Residue Leu973 of the Rat α1 Subunit of the Na,K-ATPase Is Located on the Cytoplasmic Side of the Plasma Membrane

Lee, Kyunglim; Guidotti, Guido

doi:10.1006/bbrc.1998.9534

Cited by 4 publications

(5 citation statements)

References 21 publications

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“…Evidence was lacking for the final two segments after Val 939 ; thus it was concluded that the ␣-subunit had eight transmembrane segments. This conclusion was repeated in a more recent work from Lee and Guidotti (12) who inserted a tag between residues 973 and 974 and determined, using immunofluorescence, that residue 973 was internal. This recent finding is contradicted by our current observation that Met 973 is exposed at the extracellular surface, as well as by our earlier suggestion about the membrane location of a segment beginning at Met 973 based on tryptic digestion studies of membrane-associated peptides (8).…”

Section: Figmentioning

confidence: 76%

“…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.…”

Section: Figmentioning

confidence: 99%

“…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).…”

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confidence: 99%

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

Kaplan

2000

Journal of Biological Chemistry

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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...

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Section: Figmentioning

confidence: 76%

Section: Figmentioning

confidence: 99%

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confidence: 99%

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

Kaplan

2000

Journal of Biological Chemistry

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“…and Val*$* [48] ; and Ser""%, Arg"') and Leu$"$ [49]. Other insertions gave topologies incompatible with the SERCA structure : Arg*$' and Leu*($ [47] ; Phe)(' and Thr*)" [49] ; and Leu*($ [50]. Note that one laboratory numbered residues from the prosequence of rat α1 [47,49], and so numbers in their papers are larger by 5.…”

Section: Topologymentioning

confidence: 99%

Structural similarities of Na,K-ATPase and SERCA, the Ca2+-ATPase of the sarcoplasmic reticulum

Sweadner

Donnet

2001

Biochemical Journal

147

110

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The crystal structure of SERCA1a (skeletal-muscle sarcoplasmic-reticulum/endoplasmic-reticulum Ca2+-ATPase) has recently been determined at 2.6 Å (note 1 Å = 0.1nm) resolution [Toyoshima, Nakasako, Nomura and Ogawa (2000) Nature (London) 405, 647–655]. Other P-type ATPases are thought to share key features of the ATP hydrolysis site and a central core of transmembrane helices. Outside of these most-conserved segments, structural similarities are less certain, and predicted transmembrane topology differs between subclasses. In the present review the homologous regions of several representative P-type ATPases are aligned with the SERCA sequence and mapped on to the SERCA structure for comparison. Homology between SERCA and the Na,K-ATPase is more extensive than with any other ATPase, even PMCA, the Ca2+-ATPase of plasma membrane. Structural features of the Na,K-ATPase are projected on to the Ca2+-ATPase crystal structure to assess the likelihood that they share the same fold. Homology extends through all ten transmembrane spans, and most insertions and deletions are predicted to be at the surface. The locations of specific residues are examined, such as proteolytic cleavage sites, intramolecular cross-linking sites, and the binding sites of certain other proteins. On the whole, the similarity supports a shared fold, with some particular exceptions.

show abstract

Section: Topologymentioning

confidence: 99%

Structural similarities of Na,K-ATPase and SERCA, the Ca2+-ATPase of the sarcoplasmic reticulum

2001

View full text Add to dashboard Cite

The crystal structure of SERCA1a (skeletal-muscle sarcoplasmic-reticulum/endoplasmic-reticulum Ca(2+)-ATPase) has recently been determined at 2.6 A (note 1 A = 0.1 nm) resolution [Toyoshima, Nakasako, Nomura and Ogawa (2000) Nature (London) 405, 647-655]. Other P-type ATPases are thought to share key features of the ATP hydrolysis site and a central core of transmembrane helices. Outside of these most-conserved segments, structural similarities are less certain, and predicted transmembrane topology differs between subclasses. In the present review the homologous regions of several representative P-type ATPases are aligned with the SERCA sequence and mapped on to the SERCA structure for comparison. Homology between SERCA and the Na,K-ATPase is more extensive than with any other ATPase, even PMCA, the Ca(2+)-ATPase of plasma membrane. Structural features of the Na,K-ATPase are projected on to the Ca(2+)-ATPase crystal structure to assess the likelihood that they share the same fold. Homology extends through all ten transmembrane spans, and most insertions and deletions are predicted to be at the surface. The locations of specific residues are examined, such as proteolytic cleavage sites, intramolecular cross-linking sites, and the binding sites of certain other proteins. On the whole, the similarity supports a shared fold, with some particular exceptions.

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Residue Leu973 of the Rat α1 Subunit of the Na,K-ATPase Is Located on the Cytoplasmic Side of the Plasma Membrane

Cited by 4 publications

References 21 publications

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

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

Structural similarities of Na,K-ATPase and SERCA, the Ca2+-ATPase of the sarcoplasmic reticulum

Structural similarities of Na,K-ATPase and SERCA, the Ca2+-ATPase of the sarcoplasmic reticulum

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