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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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A chimera lacking the TM4 of the gastric H,K-ATPase is also found at the apical membrane. Cells expressing chimera VIII were fixed and stained as in Fig. 1. Chimera VIII is found predominantly at the apical membrane (A and C), demonstrating that the TM4 of the gastric H,K-ATPase is not necessary to ensure apical localization in LLC-PK1 cells.
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Figure 6: A chimera lacking the TM4 of the gastric H,K-ATPase is also found at the apical membrane. Cells expressing chimera VIII were fixed and stained as in Fig. 1. Chimera VIII is found predominantly at the apical membrane (A and C), demonstrating that the TM4 of the gastric H,K-ATPase is not necessary to ensure apical localization in LLC-PK1 cells.

Mentions: It has been shown that both the Na,K-ATPase and H,K-ATPase α subunits must associate with their respective β subunits in order to leave the ER and be transported to the plasma membrane. The α subunit chimeras presented here contain the COOH-terminal half of the Na,K-ATPase, which has been shown to determine specificity in β subunit assembly (Gottardi and Caplan 1993b). Therefore, we would expect these chimeras to dimerize with the Na,K-ATPase β polypeptide. Furthermore, no endogenous H,K-ATPase β subunit has been detected in LLC-PK1 cells by immunofluorescence. In the case of cells expressing apically localized chimeras, which incorporate the COOH-terminal half of Na,K-ATPase α subunit, we would expect that the Na,K-ATPase β subunit would assemble in the ER with both the Na,K-ATPase α subunit and chimeric α 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 β 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 β subunit protein, and like the first chimera H519N, redirect this normally basolateral protein to the apical surface.


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

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

A chimera lacking the TM4 of the gastric H,K-ATPase is also found at the apical membrane. Cells expressing chimera VIII were fixed and stained as in Fig. 1. Chimera VIII is found predominantly at the apical membrane (A and C), demonstrating that the TM4 of the gastric H,K-ATPase is not necessary to ensure apical localization in LLC-PK1 cells.
© Copyright Policy
Related In: Results  -  Collection

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Figure 6: A chimera lacking the TM4 of the gastric H,K-ATPase is also found at the apical membrane. Cells expressing chimera VIII were fixed and stained as in Fig. 1. Chimera VIII is found predominantly at the apical membrane (A and C), demonstrating that the TM4 of the gastric H,K-ATPase is not necessary to ensure apical localization in LLC-PK1 cells.
Mentions: It has been shown that both the Na,K-ATPase and H,K-ATPase α subunits must associate with their respective β subunits in order to leave the ER and be transported to the plasma membrane. The α subunit chimeras presented here contain the COOH-terminal half of the Na,K-ATPase, which has been shown to determine specificity in β subunit assembly (Gottardi and Caplan 1993b). Therefore, we would expect these chimeras to dimerize with the Na,K-ATPase β polypeptide. Furthermore, no endogenous H,K-ATPase β subunit has been detected in LLC-PK1 cells by immunofluorescence. In the case of cells expressing apically localized chimeras, which incorporate the COOH-terminal half of Na,K-ATPase α subunit, we would expect that the Na,K-ATPase β subunit would assemble in the ER with both the Na,K-ATPase α subunit and chimeric α 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 β 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 β subunit protein, and like the first chimera H519N, redirect this normally basolateral protein to the apical surface.

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