Localization of Cytoplasmic and Extracellular Domains of Na,K-ATPase by Epitope Tag Insertion

Abstract

We have used epitope tag addition to analyze the transmembrane topology of the Na,K-ATPase catalytic (alpha) subunit. An antigenic peptide derived from the hemagglutinin (HA) of influenza virus was inserted at 15 different positions within the rat Na,K-ATPase alpha 1 subunit isoform. The functional integrity of the tagged proteins was tested by their capacity to confer ouabain resistance upon human HEK 293 cells. Constructs with the tag at aa positions 119, 173, 318, 815, 881, 953, 987, and 1023 conferred ouabain resistance, and the mutant proteins were detectable in the plasma membrane of transfected cells. In contrast, alpha 1 subunits with insertions at aa positions 338, 797, 805, 868, 895, 910, and 921 were unable to confer drug resistance. Immunofluorescence analysis of permeabilized and intact cells using a monoclonal antibody specific for the HA epitope showed that double tags at positions 119 and 318 were located extracellularly, whereas single or double tags at positions 173, 815, 881, 987, and 1023 were cytoplasmically disposed. These results are consistent with an eight transmembrane domain arrangement for the alpha subunit. Epitope insertion within TM4, and the region linking transmembrane segments TM6-TM7, caused the loss of alpha subunit function, suggesting that the integrity of these domains is essential for the proper biosynthesis and/or maturation of the alpha subunit.

Site-directed Chemical Labeling of Extracellular Loops in a Membrane Protein

“…The amino-terminal third of the Na,K-ATPase ␣-subunit has been proposed to contain four membrane-spanning regions (7)(8)(9)(10), and the accessibility profiles of the P118C and T309C mutants confirmed the locations of Pro 118 and Thr 309 in the respective M1M2 and M3M4 extracellular loops (Figs. 2 and 3).…”

“…This suggests that their processing and folding is not affected by the substitutions. In contrast, previous topology studies employed ␣-subunit mutants that were less well characterized (due to endogenous Na,KATPase activity) (9,10) or nonfunctional (12,13), when assumptions about the "normal" orientations of the expressed proteins were made. This is particularly relevant to approaches that use carboxyl-terminal truncations and reporter groups (such as glycosylation status) to establish topology.…”

“…In the use of epitope accessibility, assumptions must be made about the effects of permeablilizing detergents, i.e. that such detergent treatments do not alter protein structure (9,10,16). These methods are particularly prone to provide misleading information with a protein such as the Na,K-ATPase, where abundant evidence exists demonstrating the mobility or flexibility of its carboxyl-terminal regions.…”

“…The secondary structural information on the protein, on the other hand, remains controversial despite extensive investigation; such information is essential for understanding structure/function relationships of the Na,K-ATPase. Based on hydropathy analysis (6), protease accessibility (7,8), and immunochemical studies (9,10), the amino-terminal third of the ␣-subunit appears to contain four membrane-spanning regions. These regions are followed by a large cytoplasmic loop that has been identified as the ATP-binding domain.…”

“…This loop has recently been overexpressed in Escherichia coli and exhibits the same nucleotide-binding specificities as native Na,KATPase (11). Studies on the carboxyl-terminal third of the ␣-subunit have produced conflicting results (7)(8)(9)(10)(12)(13)(14)(15). Recent data using epitope accessibility claim to establish four transmembrane segments in the carboxyl-terminal region and have a total of eight transmembrane segments (12), whereas several other studies claim to establish that there are 10 transmembrane segments (13)(14)(15).…”