The adhesion molecule on glia (AMOG/beta 2) and alpha 1 subunits assemble to functional sodium pumps in Xenopus oocytes.

Schmalzing, Günther; Kröner, Silke; Schachner, Melitta; Gloor, Sergio M.

doi:10.1016/s0021-9258(19)88688-x

Cited by 86 publications

(16 citation statements)

References 33 publications

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“…In this paper we have extended the previous finding by us (Jaisser et al, 1992a, b) and others (Schmalzing et al, 1992;Eakle et al, 1992) that the structure of the 13 subunit has an influence on the function of the Na,K pump present at the plasma membrane, and more specifically on the apparent affinity of potassium for its external binding site. The Na,K pump is a transport system that undergoes a complex cycle with at least two conformations, one of which (the E2 conformation) of 10 mM K ÷ are plotted against the membrane potential (Vm).…”

Section: Discussionsupporting

confidence: 69%

Modulation of the Na,K-pump function by beta subunit isoforms.

Jaisser¹,

Jaunin²,

Geering³

et al. 1994

The Journal of General Physiology

127

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To study the role of the Na,K-ATPase beta subunit in the ion transport activity, we have coexpressed the Bufo alpha 1 subunit (alpha 1) with three different isotypes of beta subunits, the Bufo Na,K-ATPase beta 1 (beta 1NaK) or beta 3 (beta 3NaK) subunit or the beta subunit of the rabbit gastric H,K-ATPase (beta HK), by cRNA injection in Xenopus oocyte. We studied the K+ activation kinetics by measuring the Na,K-pump current induced by external K+ under voltage clamp conditions. The endogenous oocyte Na,K-ATPase was selectively inhibited, taking advantage of the large difference in ouabain sensitivity between Xenopus and Bufo Na,K pumps. The K+ half-activation constant (K1/2) was higher in the alpha 1 beta 3NaK than in the alpha 1 beta 1NaK groups in the presence of external Na+, but there was no significant difference in the absence of external Na+. Association of alpha 1 and beta HK subunits produced active Na,K pumps with a much lower apparent affinity for K+ both in the presence and in the absence of external Na+. The voltage dependence of the K1/2 for external K+ was similar with the three beta subunits. Our results indicate that the beta subunit has a significant influence on the ion transport activity of the Na,K pump. The small structural differences between the beta 1NaK and beta 3NaK subunits results in a difference of the apparent affinity for K+ that is measurable only in the presence of external Na+, and thus appears not to be directly related to the K+ binding site. In contrast, association of an alpha 1 subunit with a beta HK subunit results in a Na,K pump in which the K+ binding or translocating mechanisms are altered since the apparent affinity for external K+ is affected even in the absence of external Na+.

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Section: Discussionsupporting

confidence: 69%

Modulation of the Na,K-pump function by beta subunit isoforms.

Jaisser¹,

Jaunin²,

Geering³

et al. 1994

The Journal of General Physiology

127

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“…In this study we were mainly concerned with characterizing structural determinants in the/3 subunit of Na,K-ATPase that are implicated in its posttranslational fate, namely in its assembly with ot subunits and its ER retention as an unassembled protein. However, the use of chimeric B-proteins permitted us also to substantiate the recent observation that/3 subunits might not only have a primary role in the structural and functional maturation of newly synthesized ot subunits, but could as well be modulators of the transport activities of mature Na,K-pumps expressed at the plasma membrane (Eakle et al, 1992;Jaisser et al, 1992;Lutsenko and Kaplan, 1992;Schmalzing et al, 1992; Jaisser, E, P. Jaunin, K. Geering, B. C. Rossier and J. H. Horisberger; manuscript submitted for publication). On the one hand, we show that Na,K-pumps composed ofaNK and/3HK exhibit a lower apparent affinity for K ÷ than otNK-/3NK complexes and on the other hand, we provide evidence that the exchange of the NH2-and the COOH-terminal in chimeric proteins of/3NK and/3HK is not sufficient to produce Na,K-pumps with an apparent K ÷ affinity of either ct-/3NK or tx-/3HK complexes (Table I).…”

Section: Both the Transraembrane And The Ectodomain Orb Subunits Are Involved In The Modulation Of The Transport Activity Of Nak-pumpmentioning

confidence: 67%

“…After the chase period, oocytes were either extracted with Triton X-100 or digitonin. Triton extracts were obtained as described (launin etal., 1992), Digitonin extracts were essentially prepared as described by Schmalzing et al (1992). 30 tLl/oocyte of a solution containing 100 mM NaC1, 20 mM Tris-HC1 (pH 7.6), 10 mM methionine, 0.5% (wt/vol) digitonin (water soluble; Fluka Chemic AG, Buchs, Switzerland), 1 mM PMSF, and 5 #g/nll of each leupeptin, pepstatin, and antipain were added.…”

Section: In Vitro Translation Expression In Xenopus Oocytes and Immunoprecipitation Of Chimeramentioning

confidence: 99%

“…In addition, as in other multimeric proteins, subunit oligomerization is needed for the ER exit of u subunits as well as of Xenopus /3,and/3risoforms expressed in the Xenopus oocyte (Jaunin et al, 1992). Finally, recent experimental evidence suggests that/~ subunits might be modulators of the transport activity of Na,K-ATPase expressed at the plasma membrane (Eakle et al, 1992;Jaisser et al, 1992;Lutsenko and Kaplan, 1992;Schmalzing et al, 1992).…”

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confidence: 99%

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Role of the transmembrane and extracytoplasmic domain of beta subunits in subunit assembly, intracellular transport, and functional expression of Na,K-pumps.

Jaunin

Jaisser

Beggah

et al. 1993

The Journal of Cell Biology

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The ubiquitous Na,K- and the gastric H,K-pumps are heterodimeric plasma membrane proteins composed of an alpha and a beta subunit. The H,K-ATPase beta subunit (beta HK) can partially act as a surrogate for the Na,K-ATPase beta subunit (beta NK) in the formation of functional Na,K-pumps (Horisberger et al., 1991. J. Biol. Chem. 257:10338-10343). We have examined the role of the transmembrane and/or the ectodomain of beta NK in (a) its ER retention in the absence of concomitant synthesis of Na,K-ATPase alpha subunits (alpha NK) and (b) the functional expression of Na,K-pumps at the cell surface and their activation by external K+. We have constructed chimeric proteins between Xenopus beta NK and rabbit beta HK by exchanging their NH2-terminal plus transmembrane domain with their COOH-terminal ectodomain (beta NK/HK, beta HK/NK). We have expressed these constructs with or without coexpression of alpha NK in the Xenopus oocyte. In the absence of alpha NK, Xenopus beta NK and all chimera that contained the ectodomain of beta NK were retained in the ER while beta HK and all chimera with the ectodomain of beta HK could leave the ER suggesting that ER retention of unassembled Xenopus beta NK is mediated by a retention signal in the ectodomain. When coexpressed with alpha NK, only beta NK and beta NK/HK chimera assembled efficiently with alpha NK leading to similar high expression of functional Na,K-pumps at the cell surface that exhibited, however, a different apparent K+ affinity. beta HK or chimera with the transmembrane domain of beta HK assembled less efficiently with alpha NK leading to lower expression of functional Na,K-pumps with a different apparent K+ affinity. The data indicate that the transmembrane domain of beta NK is important for efficient assembly with alpha NK and that both the transmembrane and the ectodomain of beta subunits play a role in modulating the transport activity of Na,K-pumps.

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“…Association of the [3 subunit with the newly synthesized, catalytic c~ subunit at the level of the ER is necessary for its structural maturation, which is characterized by an increase in the trypsin resistance (10) and/or the half-life as well as the ER exit of the a subunit (1,19). Finally, in addition to its function in the early maturation, the 13 subunit plays a role in the mature enzyme and affects the transport activity, in particular the K+-activation of Na,Kpumps expressed in the plasma membrane of Xenopus oocytes (18,19,34) or of yeast (6).…”

mentioning

confidence: 99%

Oligomerization and maturation of Na,K-ATPase: functional interaction of the cytoplasmic NH2 terminus of the beta subunit with the alpha subunit.

Geering

Beggah

Good

et al. 1996

The Journal of Cell Biology

133

154

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Abstract. Subunit assembly plays an essential role in the maturation of oligomeric proteins. In this study, we have characterized the main structural and functional consequences of the assembly of a and [3 subunits of Na,K-ATPase. Xenopus oocytes injected with c~ and/or 13 cRNA were treated with brefeldin A, which permitted the accumulation of individual subunits or a-[3 complexes in the ER. Only ot subunits that are associated with 13 subunits become resistant to trypsin digestion and cellular degradation. Similarly, assembly with 13 subunits is necessary and probably sufficient for the catalytic et subunit to acquire its main functional properties at the level of the ER, namely the ability to adopt different ligand-dependent conformations and to hydrolyze ATP in an Na ÷-and K÷-dependent, ouabaininhibitable fashion. Not only the a but also the [3 subunit undergoes a structural change after assembly, which results in a global increase in its protease resistance. Furthermore, extensive and controlled proteolysis assays on wild-type and NHz-terminally modified 13 subunits revealed a K+-dependent interaction of the cytoplasmic NH2 terminus of the [3 subunit with the subunit, which is likely to be involved in the modulation of the K÷-activation of the Na,K-pump transport activity. Thus, we conclude that the ER assembly process not only establishes the basic structural interactions between individual subunits, which are required for the maturation of oligomeric proteins, but also distinct, functional interactions, which are involved in the regulation of functional properties of mature proteins.M ANY plasma membrane and secretory proteins are oligomeric. The subunits of these proteins are synthesized independenly of each other and are inserted into the ER membrane or the lumen during their synthesis. They are subjected to cotranslational modifications, fold, and then oligomerize. Once correctly assembled, the proteins leave the ER and are targeted to their final cellular site of action. Misfolded or unassembled subunits are retained in the ER and are degraded (15). Although it is increasingly clear that the oligomeric state controls the proper function of the protein, little is known about the nature of interactions that are involved in subunit assembly and about the structural and functional consequences of subunit oligomerization. In this study, we investigate several aspects of this question by analyzing the oligomerization and the functional maturation of Na,KATPase.The ubiquitous Na,K-ATPase is responsible for the maintenance of the sodium and potassium gradients between the intra-and extracellular milieu. The enzyme is composed of two heterologous subunits. The ct subunit is a

show abstract

The adhesion molecule on glia (AMOG/beta 2) and alpha 1 subunits assemble to functional sodium pumps in Xenopus oocytes.

Cited by 86 publications

References 33 publications

Modulation of the Na,K-pump function by beta subunit isoforms.

Modulation of the Na,K-pump function by beta subunit isoforms.

Role of the transmembrane and extracytoplasmic domain of beta subunits in subunit assembly, intracellular transport, and functional expression of Na,K-pumps.

Oligomerization and maturation of Na,K-ATPase: functional interaction of the cytoplasmic NH2 terminus of the beta subunit with the alpha subunit.

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