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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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Detergent solubility of an apical pump chimera. A detergent solubility assay was performed to determine whether the apically located chimera H519N is associated with glycolipid-rich membrane domains. Cells were lysed on ice with Triton X-100 and loaded onto a sucrose floatation gradient. Fractions were collected and examined for the presence of alkaline phosphatase activity, the chimera, and Na,K-ATPase. As shown in A, the endogenous GPI-linked alkaline phosphatase is found in the lighter fractions of the gradient 2–4, as is typical for proteins associated with glycolipid-rich membranes (Arreaza et al. 1994). Western blotting reveals that both the chimera and the endogenous Na,K-ATPase appear in the heavier fractions 6–10 (B), which is characteristic of soluble proteins. The chimera α subunit runs as both a monomer (lower band) and a higher molecular weight α/β dimer. Densitometric quantification of the blots (A) clearly demonstrates that the chimera (squares) does not colocalize with the alkaline phosphatase (diamonds), and therefore is probably not associated with GSL-rich membrane domains; instead, the chimera is found in the same fractions as the Na,K-ATPase (triangles). The experiment presented in this figure is typical of three independent trials.
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Figure 5: Detergent solubility of an apical pump chimera. A detergent solubility assay was performed to determine whether the apically located chimera H519N is associated with glycolipid-rich membrane domains. Cells were lysed on ice with Triton X-100 and loaded onto a sucrose floatation gradient. Fractions were collected and examined for the presence of alkaline phosphatase activity, the chimera, and Na,K-ATPase. As shown in A, the endogenous GPI-linked alkaline phosphatase is found in the lighter fractions of the gradient 2–4, as is typical for proteins associated with glycolipid-rich membranes (Arreaza et al. 1994). Western blotting reveals that both the chimera and the endogenous Na,K-ATPase appear in the heavier fractions 6–10 (B), which is characteristic of soluble proteins. The chimera α subunit runs as both a monomer (lower band) and a higher molecular weight α/β dimer. Densitometric quantification of the blots (A) clearly demonstrates that the chimera (squares) does not colocalize with the alkaline phosphatase (diamonds), and therefore is probably not associated with GSL-rich membrane domains; instead, the chimera is found in the same fractions as the Na,K-ATPase (triangles). The experiment presented in this figure is typical of three independent trials.

Mentions: GPI-linked proteins that have become associated with GSL-rich membrane domains are insoluble in 1% Triton X-100 at 4°C. When a cell lysate prepared in this fashion is fractionated on a sucrose floatation gradient, insoluble proteins are found near the top of the gradient, whereas soluble proteins remain in the heavier fractions (Arreaza et al. 1994). We lysed LLC-PK1 cells expressing the apically located chimera H519N on ice with 1% Triton X-100, and examined the distribution of the chimera, the endogenous Na,K-ATPase, and the endogenous GPI-linked alkaline phosphatase in fractions collected from a sucrose floatation gradient. Lysis took place in the presence of sodium carbonate to disrupt any cytoskeletal associations that could influence the solubility of the ion pumps. An alkaline phosphatase assay performed on each fraction shows that the enzymatic activity of this endogenous GPI-linked protein is restricted to fractions 2–4 (Fig. 5 A). These fractions correspond to the interface between the 5 and 35% sucrose layers, where GPI-linked proteins are expected to be found based on published reports (Arreaza et al. 1994). The endogenous Na,K-ATPase on the other hand, appears in the heavier fractions 6–10, a pattern typical for detergent-soluble membrane proteins (Fig. 5A and Fig. B). If the chimera containing TM4 of the gastric H,K-ATPase partitions into insoluble glycolipid patches, we would have expected it to codistribute with the GPI-linked alkaline phosphatase. Instead, the chimera is found in the same fractions as the Na,K-ATPase, and is not present in any significant amounts in the fractions containing alkaline phosphatase activity (Fig. 5A and Fig. B). Thus, under steady-state conditions, an apically located chimera containing the TM4 of the H,K-ATPase exhibits no difference in its detergent solubility characteristics as compared with the basolaterally located Na,K-ATPase. Furthermore, no difference in solubility between the endogenous Na,K-ATPase and apically located chimeras is detected when lower concentrations of detergent are used (data not shown). Finally, the endogenous gastric H,K-ATPase of mouse stomach mucosa is also fully soluble under these detergent extraction conditions. These results suggest that mechanisms other than lipid association may be responsible for the sorting function of the transmembrane domain.


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

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

Detergent solubility of an apical pump chimera. A detergent solubility assay was performed to determine whether the apically located chimera H519N is associated with glycolipid-rich membrane domains. Cells were lysed on ice with Triton X-100 and loaded onto a sucrose floatation gradient. Fractions were collected and examined for the presence of alkaline phosphatase activity, the chimera, and Na,K-ATPase. As shown in A, the endogenous GPI-linked alkaline phosphatase is found in the lighter fractions of the gradient 2–4, as is typical for proteins associated with glycolipid-rich membranes (Arreaza et al. 1994). Western blotting reveals that both the chimera and the endogenous Na,K-ATPase appear in the heavier fractions 6–10 (B), which is characteristic of soluble proteins. The chimera α subunit runs as both a monomer (lower band) and a higher molecular weight α/β dimer. Densitometric quantification of the blots (A) clearly demonstrates that the chimera (squares) does not colocalize with the alkaline phosphatase (diamonds), and therefore is probably not associated with GSL-rich membrane domains; instead, the chimera is found in the same fractions as the Na,K-ATPase (triangles). The experiment presented in this figure is typical of three independent trials.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2169368&req=5

Figure 5: Detergent solubility of an apical pump chimera. A detergent solubility assay was performed to determine whether the apically located chimera H519N is associated with glycolipid-rich membrane domains. Cells were lysed on ice with Triton X-100 and loaded onto a sucrose floatation gradient. Fractions were collected and examined for the presence of alkaline phosphatase activity, the chimera, and Na,K-ATPase. As shown in A, the endogenous GPI-linked alkaline phosphatase is found in the lighter fractions of the gradient 2–4, as is typical for proteins associated with glycolipid-rich membranes (Arreaza et al. 1994). Western blotting reveals that both the chimera and the endogenous Na,K-ATPase appear in the heavier fractions 6–10 (B), which is characteristic of soluble proteins. The chimera α subunit runs as both a monomer (lower band) and a higher molecular weight α/β dimer. Densitometric quantification of the blots (A) clearly demonstrates that the chimera (squares) does not colocalize with the alkaline phosphatase (diamonds), and therefore is probably not associated with GSL-rich membrane domains; instead, the chimera is found in the same fractions as the Na,K-ATPase (triangles). The experiment presented in this figure is typical of three independent trials.
Mentions: GPI-linked proteins that have become associated with GSL-rich membrane domains are insoluble in 1% Triton X-100 at 4°C. When a cell lysate prepared in this fashion is fractionated on a sucrose floatation gradient, insoluble proteins are found near the top of the gradient, whereas soluble proteins remain in the heavier fractions (Arreaza et al. 1994). We lysed LLC-PK1 cells expressing the apically located chimera H519N on ice with 1% Triton X-100, and examined the distribution of the chimera, the endogenous Na,K-ATPase, and the endogenous GPI-linked alkaline phosphatase in fractions collected from a sucrose floatation gradient. Lysis took place in the presence of sodium carbonate to disrupt any cytoskeletal associations that could influence the solubility of the ion pumps. An alkaline phosphatase assay performed on each fraction shows that the enzymatic activity of this endogenous GPI-linked protein is restricted to fractions 2–4 (Fig. 5 A). These fractions correspond to the interface between the 5 and 35% sucrose layers, where GPI-linked proteins are expected to be found based on published reports (Arreaza et al. 1994). The endogenous Na,K-ATPase on the other hand, appears in the heavier fractions 6–10, a pattern typical for detergent-soluble membrane proteins (Fig. 5A and Fig. B). If the chimera containing TM4 of the gastric H,K-ATPase partitions into insoluble glycolipid patches, we would have expected it to codistribute with the GPI-linked alkaline phosphatase. Instead, the chimera is found in the same fractions as the Na,K-ATPase, and is not present in any significant amounts in the fractions containing alkaline phosphatase activity (Fig. 5A and Fig. B). Thus, under steady-state conditions, an apically located chimera containing the TM4 of the H,K-ATPase exhibits no difference in its detergent solubility characteristics as compared with the basolaterally located Na,K-ATPase. Furthermore, no difference in solubility between the endogenous Na,K-ATPase and apically located chimeras is detected when lower concentrations of detergent are used (data not shown). Finally, the endogenous gastric H,K-ATPase of mouse stomach mucosa is also fully soluble under these detergent extraction conditions. These results suggest that mechanisms other than lipid association may be responsible for the sorting function of the transmembrane domain.

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