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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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Amino acid sequence comparison. The linear sequences of the predicted second ectodomains (between TM3 and TM4) and the TM4 of the gastric H,K-ATPase and Na,K-ATPase α subunits are shown in A. Nonidentical amino acids within the ectodomain and TM4 are boxed. The amino acids corresponding to the junction points of the chimeras are shown by arrowheads. The locations of the eight nonidentical amino acids (black bars) within TM4 are shown in the context of the α subunit's predicted membrane topology in B.
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Figure 4: Amino acid sequence comparison. The linear sequences of the predicted second ectodomains (between TM3 and TM4) and the TM4 of the gastric H,K-ATPase and Na,K-ATPase α subunits are shown in A. Nonidentical amino acids within the ectodomain and TM4 are boxed. The amino acids corresponding to the junction points of the chimeras are shown by arrowheads. The locations of the eight nonidentical amino acids (black bars) within TM4 are shown in the context of the α subunit's predicted membrane topology in B.

Mentions: Comparison of the sequences of the TM4s of the two ion pump α subunits shows surprisingly little nonhomology, considering that this region can mediate the strikingly different membrane distributions exhibited by these proteins (Fig. 4 A). Of the 28 amino acids that comprise the putative transmembrane domain, only 8 are nonidentical. Seven of the nonconserved amino acids in this segment are found in the portion of the transmembrane domain that is predicted to pass through the outer leaflet of the lipid bilayer (Fig. 4 B). The outer leaflet of the apical plasma membrane of many epithelial cell types is enriched in glycosphingolipids (GSLs). Furthermore, there is evidence that GPI-linked proteins, as well as other apical polypeptides, are incorporated into GSL-rich domains during their biosynthetic passage through the Golgi complex (Brown and Rose 1992). This interaction has been proposed to mediate their targeting to the apical plasma membrane (Simons and Wandinger-Ness 1990). It has also been suggested that glycolipid sorting may be involved in the polarized delivery of the Na,K-ATPase to the plasma membrane (Mays et al. 1995). An MDCK line that lacks GSL polarity also mistargets the Na,K-ATPase. In this cell line, the Na,K-ATPase is delivered equally to both the apical and basolateral membrane. The population at the apical membrane is subsequently degraded, whereas the cohort delivered to the basolateral surface is stabilized, apparently through interactions with the cytoskeleton. The mistargeting of the Na,K-ATPase seen in this cell line may be directly linked to the lack of GSL polarity, since fumonisin, a drug that disrupts GSL synthesis, causes the same random pump delivery when applied to MDCK cell lines that ordinarily have a polarized distribution of GSLs. These experiments suggest that the Na,K-ATPase is normally exclusively sorted to the basolateral membrane by virtue of its exclusion from glycosphingolipid-rich membrane patches. We wondered whether TM4 of the H,K-ATPase exerts its effect on sorting by allowing the protein to partition into these GSL-rich domains.


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

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

Amino acid sequence comparison. The linear sequences of the predicted second ectodomains (between TM3 and TM4) and the TM4 of the gastric H,K-ATPase and Na,K-ATPase α subunits are shown in A. Nonidentical amino acids within the ectodomain and TM4 are boxed. The amino acids corresponding to the junction points of the chimeras are shown by arrowheads. The locations of the eight nonidentical amino acids (black bars) within TM4 are shown in the context of the α subunit's predicted membrane topology in B.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 4: Amino acid sequence comparison. The linear sequences of the predicted second ectodomains (between TM3 and TM4) and the TM4 of the gastric H,K-ATPase and Na,K-ATPase α subunits are shown in A. Nonidentical amino acids within the ectodomain and TM4 are boxed. The amino acids corresponding to the junction points of the chimeras are shown by arrowheads. The locations of the eight nonidentical amino acids (black bars) within TM4 are shown in the context of the α subunit's predicted membrane topology in B.
Mentions: Comparison of the sequences of the TM4s of the two ion pump α subunits shows surprisingly little nonhomology, considering that this region can mediate the strikingly different membrane distributions exhibited by these proteins (Fig. 4 A). Of the 28 amino acids that comprise the putative transmembrane domain, only 8 are nonidentical. Seven of the nonconserved amino acids in this segment are found in the portion of the transmembrane domain that is predicted to pass through the outer leaflet of the lipid bilayer (Fig. 4 B). The outer leaflet of the apical plasma membrane of many epithelial cell types is enriched in glycosphingolipids (GSLs). Furthermore, there is evidence that GPI-linked proteins, as well as other apical polypeptides, are incorporated into GSL-rich domains during their biosynthetic passage through the Golgi complex (Brown and Rose 1992). This interaction has been proposed to mediate their targeting to the apical plasma membrane (Simons and Wandinger-Ness 1990). It has also been suggested that glycolipid sorting may be involved in the polarized delivery of the Na,K-ATPase to the plasma membrane (Mays et al. 1995). An MDCK line that lacks GSL polarity also mistargets the Na,K-ATPase. In this cell line, the Na,K-ATPase is delivered equally to both the apical and basolateral membrane. The population at the apical membrane is subsequently degraded, whereas the cohort delivered to the basolateral surface is stabilized, apparently through interactions with the cytoskeleton. The mistargeting of the Na,K-ATPase seen in this cell line may be directly linked to the lack of GSL polarity, since fumonisin, a drug that disrupts GSL synthesis, causes the same random pump delivery when applied to MDCK cell lines that ordinarily have a polarized distribution of GSLs. These experiments suggest that the Na,K-ATPase is normally exclusively sorted to the basolateral membrane by virtue of its exclusion from glycosphingolipid-rich membrane patches. We wondered whether TM4 of the H,K-ATPase exerts its effect on sorting by allowing the protein to partition into these GSL-rich domains.

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