This paperís goal is to identify in the H, K- adenosine triphosphatase of gastric parietal cells motifs responsible for localization in the apical membrane domain.  The researchers use a functional approach involving the creation of chimeric proteins and ultimately implicate the fourth transmembrane domain of the H,K- ATPase as being sufficient but not necessary for apical membrane localization.
     The researchers derive their chimeras from the coding sequences of H,K- ATPase and Na,K- ATPase.  H,K- ATPase is normally directed to the apical domain while the Na,K- ATPase is directed towards the basolateral membrane domain.  Both proteins are P-type ATPases that have a catalytic alpha subunit which crosses the membrane ten times as well as a beta subunit consisting of a single transmembrane domain which must complex with an alpha subunit in order for both to exit the ER.  In addition, the beta subunit is believed to regulate the cycling of the H,K- ATPase complex between the apical domain and sub-apical compartments.  Because chimeras are transfected into the pig kidney epithelial cell line LLC-PK1 which does not contain a sub-apical compartment, the researchers are able to focus solely on apical versus basolateral localization.
     Previous research had demonstrated that the first 519 amino acids of H,K- ATPase were able to induce apical localization when paired with the carboxy terminus half of Na,K- ATPase.  A set of seven chimeras were transfected via the mammalian expression vector pCB6 into LLC-PK1 cells and chimeric localization assayed by immunofluoresence.  Figure 1 depicts the results of immunofluoresence for chimeras I, II and III.  Chimera I consisted of only the first 85 amino acids of gastric H,K- ATPase fused to a segment corresponding to the remainder of a complete P-type ATPase as determined by homology.  Chimera I localized in the basolateral membrane as it displayed the same pattern of localization as endogenous Na,K- ATPase.  The Na,K- ATPase was visualized with an antibody recognizing an epitope spanning amino acids 1 through 21.  The chimera was also visualized with an antibody detecting an amino terminus epitope between amino acids 3 and 23 of H,K- ATPase.  The researchers harnessed the inability of amino acids 1 through 85 of H,K- ATPase to confer apical distribution by tagging all subsequent chimeras with this sequence.  This addition permitted visualization specifically of the chimera yet presumably did not affect localization.  The first 324 amino acids of H,K- ATPase (Chimera II) were also unable to direct a chimera to the apical domain.  However, chimera III, containing amino acids 324-519, produced an apical staining pattern in contrast to the basolateral staining pattern produced in the same cell lines when stained for endogenous Na,K- ATPase.
     Figure 2 depicts localization results for four more chimeras.  Chimeras IV and V subdivide the H,K- ATPase chimeric region from chimera III into amino acids 356-519 and 324-356 respectively.  Chimera IV displays basolateral distribution while chimera V shows apical distribution as seen by immunofluoresence results achieved in the same manner as figure 1.  The H,K- ATPase region of chimera V constitutes the second ectodomain and the fourth transmembrane domain of the alpha subunit.  Chimeras VI and VII further define this region defined by chimera V.  Chimera VI contains only the second ectodomain and localizes basolaterally.  Chimera VII contains only the fourth transmembrane domain but it shows clearly distinguishable apical distribution in comparison to endogenous Na,K- ATPase.  The researchers conclude that the fourth transmembrane domain is sufficient for apical localization of a H,K- ATPase/ Na,K- ATPase chimeric protein in polar, LLC-PK1 epithelial cells.
     As it had been previously demonstrated that complexation with the beta subunit is necessary for ER export, the investigators were interested in establishing the identity of the beta subunit which colocalized with their chimeras.  Figure 3 depicts immunofluoresence results from LLC-PK1 cells transfected with chimera IV and stained with both the antibody which recognizes the chimeras as well as an antibody specific to the Na,K- ATPase beta subunit.  Chimera IV, as expected, exhibited an apical distribution.  The beta subunit was present at both the basolateral and apical membrane domains for the transfected cell line.  This result supported the investigatorsí prediction that the endogenous beta subunit would colocalize with chimera IV since it contained the carboxyl terminus half of Na,K- ATPase which had been shown to specify interaction with the beta subunit.  In addition, expression of H,K- ATPase beta subunit in LLC-PK1 cells had never been detected.
     A final chimera was constructed to see if other portions of the gastric H,K- ATPase might also induce apical distribution.  A chimera containing the second ectodomain and part of the second cytoplasmic loop but lacking the fourth transmembrane domain of H,K- ATPase exhibited apical distribution in contrast to endogenous Na,K- ATPase as illustrated by the immunofluoresence results shown in Figure 6.  From this result, the investigators draw the conclusion that H,K- ATPaseís fourth transmembrane domain is sufficient but not necessary for apical localization in these chimeras.
     Only eight of the twenty eight amino acids which form the fourth transmembrane domain differ between the apically and basolaterally directed ATPases.  Seven of these non-conserved positions were positioned at the outer leaflet of the apical membrane domain.  The outer leaflet is known to be rich in glycosphingolipids (GSLs).  Loss of GSL polarity was known to result in failure of the Na,K- ATPase delivery system to distinguish between apical and basolateral domains.  As the outer leaflet of the apical membrane was rich in GSLs and the positioning of glycophosphatidylinositol (GPI)-linked proteins had been traced to this aspect of membrane polarity, the investigators tested to see if a chimera containing the first 519 amino acids of H,K- ATPase was, like GPI-linked proteins, insoluble in Triton X-100.  As shown in figure 5a, the chimera H519n as well as endogenous Na,K- ATPase were detected in insoluble fractions of the sucrose gradient used to assay their detergent solubility while activity of GPI-linked alkaline phosphatase was detected in soluble fractions.  Figure 5b affirms, via a western blot of loaded fractions, that both endogenous and chimeric proteins resided in Triton X-100 insoluble fractions.  This result failed to support the researcherís hypothesis that H,K-ATPase localization resulted from partitioning within GSL-rich domains.
     The final set of results in this report examines the enzymatic properties of the chimeras.  The rat Na,K- ATPase used in the construction of the chimeras has a four-fold lower affinity than the endogenous protein for ouabain which inhibits the activity of Na,K- ATPase.  Cells expressing chimeras I-VII were exposed to a concentration of ouabain which is lethal to untransfected LLC-PK1 cells.  Every basolaterally distributed chimera and one apically distributed chimera, VIII, conferred ouabain resistance demonstrating that basolateral localization was not necessary for Na, K pump activity as shown in Figure 7a.  In addition, apical distribution did not inhibit all enzymatic function.  Figure 7b illustrates an enzymatic assay showing that the apical domains of cells transfected with chimera III were able to acidify their extracellular environment while basolateral domains, untransfected LLC-PK1 cells, or any inhibited with ouabain were unable to lower pH.
     The content of this report is generally convincing.  The investigators use established, well controlled methods and draw conservative conclusions from the results.  The main conclusion that the fourth transmembrane domain of H,K- ATPase is sufficient but not necessary to induce apical localization of a H,K- ATPase/ Na,K- ATPase chimera is convincing to the point that one is hard pressed to raise any serious objections.  The chimeric proteins differ from endogenous proteins in well-defined respects and immunofluoresence is conducted in a consistent manner with a basolateral marker, endogenous Na,K- ATPase, included in every panel.  However, certain aspects of the report invite criticism.  The investigators should have demonstrated that rat Na,K- ATPase, from which they derived the chimeras, localizes basolaterally since it differs in sequence from the endogenous version.  Considering that chimera VIII demonstrates that a non-contiguous region could be sufficient for apical localization, the inclusion of the eighty-five amino acid epitope tag should be acknowledged in every claim of sufficiency.
     The investigators admit that the detergent solubility assay might not remain informative when applied to multipass transmembrane proteins such as the P-type ATPases.  The failure of both the chimera and endogenous proteins to appear in detergent soluble fractions does not rule out protein/ lipid interactions.  Furthermore, the justification of this assay is questionable.  The prediction that the chimera or endogenous protein would be detergent soluble is based on the assumption that the ways in which GPI-linked proteins and P-type ATPases interact with GSLs are similar.  If GSL-rich domains are responsible for the localization of chimera 519n or any other P type ATPase, then protein transmembrane domain/ lipid interactions are expected to be responsible.  By contrast, GPI-linked protein localization in GSL-rich domains has been proposed to result from lipid/ lipid interactions.
     The research presented in this report addresses the existence of an apical sorting signal present in H,K- ATPase but absent in Na,K- ATPase.  However, as noted in the discussion, the possibility remains that apical distribution represents the default pathway for P-type ATPases and that chimeras containing either the fourth transmembrane domain of H,K- ATPase or adjoining fragments disrupt the function of a basolateral signal.  Future research might attempt to identify such a signal using a similar, chimeric approach.  However, the H,K- ATPase and Na,K- ATPase used in the construction of the chimeras both participate in the functional relationship being examined.  A negative of chimera VII in which the fourth transmembrane of H,K- ATPase is replaced by that of Na,K- ATPase would consequently still produce ambiguous results which would conclusively demonstrate neither gain nor loss of function.  A chimera in which the first 519 amino acids of H,K- ATPase or a subset of this fragment are fused to a generally directed plasma membrane protein in such a way so that the multipass configuration of the putative signal is not disturbed would validate the existence of the signal if it exhibited apical localization.  However, if the apical signal resulted from interactions between the putative signal and other parts of the alpha subunit, then the neutral portion of the chimera would not likely induce apical distribution.
     Chimeras IV and VII differ only by the source of their second ectodomain.  Chimera VIII contains the H,K- ATPase ectodomain while chimera IV contains that of Na,K- ATPase yet this segment differs at only two residues between the two source proteins.  By using PCR-based, site-directed mutagenesis to create substitutions in the chimeric ORFs in order induce a change to the alternate identity for only one residue at a time, one could identify whether a single change in one and/or the other amino acid is sufficient for apical localization or whether a change in both amino acids is required.
     A broader direction for future research might involve investigating a possible role of exclusion rather than retention signals in the maintenance of compartmental diversity for the golgi apparatus.  Specific retention signals are known to be responsible for the localization of golgi proteins in a single compartment.  However, a retention signal necessitates the existence of a receptor protein which itself must cycle between the location where it retrieves its target from and the location to which it returns its target.  The investigation of a signal which inhibits inclusion in certain membrane domains or within certain vesicles might lead to a better understanding of these processes.

     Dunbar, Lisa A., Paul Aronson, Michael J. Caplan.  2000.  A Transmembrane Segment Determines the Steady-state Localization of an Ion-transporting Adenosine Triphosphatase. J. Cell Biol.  148:769-778.
 
 
 
 

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