Specific Cross-Links between Fragments of Proteolyzed Na,K-ATPase Induced by <i>o</i>-Phthalaldehyde

Or, Eran; Goldshleger, Rivka; Shainskaya, and Alla; Karlish, Steven J. D.

doi:10.1021/bi9730442

Cited by 15 publications

(19 citation statements)

References 53 publications

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“…This loop appears to be the focal region for ␥-␣-␤ interactions. Accordingly, at the extramembranous extracellular region, the ␤ subunit interacts with L7/8 of ␣1 as shown in several earlier studies (19,(33)(34)(35)(36), and ␥ can also be cross-linked to ␤ (19), and as already suggested (19), the site of ␥-␤ interaction is probably close to the site of ␣-␤ interaction.…”

Section: ␥B-transfectedsupporting

confidence: 65%

Regions of the Catalytic α Subunit of Na,K-ATPase Important for Functional Interactions with FXYD 2

Zouzoulas¹,

Blostein

2006

Journal of Biological Chemistry

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The ␥ modulator (FXYD 2) is a member of the FXYD family of single transmembrane proteins that modulate the kinetic behavior of Na,K-ATPase. This study concerns the identification of regions in the ␣ subunit that are important for its functional interaction with ␥. An important effect of ␥ is to increase K ؉ antagonism of cytoplasmic Na ؉ activation apparent as an increase in K Na at high. We show that although ␥ associates with ␣1, ␣2, and ␣3 isoforms, it increases the K Na of ␣1 and ␣3 but not ␣2. Accordingly, chimeras of ␣1 and ␣2 were used to identify regions of ␣ critical for the increased K Na . As with ␣1 and ␣2, all chimeras associate with ␥. Kinetic analysis of ␣2 front /␣1 back chimeras indicate that the C-terminal (Lys 907 -Tyr 1018 ) region of ␣1, which includes transmembrane (TM)9 close to ␥, is important for the increase in K Na . However, similar experiments with ␣1 front /␣2 back chimeras indicate a modulatory role of the loop between TMs 7 and 8. Thus, as long as the ␣1 L7/8 loop is present, replacement of TM9 of ␣1 with that of ␣2 does not abrogate the ␥ effect on K Na . In contrast, as long as TM9 is that of ␣1, replacement of L7/8 of ␣1 with that of ␣2 does not abolish the effect. It is suggested that structural association of the TM regions of ␣ and FXYD 2 is not the sole determinant of this effect of FXYD on K Na but is subject to long range modulation by the extramembranous L7/8 loop of ␣.The Na,K-ATPase or sodium pump is an integral membrane protein found in the cells of virtually all higher eukaryotes. It couples the hydrolysis of one molecule of ATP to the electrogenic exchange of three intracellular Na ϩ for two extracellular K ϩ ions against their electrochemical gradients.The sodium pump is an oligomer of two subunits, a large catalytic ␣ subunit and a smaller, highly glycosylated ␤ subunit whose role is to ensure the proper folding, insertion, and maturation of ␣ in the plasma membrane. Four isoforms of ␣ and three of ␤ are expressed in a tissue-and development-specific manner. (for reviews, see Refs. 1 and 2). The sodium pump is subject to diverse modes of regulation by substrates, membrane-associated components, hormones, and neurotransmitters (3). In recent years, considerable attention has been focused on tissue-specific regulation by small transmembrane proteins referred to as FXYD proteins (for reviews, see Refs. 4 -7). These proteins belong to a gene family of small, single transmembrane proteins, with the exception of MAT-8 which has two transmembranes (TM(s)).3 There are at least seven members of which several (FXYDs 1-4 and 7) appear to regulate the kinetic behavior of the sodium pump in distinct and specific ways, in particular the apparent affinity of the enzyme for ligands (Na ϩ , K ϩ , and ATP). Members possess a 35-amino-acid signature sequence that includes 6 invariant amino acids before, in, and after the TM domain. The N-terminal FXYD (Phe-Xaa-Tyr-Asp) motif is invariant, along with two glycines in the TM domain, and a serine in the C-terminal domain. The first FXYD...

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Section: ␥B-transfectedsupporting

confidence: 65%

Regions of the Catalytic α Subunit of Na,K-ATPase Important for Functional Interactions with FXYD 2

Zouzoulas¹,

Blostein

2006

Journal of Biological Chemistry

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“…OPA reacts with thiols and primary amines yielding highly fluorescent 1-alkylthio-2-alkylisoindoles. The compound has been successfully used to determine the interaction between the subunits of Na + , K + -ATPases (Khan et al, 1996;Or et al, 1998). In this study, we have treated solubilized PMs from potato wild-type plants with OPA.…”

Section: Confirmation Of St Sut1 Dimerization By Alternative Methodsmentioning

confidence: 99%

Transport and Sorting of the Solanum tuberosum Sucrose Transporter SUT1 Is Affected by Posttranslational Modification

et al. 2008

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The plant sucrose transporter SUT1 from Solanum tuberosum revealed a dramatic redox-dependent increase in sucrose transport activity when heterologously expressed in Saccharomyces cerevisiae. Plant plasma membrane vesicles do not show any change in proton flux across the plasma membrane in the presence of redox reagents, indicating a SUT1-specific effect of redox reagents. Redox-dependent sucrose transport activity was confirmed electrophysiologically in Xenopus laevis oocytes with SUT1 from maize (Zea mays). Localization studies of green fluorescent protein fusion constructs showed that an oxidative environment increased the targeting of SUT1 to the plasma membrane where the protein concentrates in 200-to 300-nm raft-like microdomains. Using plant plasma membranes, St SUT1 can be detected in the detergent-resistant membrane fraction. Importantly, in yeast and in plants, oxidative reagents induced a shift in the monomer to dimer equilibrium of the St SUT1 protein and increased the fraction of dimer. Biochemical methods confirmed the capacity of SUT1 to form a dimer in plants and yeast cells in a redox-dependent manner. Blue native PAGE, chemical cross-linking, and immunoprecipitation, as well as the analysis of transgenic plants with reduced expression of St SUT1, confirmed the dimerization of St SUT1 and Sl SUT1 (from Solanum lycopersicum) in planta. The ability to form homodimers in plant cells was analyzed by the split yellow fluorescent protein technique in transiently transformed tobacco (Nicotiana tabacum) leaves and protoplasts. Oligomerization seems to be cell type specific since under native-like conditions, a phloem-specific reduction of the dimeric form of the St SUT1 protein was detectable in SUT1 antisense plants, whereas constitutively inhibited antisense plants showed reduction only of the monomeric form. The role of redox control of sucrose transport in plants is discussed.

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“…This specific arrangement is not compatible with membrane insertion mediated only by intramolecular interactions and has required, during evolution, the association of a helper protein that assists the correct packing of K ϩ -transporting P-type ATPases. Or et al (48) previously suggested that the M7-M8 loop interacts with M4-M6 containing the cation sites so that disruption of the ␣-␤ interaction alters the disposition of this loop and inactivates cation occlusion. Our results strengthen this hypothesis, that is we provide evidence that the C-terminal regions of Na ϩ ,K ϩ -ATPase and H ϩ ,K ϩ -ATPase are likely to be involved in K ϩ de-occlusion.…”

mentioning

confidence: 99%

Chimeras of X+,K+-ATPases

Koenderink

Swarts

Stronks

et al. 2001

Journal of Biological Chemistry

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In this study we reveal regions of Na+,K+-ATPase and H+,K+-ATPase that are involved in cation selectivity. A chimeric enzyme in which transmembrane hairpin M5-M6 of H+,K+-ATPase was replaced by that of Na+,K+-ATPase was phosphorylated in the absence of Na+and showed no K+-dependent reactions. Next, the part originating from Na+,K+-ATPase was gradually increased in the N-terminal direction. We demonstrate that chimera HN16, containing the transmembrane segments one to six and intermediate loops of Na+,K+-ATPase, harbors the amino acids responsible for Na+specificity. Compared with Na+,K+-ATPase, this chimera displayed a similar apparent Na+affinity, a lower apparent K+affinity, a higher apparent ATP affinity, and a lower apparent vanadate affinity in the ATPase reaction. This indicates that theE2K form of this chimera is less stable than that of Na+,K+-ATPase, suggesting that it, like H+,K+-ATPase, de-occludes K+ions very rapidly. Comparison of the structures of these chimeras with those of the parent enzymes suggests that the C-terminal 187 amino acids and the β-subunit are involved in K+occlusion. Accordingly, chimera HN16 is not only a chimeric enzyme in structure, but also in function. On one hand it possesses the Na+-stimulated ATPase reaction of Na+,K+-ATPase, while on the other hand it has the K+occlusion properties of H+,K+-ATPase.

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Specific Cross-Links between Fragments of Proteolyzed Na,K-ATPase Induced by o-Phthalaldehyde

Cited by 15 publications

References 53 publications

Regions of the Catalytic α Subunit of Na,K-ATPase Important for Functional Interactions with FXYD 2

Regions of the Catalytic α Subunit of Na,K-ATPase Important for Functional Interactions with FXYD 2

Transport and Sorting of the Solanum tuberosum Sucrose Transporter SUT1 Is Affected by Posttranslational Modification

Chimeras of X+,K+-ATPases

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