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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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Several chimeras appear to be enzymatically active. The ability of chimeras to substitute for the sodium transporting activity of the endogenous Na,K-ATPase was assessed by determining whether expression of the chimera conferred ouabain resistance. All of the basolateral chimeras appears to be active as sodium pumps. However, the presence of Na,K-ATPase activity does not correlate with the steady-state distribution of the chimeras, since the apical chimera VIII confers ouabain resistance (A). The apically located chimera III also appears to be active, although it does not confer ouabain resistance. Expression of this apical chimera results in the acidification of the apical media when cells expressing this protein are grown on porous filters (B). Proton efflux was assessed by monitoring the change in pH of weakly buffered media bathing the apical or basolateral surfaces of transfected or untransfected LLC-PK1 cells after 2 (open bars) or 4 h (filled bars) of incubation. The acidification appears to be due to expression of the chimera, since the magnitude of the acidification of the apical media is not seen in untransfected LLC-PK1 cells and it is inhibited by high concentrations of ouabain added to the apical media as shown on the right.
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Figure 7: Several chimeras appear to be enzymatically active. The ability of chimeras to substitute for the sodium transporting activity of the endogenous Na,K-ATPase was assessed by determining whether expression of the chimera conferred ouabain resistance. All of the basolateral chimeras appears to be active as sodium pumps. However, the presence of Na,K-ATPase activity does not correlate with the steady-state distribution of the chimeras, since the apical chimera VIII confers ouabain resistance (A). The apically located chimera III also appears to be active, although it does not confer ouabain resistance. Expression of this apical chimera results in the acidification of the apical media when cells expressing this protein are grown on porous filters (B). Proton efflux was assessed by monitoring the change in pH of weakly buffered media bathing the apical or basolateral surfaces of transfected or untransfected LLC-PK1 cells after 2 (open bars) or 4 h (filled bars) of incubation. The acidification appears to be due to expression of the chimera, since the magnitude of the acidification of the apical media is not seen in untransfected LLC-PK1 cells and it is inhibited by high concentrations of ouabain added to the apical media as shown on the right.

Mentions: 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 (Blostein et al. 1999).


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

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

Several chimeras appear to be enzymatically active. The ability of chimeras to substitute for the sodium transporting activity of the endogenous Na,K-ATPase was assessed by determining whether expression of the chimera conferred ouabain resistance. All of the basolateral chimeras appears to be active as sodium pumps. However, the presence of Na,K-ATPase activity does not correlate with the steady-state distribution of the chimeras, since the apical chimera VIII confers ouabain resistance (A). The apically located chimera III also appears to be active, although it does not confer ouabain resistance. Expression of this apical chimera results in the acidification of the apical media when cells expressing this protein are grown on porous filters (B). Proton efflux was assessed by monitoring the change in pH of weakly buffered media bathing the apical or basolateral surfaces of transfected or untransfected LLC-PK1 cells after 2 (open bars) or 4 h (filled bars) of incubation. The acidification appears to be due to expression of the chimera, since the magnitude of the acidification of the apical media is not seen in untransfected LLC-PK1 cells and it is inhibited by high concentrations of ouabain added to the apical media as shown on the right.
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Related In: Results  -  Collection

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Figure 7: Several chimeras appear to be enzymatically active. The ability of chimeras to substitute for the sodium transporting activity of the endogenous Na,K-ATPase was assessed by determining whether expression of the chimera conferred ouabain resistance. All of the basolateral chimeras appears to be active as sodium pumps. However, the presence of Na,K-ATPase activity does not correlate with the steady-state distribution of the chimeras, since the apical chimera VIII confers ouabain resistance (A). The apically located chimera III also appears to be active, although it does not confer ouabain resistance. Expression of this apical chimera results in the acidification of the apical media when cells expressing this protein are grown on porous filters (B). Proton efflux was assessed by monitoring the change in pH of weakly buffered media bathing the apical or basolateral surfaces of transfected or untransfected LLC-PK1 cells after 2 (open bars) or 4 h (filled bars) of incubation. The acidification appears to be due to expression of the chimera, since the magnitude of the acidification of the apical media is not seen in untransfected LLC-PK1 cells and it is inhibited by high concentrations of ouabain added to the apical media as shown on the right.
Mentions: 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 (Blostein et al. 1999).

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