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A transmembrane segment determines the steady-state localization of an ion-transporting adenosine triphosphatase.

Dunbar LA, Aronson P, Caplan MJ - J. Cell Biol. (2000)

Bottom Line: 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 alpha 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, Connecticut 06510, USA. ldunbar@biomed.med.yale.edu

ABSTRACT
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 alpha subunit of the H, K-ATPase encodes localization information responsible for this pump's apical distribution, whereas the beta 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 alpha subunit and the highly homologous Na,K-ATPase alpha 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 alpha 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 alpha 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.

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Localization of chimeras I–III in LLC-PK1 cells. Immunofluorescence was performed on LLC-PK1 cell lines stably expressing each chimera using antibodies that recognize the chimera (A, C, E, G, I, and K) or the endogenous Na,K-ATPase (B, D, F, H, J, and L). The structure of each chimera is shown on the left of the panel in which its localization is depicted. The Na,K-ATPase portions are shown in gray and the H,K-ATPase portions are shown in black. As can be seen above, the Na,K-ATPase is basolateral in all of the cell lines when viewed en face (B, F, and J) and in xz cross-section (D, H, and L). Chimeras I and II are located at the basolateral membrane as seen en face (A and E) and in xz cross section (C and G). However, chimera III demonstrates an apical staining pattern shown en face (I) and in xz cross section (K), suggesting that the H,K-ATPase sequence incorporated in this chimera contains apical localization information.
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Figure 1: Localization of chimeras I–III in LLC-PK1 cells. Immunofluorescence was performed on LLC-PK1 cell lines stably expressing each chimera using antibodies that recognize the chimera (A, C, E, G, I, and K) or the endogenous Na,K-ATPase (B, D, F, H, J, and L). The structure of each chimera is shown on the left of the panel in which its localization is depicted. The Na,K-ATPase portions are shown in gray and the H,K-ATPase portions are shown in black. As can be seen above, the Na,K-ATPase is basolateral in all of the cell lines when viewed en face (B, F, and J) and in xz cross-section (D, H, and L). Chimeras I and II are located at the basolateral membrane as seen en face (A and E) and in xz cross section (C and G). However, chimera III demonstrates an apical staining pattern shown en face (I) and in xz cross section (K), suggesting that the H,K-ATPase sequence incorporated in this chimera contains apical localization information.

Mentions: A comparison of the amino acid sequences of the Na,K- and H,K-ATPase α 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 α subunit is 13 amino acids longer than that of the Na,K-ATPase α 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.


A transmembrane segment determines the steady-state localization of an ion-transporting adenosine triphosphatase.

Dunbar LA, Aronson P, Caplan MJ - J. Cell Biol. (2000)

Localization of chimeras I–III in LLC-PK1 cells. Immunofluorescence was performed on LLC-PK1 cell lines stably expressing each chimera using antibodies that recognize the chimera (A, C, E, G, I, and K) or the endogenous Na,K-ATPase (B, D, F, H, J, and L). The structure of each chimera is shown on the left of the panel in which its localization is depicted. The Na,K-ATPase portions are shown in gray and the H,K-ATPase portions are shown in black. As can be seen above, the Na,K-ATPase is basolateral in all of the cell lines when viewed en face (B, F, and J) and in xz cross-section (D, H, and L). Chimeras I and II are located at the basolateral membrane as seen en face (A and E) and in xz cross section (C and G). However, chimera III demonstrates an apical staining pattern shown en face (I) and in xz cross section (K), suggesting that the H,K-ATPase sequence incorporated in this chimera contains apical localization information.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2169368&req=5

Figure 1: Localization of chimeras I–III in LLC-PK1 cells. Immunofluorescence was performed on LLC-PK1 cell lines stably expressing each chimera using antibodies that recognize the chimera (A, C, E, G, I, and K) or the endogenous Na,K-ATPase (B, D, F, H, J, and L). The structure of each chimera is shown on the left of the panel in which its localization is depicted. The Na,K-ATPase portions are shown in gray and the H,K-ATPase portions are shown in black. As can be seen above, the Na,K-ATPase is basolateral in all of the cell lines when viewed en face (B, F, and J) and in xz cross-section (D, H, and L). Chimeras I and II are located at the basolateral membrane as seen en face (A and E) and in xz cross section (C and G). However, chimera III demonstrates an apical staining pattern shown en face (I) and in xz cross section (K), suggesting that the H,K-ATPase sequence incorporated in this chimera contains apical localization information.
Mentions: A comparison of the amino acid sequences of the Na,K- and H,K-ATPase α 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 α subunit is 13 amino acids longer than that of the Na,K-ATPase α 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.

Bottom Line: 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 alpha 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, Connecticut 06510, USA. ldunbar@biomed.med.yale.edu

ABSTRACT
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 alpha subunit of the H, K-ATPase encodes localization information responsible for this pump's apical distribution, whereas the beta 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 alpha subunit and the highly homologous Na,K-ATPase alpha 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 alpha 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 alpha 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.

Show MeSH
Related in: MedlinePlus