Studies of epithelial membrane polarity have been greatly facilitated through the use of the N-hydroxysuccinimide-biotin surface labeling technique (M. Sargiacomo, M. Lisanti, L. Graeve, A. Le Bivic, and E. Rodriguez-Boulan. J. Membr. Biol. 107: 277-286, 1989). We have used this technique in studies on the sorting and targeting of ion-transporting adenosinetriphosphatase molecules in polarized epithelial cells. Through efforts to optimize this technique in our experimental system, we have encountered several experimental conditions and circumstances where biotinylation is extremely inefficient and the assessment of membrane polarity which it provides is misleading. We demonstrate that the pH and ionic strength of the biotinylation buffer can dramatically affect biotin incorporation and that protocol-dependent variations in the recovery of biotinylated proteins can result in misrepresentation of the actual apical/basolateral distribution of a protein. Conditions and protocols that may improve the sensitivity and accuracy of this technique are discussed.
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 α subunit of the H,K-ATPase encodes localization information responsible for this pump's apical distribution, whereas the β 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 α subunit and the highly homologous Na,K-ATPase α 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 α 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 α 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.
The physiologic function of an ion transport protein is determined, in part, by its subcellular localization and by the cellular mechanisms that modulate its activity. The Na ؉ ,K ؉ -ATPase and the H ؉ ,K ؉ -ATPases are closely related members of the P-type family of ion transporting ATPases. Despite their homology, these pumps are sorted to different domains in polarized epithelial cells, and their enzymatic activities are subject to distinct regulatory pathways. The molecular signals responsible for these properties have begun to be elucidated. It appears that a complex array of inter-and intramolecular interactions govern trafficking, distribution, and catalytic capacities of these proteins.
We have generated protein chimeras to investigate the role of the fourth transmembrane segments (TM4) of the Na,K-and gastric H,K-ATPases in determining the distinct cation selectivities of these two pumps. Based on a helical wheel analysis, three residues of TM4 of the Na,K-ATPase were changed to their H,K-counterparts. A construct carrying three mutations in TM4 (L319F, N326Y, and T340S) and two control constructs were heterologously expressed in Xenopus laevis oocytes and in the pig kidney epithelial cell line LLC-PK 1 . Biochemical ATPase assays demonstrated a large sodium-independent ATPase activity at pH 6.0 for the pump carrying the TM4 substitutions, whereas the control constructs exhibited little or no activity in the absence of sodium. Furthermore, at pH 6.0 the K 1 ⁄2(Na ؉ ) shifted to 1.5 mM for the TM4 construct compared with 9.4 and 5.9 mM for the controls. In contrast, at pH 7.5 all three constructs had characteristics similar to wild type Na,K-ATPase. Large increases in K 1 ⁄2(K ؉ ) were observed for the TM4 construct compared with the control constructs both in two-electrode voltage clamp experiments in Xenopus oocytes and in ATPase assays. ATPase assays also revealed a 10-fold shift in vanadate sensitivity for the TM4 construct. Based on these findings, it appears that the three identified TM4 residues play an important role in determining both the specific cation selectivities and the E 1 /E 2 conformational equilibria of the Na,K-and H,K-ATPase.
Chimeras of the catalytic subunits of the gastric H,KATPase and Na,K-ATPase were constructed and expressed in LLC-PK 1 cells. The chimeras included the following: (i) a control, H85N (the first 85 residues comprising the cytoplasmic N terminus of Na,K-ATPase replaced by the analogous region of H,K-ATPase); (ii) H85N/H356 -519N (the N-terminal half of the cytoplasmic M4 -M5 loop also replaced); and (iii) H519N (the entire front half replaced). The latter two replacements confer a decrease in apparent affinity for extracellular K /K؉ exchange even at neutral pH. Overall, this study provides evidence for important roles in cation selectivity for both the N-terminal half of the M4 -M5 loop and the adjacent transmembrane helice(s).The gastric H,K-ATPase and ubiquitous Na,K-ATPase have the highest sequence similarity (62% amino acid sequence homology) of the phosphorylating class (P-type) of ion motive ATPases. The reaction sequence catalyzed by these enzymes involves ATP binding followed by cation-dependent phosphorylation and dephosphorylation of an aspartyl residue at the active site, as well as conformational and vectorial transitions of phospho-and dephosphoenzyme. These reactions transduce the chemical energy of ATP hydrolysis into cation binding at one side of the membrane followed by occlusion in an ionbinding pocket and then cation release at the opposite side of the membrane. Sequence similarity between these enzymes is greatest in the regions associated with ATP binding and phosphorylation (for review see Ref.
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