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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 IV–VII. LLC-PK1 cells stably expressing chimeras IV–VII were stained as in Fig. 1. Confocal images were generated to show the localization of the chimeras (A, C, E, G, I, K, M, and O) or the endogenous Na,K-ATPase (B, D, F, H, J, L, N, and P). The Na,K-ATPase is localized to the basolateral membrane in all cell lines as shown in en face (B, F, J, and N) and xz cross sections (D, H, L, and P). Chimeras IV and VI are also localized to the basolateral membrane seen en face (A and I) and in xz cross section (C and K). In contrast, chimeras V and VII display a predominantly apical distribution when viewed en face (E and M) and in xz cross section (G and O), demonstrating that the TM4 of the H,K-ATPase is sufficient for apical localization in LLC-PK1 cells.
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Figure 2: Localization of chimeras IV–VII. LLC-PK1 cells stably expressing chimeras IV–VII were stained as in Fig. 1. Confocal images were generated to show the localization of the chimeras (A, C, E, G, I, K, M, and O) or the endogenous Na,K-ATPase (B, D, F, H, J, L, N, and P). The Na,K-ATPase is localized to the basolateral membrane in all cell lines as shown in en face (B, F, J, and N) and xz cross sections (D, H, L, and P). Chimeras IV and VI are also localized to the basolateral membrane seen en face (A and I) and in xz cross section (C and K). In contrast, chimeras V and VII display a predominantly apical distribution when viewed en face (E and M) and in xz cross section (G and O), demonstrating that the TM4 of the H,K-ATPase is sufficient for apical localization in LLC-PK1 cells.

Mentions: We further dissected the region between amino acids 324 and 519 by examining two chimeras consisting of the 85–amino acid epitope tag and H,K-ATPase sequence between either amino acids 356 and 519 (Fig. 2, chimera IV) or amino acids 324 through 356 (Fig. 2, chimera V). The chimera containing H,K-ATPase sequence between amino acids 356 and 519, which corresponds to part of the large cytoplasmic loop, resides at the basolateral membrane in transfected LLC-PK1 cells (Fig. 2A and Fig. C). The second chimera (Fig. 2, chimera V) on the other hand, is predominantly localized to the apical membrane when it is expressed in LLC-PK1 cells (Fig. 2E and Fig. G) as seen from the microvillar staining. Therefore, the second ectodomain loop and TM4 of the gastric proton pump appear to contain information that is sufficient to allow the chimeric ATPase to reach the apical membrane.


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 IV–VII. LLC-PK1 cells stably expressing chimeras IV–VII were stained as in Fig. 1. Confocal images were generated to show the localization of the chimeras (A, C, E, G, I, K, M, and O) or the endogenous Na,K-ATPase (B, D, F, H, J, L, N, and P). The Na,K-ATPase is localized to the basolateral membrane in all cell lines as shown in en face (B, F, J, and N) and xz cross sections (D, H, L, and P). Chimeras IV and VI are also localized to the basolateral membrane seen en face (A and I) and in xz cross section (C and K). In contrast, chimeras V and VII display a predominantly apical distribution when viewed en face (E and M) and in xz cross section (G and O), demonstrating that the TM4 of the H,K-ATPase is sufficient for apical localization in LLC-PK1 cells.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 2: Localization of chimeras IV–VII. LLC-PK1 cells stably expressing chimeras IV–VII were stained as in Fig. 1. Confocal images were generated to show the localization of the chimeras (A, C, E, G, I, K, M, and O) or the endogenous Na,K-ATPase (B, D, F, H, J, L, N, and P). The Na,K-ATPase is localized to the basolateral membrane in all cell lines as shown in en face (B, F, J, and N) and xz cross sections (D, H, L, and P). Chimeras IV and VI are also localized to the basolateral membrane seen en face (A and I) and in xz cross section (C and K). In contrast, chimeras V and VII display a predominantly apical distribution when viewed en face (E and M) and in xz cross section (G and O), demonstrating that the TM4 of the H,K-ATPase is sufficient for apical localization in LLC-PK1 cells.
Mentions: We further dissected the region between amino acids 324 and 519 by examining two chimeras consisting of the 85–amino acid epitope tag and H,K-ATPase sequence between either amino acids 356 and 519 (Fig. 2, chimera IV) or amino acids 324 through 356 (Fig. 2, chimera V). The chimera containing H,K-ATPase sequence between amino acids 356 and 519, which corresponds to part of the large cytoplasmic loop, resides at the basolateral membrane in transfected LLC-PK1 cells (Fig. 2A and Fig. C). The second chimera (Fig. 2, chimera V) on the other hand, is predominantly localized to the apical membrane when it is expressed in LLC-PK1 cells (Fig. 2E and Fig. G) as seen from the microvillar staining. Therefore, the second ectodomain loop and TM4 of the gastric proton pump appear to contain information that is sufficient to allow the chimeric ATPase to reach the apical membrane.

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