Membrane Topology of the α Subunit of Na,K-ATPase

Mohraz, Manijeh; Arystarkhova, Elena; Sweadner, Kathleen J.

doi:10.1007/978-3-642-72511-1_58

Cited by 8 publications

(17 citation statements)

References 8 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…5). The amino acid Met 809 is localized within the sequence 809 MVPAISLAYEQAESDIM 825 , which was shown previously by the use of a specific antibody to be at least in part extracellular (13,20 (12) are localized on the cytosolic side, supports the conclusion that there must be a membrane span between Met 809 and these amino acids. In our interpretation, the area between Met 809 and Asn 831 would include the M6 part of the ␣ 1 -subunit (Fig.…”

supporting

confidence: 74%

“…Although this approach is most promising for the analysis of transmembrane topology, the results obtained can be contro-versial. For example, the sequence 887 WINDVEDSYGQQW-TYEQR 904 of the ␣-subunit from pig kidney was detected on either the extra-or intracellular side of the plasma membrane using the same IIC9 antibody in different investigations (13,20). In addition, the use of antibodies against artificial epitopes that were inserted by molecular biological methods is not always conclusive.…”

Section: Namentioning

confidence: 99%

“…The transmembrane topology of the ␣-subunit has also been analyzed by the use of antibodies directed against either naturally occurring or artificially inserted epitopes (13,15,20,21). Although this approach is most promising for the analysis of transmembrane topology, the results obtained can be contro-versial.…”

Section: Namentioning

confidence: 99%

See 2 more Smart Citations

Transmembrane Topology of α- and β-Subunits of Na+,K+-ATPase Derived from β-Galactosidase Fusion Proteins Expressed in Yeast

Fiedler¹,

Scheiner‐Bobis

1996

Journal of Biological Chemistry

View full text Add to dashboard Cite

Various models of the transmembrane topology of the Na ؉ ,K ؉ -ATPase predict either 8 or 10 membrane spans for the ␣-subunit and one to three membrane spans for the ␤-subunit. Structure/function analysis, however, requires precise knowledge about the folding of enzymes. Therefore, the intention of this work was to establish a transmembrane topology model for the subunits of Na LGGYP 236 ) of the dog ␤-subunit. The fusion constructs were expressed in yeast cells for studies on the localization of the fused reporter enzyme. Activity measurements of the reporter enzyme revealed that only intracellular fusion sites lead to active ␤-galactosidase. Indirect immunofluorescence microscopy with cells expressing ␣ 1 /␤-galactosidase and ␤/␤-galactosidase hybrid proteins demonstrated that inactive ␤-galactosidase is associated with the yeast plasma membrane and can be detected from the extracellular side. The data obtained suggest that Pro 236 of the ␤-subunit is located on the extracellular surface, corresponding to a model with one transmembrane segment, and that the ␣-subunit of the Na ؉ ,K ؉ -ATPase consists of 10 membrane-associated spans. They also suggest that a stretch of the ␣ 1 -subunit between membrane spans M7 and M8 might be hidden within the membrane, surrounded by the other hydrophobic spans, in analogy to the P-loop of Na ؉ or K ؉ channels and to the "hourglass" structure of water channels.

show abstract

supporting

confidence: 74%

Section: Namentioning

confidence: 99%

See 1 more Smart Citation

Transmembrane Topology of α- and β-Subunits of Na+,K+-ATPase Derived from β-Galactosidase Fusion Proteins Expressed in Yeast

Fiedler¹,

Scheiner‐Bobis

1996

Journal of Biological Chemistry

View full text Add to dashboard Cite

show abstract

“…Gin 9°3 in the Na,K-ATPase c( subunit of basolateral membranes in intact mTAL cells, consistent with our previous report on isolated membranes [1] and some immunological data reported by others [16,17].…”

Section: P-facesupporting

confidence: 91%

Epitope topology of Na,K‐ATPase α subunit analyzed in basolateral cell membranes of rat kidney tubules

1996

View full text Add to dashboard Cite

were processed for immunocytochemistry. Immunogold labeling using antibodies against the N-termlnus (Glyl-Hisl3), Leu siSGin s28 and the C-terminus (Ilel°°2-Tyr t°°6) was superimposed on 2. Materials and methods the images of the electron microscope protoplasmic fracture face of the basolateral plasma membranes, thus demonstrating Tissue preparation and quick:[reezingThe inner stripe of the outer medulla of rat kidneys was cut into cytoplasmic locations of these epitopes. On the contrary, SDSsections less than 100 ~m thick by a Microslicer (Dosaka EM Co.,

show abstract

“…It is interesting to note that previous immunochemical labeling studies using an antibody raised against the Trp 887 -Arg 904 sequence have produced conflicting results. In one study, the antibody detected Na,K-ATPase from the extracellular domain after prolonged incubation (36). In that study three new models were suggested for the transmembrane organization of the Na,KATPase ␣-subunit, consisting of either 8 or 10 transmembrane segments.…”

Section: Figmentioning

confidence: 99%

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

Kaplan

2000

Journal of Biological Chemistry

View full text Add to dashboard Cite

We have mapped the membrane topology of the renal Na,K-ATPase ␣-subunit by using a combination of introduced cysteine mutants and surface labeling with a membrane impermeable Cys-directed reagent, N-biotinylaminoethyl methanethiosulfonate. To begin our investigation, two cysteine residues (Cys 911 and Cys 964 ) in the wild-type ␣-subunit were substituted to create a background mutant devoid of exposed cysteines (Lutsenko, S., Daoud, S., and Kaplan, J. H. (1997) J. Biol. Chem. 272, 5249 -5255). Into this background construct were then introduced single cysteines in each of the five putative extracellular loops (P118C, T309C, L793C, L876C, and M973C) and the resulting ␣-subunit mutants were co-expressed with the ␤-subunit in baculovirusinfected insect cells. All of our expressed Na,K-ATPase mutants were functionally active. Their ATPase, phosphorylation, and ouabain binding activities were measured, and the turnover of the phosphoenzyme intermediate was close to the wild-type enzyme, suggesting that they are folded properly in the infected cells. Incubation of the insect cells with the cysteine-selective reagent revealed essentially no labeling of the ␣-subunit of the background construct and labeling of all five mutants with single cysteine residues in putative extracellular loops. Two additional mutants, V969C and L976C, were created to further define the M9M10 loop. The lack of labeling for these two mutants showed that although Met 973 is apparently exposed, Val 969 and Leu 976 are not, demonstrating that this method may also be utilized to define membrane aqueous boundaries of membrane proteins. Our labeling studies are consistent with a specific 10-transmembrane segment model of the Na,KATPase ␣-subunit. This strategy utilized only functional Na,K-ATPase mutants to establish the membrane topology of the entire ␣-subunit, in contrast to most previously applied methods. Na,K-ATPase (sodium pump) is an integral membrane protein that is present in most animal cells. The enzyme consists of two subunits, a large catalytic ␣-subunit (about 110 kDa) and a glycosylated ␤-subunit (ϳ55 kDa); both subunits are required for enzymatic activity (1-3). Na,K-ATPase utilizes the energy derived from ATP hydrolysis to transport Na ϩ and K ϩ ions across plasma membranes against their electrochemical gradients and is a member of the P 2 -ATPase family (4). The ion gradients generated by the sodium pump are important for regulating a variety of physiological functions such as cell excitability, contractility, and secondary active transport. Several isoforms of the ␣-and ␤-subunits have been cloned from various species, and the primary sequences have been described (5). 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 ␣-subun...

show abstract

Membrane Topology of the α Subunit of Na,K-ATPase

Cited by 8 publications

References 8 publications

Transmembrane Topology of α- and β-Subunits of Na+,K+-ATPase Derived from β-Galactosidase Fusion Proteins Expressed in Yeast

Transmembrane Topology of α- and β-Subunits of Na+,K+-ATPase Derived from β-Galactosidase Fusion Proteins Expressed in Yeast

Epitope topology of Na,K‐ATPase α subunit analyzed in basolateral cell membranes of rat kidney tubules

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

Contact Info

Product

Resources

About