inhibits GSL synthesis. However, in both cases, the apical membrane populations of Na,K-ATPase become degraded while the basolateral populations become stabilized. Therefore, it seemed likely that Na,K-ATPase is localized to the basolateral membrane as a result of its exclusion from the GSL pathway, while H,K-ATPase is localized to the apical membrane as a result of its inclusion in the pathway.
To test this hypothesis, the authors used a sucrose floatation gradient, which determines the solubility of proteins in GSL. This procedure involves treatment of the proteins with Triton X-100, in which GSL-rich membrane domains are insoluble and those that are not GSL-associated are soluble. Sucrose floatation fractions were determined for chimera H519N (containing the amino-terminus half of H,K-ATPase), endogenous Na,K-ATPase, and GPI-linked alkaline phosphatase as a positive control. Because the alkaline phosphatase is a GSL-rich protein, it was found to be insoluble in Triton and was most active within the lighter fractions (2-4) at the top of the gradient (Figure 5A). Na,K-ATPase appeared in the heavier fractions, indicating that it is soluble and therefore is not associated with GSL, as was expected (Fig. 5A). If H,K-ATPase localizes to the apical membrane through association with GSL, the H519N chimera would be expected to appear in the insoluble top region of the fraction gradient with alkaline phosphatase. However, it appeared in the same region as the Na,K-ATPase, and demonstrated virtually the same activity, indicating that a mechanism other than GSL lipid association is responsible for localization of H,K-ATPase to the apical membrane. The presence of both the Na,K-ATPase and H,K-ATPase in the heavier, soluble fractions of the sucrose gradient is confirmed by a western blot (Figure 5B). Two bands appear for the chimera, representing ? subunit with and without ? subunit.
The failure of TM4 to associate with GSL prompted the authors to question whether it is not only sufficient, but also necessary, for apical localization. To determine this they constructed an eighth chimera consisting of Na,K-ATPase TM4 flanked on either side by the H,K-ATPase ectodomain residing between transmembrane domains 3 and 4, and half of the large cytoplasmic domain of H,K-ATPase. Surprisingly, this chimera localized to the apical membrane, as indicated by confocal en face and xz section images, using the standard endogenous Na,K-ATPase antibody as a control (Figure 6). Therefore, TM4 of H,K-ATPase is sufficient, but not necessary, for apical localization. The same is true of the TM4 flanking sequences of H,K-ATPase.
In order to determine whether the localization of chimeras correlated with the expected enzymatic activity of the pumps, the authors tested the functionality of the chimeric pumps in the LLC-PK1 cells by blocking the activity of endogenous Na,K-ATPase. Ouabain, a specific inhibitor of Na,K-ATPase, is lethal to LLC-PK1 cells because it inhibits the essential sodium-pumping activity of Na,K-ATPase. All 8 chimeras were exposed to ouabain, and all but 3 conferred resistance (Figure 7A), indicating that they were not susceptible to ouabain and were enzymatically active enough to maintain the intercellular sodium concentration necessary for cell survival. The 5 that conferred resistance were the chimeras that exhibited basolateral localization, with exception of chimera VIII, which conferred resistance from an apical position. Oddly, chimera VIII was the only apical chimera to display enzymatic sodium-pumping abilities. In order to determine whether an apical chimera was enzymatically active, cells expressing chimera III were shown to acidify the apical media, as demonstrated by a gradual decrease of the pH over four hours (Figure 7B). Chimera III was better able to acidify the media than untransfected LLC-PK1 cells, and seems to be dependent on the presence of chimera III due to the inhibited acidification in the presence of ouabain.
At this point it is unclear what is responsible for localization of H,K-ATPase to the apical membrane of gastric cells. While TM4 is sufficient for localization in three cases, the protein localized to the apical membrane in another case when Na,K-ATPase TM4 was flanked by extramembranous sequences from H,K-ATPase (chimera VIII). Elucidation of the roles of these regions would provide better insight into the factors that determine localization of ATPase. It has been shown that H,K-ATPase TM4 consists of the necessary sequence to localize apically by itself, but the similar Na,K-ATPase TM4 requires H,K-ATPase flanking regions on either end to localize to the apical membrane. It could be that this is a concidence resulting from the sequence similarity of the TM4 regions, in which the Na,K-ATPase TM4 region is bent into the proper conformation for apical localization by the flanking H,K-ATPase regions. Further elucidation of the regions that participate in apical localization could lead to location of a region that is necessary for cellular localization.
It is also possible that TM4 confers unique functional properties on
these proteins. Of the four chimeras that exhibited apical localization,
three were unable to confer ouabain resistance. All three of these
chimeras contained H,K-ATPase TM4, while the fourth contained H,K-ATPase
elements flanking a Na,K-ATPase TM4 region. This result indicates
that this TM4 may be partially responsible for differentiating the enzymatic
activity of H,K-ATPase and Na,K-ATPase, as well as playing a role in determining
cellular localization. The ability of chimera III to acidify the
apical media and the ability of chimera VIII (same as chimera III, but
without H,K-ATPase TM4) to confer ouabain resistance demonstrates that
TM4 is sufficient for the proton-pumping enzymatic activity of H,K-ATPase.
The authors could direct future work in this area to determining whether
TM4 is necessary for the apical acidification ability of H,K-ATPase.
By testing the acidifying abilities of chimeras V and VII, the doors might
be open to concluding that TM4 is necessary for the enzymatic acidification
function of H,K-ATPase. Perhaps it is also possible that a combination
of localization sequences and functionality-determining sequences determine
the ultimate fate of adenosine triphosphatase as either an apical H,K-ATPase
or a basolateral Na,K-ATPase.
*Dunbar, Lisa A., Paul Aronson, and Michael J. Caplan. “A Transmembrane
Segment Determines the Steady-state Localization of an Ion-transporting
Adenosine Triphosphatase.” Journal of Cell Biology 148 (2000): 769-778.
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