The functional roles of disulfide bonds in the β‐subunit of (Na,K)ATPase as studied by site‐directed mutagenesis

Noguchi, Shunsuke; Mutoh, Yoshinobu; Kawamura, Masaru

doi:10.1016/0014-5793(94)80463-x

Cited by 30 publications

(19 citation statements)

References 41 publications

(25 reference statements)

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“…Although the extracellular domains of Na,K-ATPase ␤-subunits have previously been shown to mediate ␣-subunit ion-transport activity (Laughery et al, 2003;Noguchi et al, 1994) and cell-cell adhesion interactions (Contreras et al, 1999;Muller-Husmann et al, 1993), to our knowledge this is the first demonstration that the extracellular domain of the ␤-subunit organizes a junctional complex rather than simply acting as a cell adhesion molecule. Although the extracellular domain could simply serve to localize the Na,K-ATPase to the SJ, evidence that the Nrv2 extracellular domain has additional roles in junctional activity is provided by the observation that the Nrv2IT/3E chimera localized to the SJ but did not provide junctional activity.…”

Section: Discussionmentioning

confidence: 66%

A pump-independent function of the Na,K-ATPase is required for epithelial junction function and tracheal tube-size control

2007

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The heterodimeric Na,K-ATPase has been implicated in vertebrate and invertebrate epithelial cell junctions, morphogenesis and oncogenesis, but the mechanisms involved are unclear. We previously showed that the Drosophila Na,K-ATPase is required for septate junction (SJ)formation and that of the three β-subunit loci, only Nrv2 isoforms support epithelial SJ barrier function and tracheal tube-size control. Here we show that Nrv1 is endogenously co-expressed with Nrv2 in the epidermis and tracheal system, but Nrv1 has a basolateral localization and appears to be excluded from the Nrv2-containing SJs. When the normally neuronal Nrv3 is expressed in epithelial cells, it does not associate with SJs. Thus, theβ-subunit is a key determinant of Na,K-ATPase subcellular localization as well as function. However, localization of the Na,K-ATPase to SJs is not sufficient for junctional activity because although several Nrv2/Nrv3 chimericβ-subunits localize to SJs, only those containing the extracellular domain of Nrv2 have junctional activity. Junctional activity is also specific to different α-subunit isoforms, with only some isoforms from the majorα-subunit locus being able to provide full barrier function and produce normal tracheal tubes. Importantly, mutations predicted to inactivate ATPα catalytic function do not compromise junctional activity,demonstrating that the Drosophila Na,K-ATPase has an ion-pump-independent role in junction formation and tracheal morphogenesis. These results define new functions for the intensively studied Na,K-ATPase. Strikingly, the rat α1 isoform has full junctional activity and can rescue Atpα-null mutants to viability, suggesting that the Na,K-ATPase has an evolutionarily conserved role in junction formation and function.

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Section: Discussionmentioning

confidence: 66%

A pump-independent function of the Na,K-ATPase is required for epithelial junction function and tracheal tube-size control

2007

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“…Previous work has implicated some or all of these disulfides as being essential for function, assembly, and/or expression (6,14,17,26). Disruption of these bridges through site-directed mutagenesis has consistently shown that blocking the formation of the second and third bridges causes a more severe disruption than the mutation of the first bridge (14, 17, 26).…”

Section: Fig 10mentioning

confidence: 99%

Mutational Analysis of α-β Subunit Interactions in the Delivery of Na,K-ATPase Heterodimers to the Plasma Membrane

Laughery

Todd

Kaplan

2003

Journal of Biological Chemistry

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The ␤-subunit of the Na,K-ATPase is required to deliver functional ␣␤؊heterodimers to the plasma membrane (PM) of baculovirus-infected insect cells. We have investigated the molecular determinants in the ␤-subunit for the assembly and delivery processes. Trafficking of both subunits was analyzed by Western blots of fractionated membranes enriched in endoplasmic reticulum (ER), Golgi, and PM. Heterodimer assembly was evaluated by co-immunoprecipitation, and enzymatic activity was measured by ATPase assay. Elimination of enzymatic activity by D369A point mutation of the ␣-subunit had no effect on the compartmental distribution of the Na,K-ATPase, demonstrating that enzymatic functioning is not a prerequisite for PM delivery. Replacement of all three N-glycosylation site asparagines with glutamines produced no effect on the delivery to the PM or the activity of the enzyme, but increased susceptibility to degradation was observed. Analysis of ␤-subunits in which the disulfide bonds were removed through substitution reveals that the bridges are important for PM targeting but not for assembly of the heterodimer. Assembly is supported by ␤-subunits with greatly truncated extracellular domains. The presence of the amino-terminal domain and transmembrane segment is sufficient for assembly and PM delivery. Intermediate length truncated ␤-subunits and some disulfide bridge substitution mutants assemble with the ␣-subunit but are not able to exit the ER. We conclude that there are different and separable requirements for the assembly of Na,K-ATPase heterodimer complexes, exit of the dimer from the ER, delivery to the PM, and catalytic activity of the dimer.

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“…It is clearly involved in the transport cycle, as the reductive loss of its intramolecular Cys-Cys bonds results in the loss of the ability to occlude K ϩ ions and the associated loss of Na-K-ATPase activity (29), and it undergoes conformational transitions in concert with the ␣-subunit during the reaction cycle (8,25). Studies in Madin-Darby canine kidney (MDCK) cells (17) confirmed the 1:1 ␤-to-␣ ratio previously reported for purified enzyme (7).…”

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confidence: 99%

β-Subunit overexpression alters the stoicheometry of assembled Na-K-ATPase subunits in MDCK cells

Clifford¹,

Kaplan²

2008

American Journal of Physiology-Renal Physiology

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Clifford RJ, Kaplan JH. ␤-Subunit overexpression alters the stoicheometry of assembled Na-K-ATPase subunits in MDCK cells. Am J Physiol Renal Physiol 295: F1314 -F1323, 2008. First published August 13, 2008 doi:10.1152/ajprenal.90406.2008In eukaryotic cells, the apparent maintenance of 1:1 stoicheometry between the Na-K-ATPase ␣-and ␤-subunits led us to question whether this was alterable and thus if some form of regulation was involved. We have examined the consequences of overexpressing Na-K-ATPase ␤ 1-subunits using Madin-Darby canine kidney (MDCK) cells expressing flag-tagged ␤ 1-subunits (␤1flag) or Myc-tagged ␤1-subunits (␤1myc) under the control of a tetracycline-dependent promoter. The induction of ␤ 1flag subunit synthesis in MDCK cells, which increases ␤1-subunit expression at the plasma membrane by more than twofold, while maintaining stable ␣ 1 expression levels, revealed that all mature ␤ 1-subunits associate with ␣1-subunits, and no evidence of "free" ␤ 1-subunits was obtained. Consequently, the ratio of assembled ␤1-to ␣1-subunits is significantly increased when "extra" ␤-subunits are expressed. An increased ␤ 1/␣1 stoicheometry is also observed in cells treated with tunicamycin, suggesting that the protein-protein interactions involved in these complexes are not dependent on glycosylation. Confocal images of cocultured ␤ 1myc-expressing and ␤1flag-expressing MDCK cells show colocalization of ␤1myc and ␤1flag subunits at the lateral membranes of neighboring cells, suggesting the occurrence of intercellular interactions between the ␤-subunits. Immunoprecipitation using MDCK cells constitutively expressing ␤1myc and tetracycline-regulated ␤1flag subunits confirmed ␤-␤-subunit interactions. These results demonstrate that the equimolar ratio of assembled ␤1/

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The functional roles of disulfide bonds in the β‐subunit of (Na,K)ATPase as studied by site‐directed mutagenesis

Cited by 30 publications

References 41 publications

A pump-independent function of the Na,K-ATPase is required for epithelial junction function and tracheal tube-size control

A pump-independent function of the Na,K-ATPase is required for epithelial junction function and tracheal tube-size control

Mutational Analysis of α-β Subunit Interactions in the Delivery of Na,K-ATPase Heterodimers to the Plasma Membrane

β-Subunit overexpression alters the stoicheometry of assembled Na-K-ATPase subunits in MDCK cells

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