Evidence for intracellular sodium pumps in permeabilized <i>Xenopus laevis</i> oocytes

Schmalzing, Günther; Kröner, Silke; Passow, Hermann

doi:10.1042/bj2600395

Cited by 23 publications

(26 citation statements)

References 13 publications

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“…Because N-glycans may play a role in the assembly with ␣1 subunits or in the routing of the holoenzyme through the cell, we examined whether ␣1͞␤2 Na,K-pumps remain in internal membranes of tunicamycintreated oocytes. Usually, about half of the Na,K-pumps synthesized in Xenopus oocytes reside in the cell interior (23,27). Two days after cRNA injection, oocytes were permeabilized with detergents and incubated with [ 3 H]ouabain to estimate the total number of Na,K-pumps (surface plus internal).…”

Section: Resultsmentioning

confidence: 99%

Isoform-specific interactions of Na,K-ATPase subunits are mediated via extracellular domains and carbohydrates

Schmalzing

Ruhl

Gloor

1997

Proc. Natl. Acad. Sci. U.S.A.

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The functional unit of the Na,K-ATPase consists of a catalytic ␣ subunit noncovalently linked with a glycoprotein subunit, ␤. Using ouabain binding assays and immunoprecipitation of rodent ␣͞␤ complexes, we show here that all six possible isozymes between three ␣ and two ␤ isoforms can be formed in Xenopus oocytes. Two isoformspecific differences in ␣͞␤ interactions are observed: (i) ␣1͞␤1 and ␣2͞␤2 complexes, in contrast to ␣1͞␤2 complexes, are stable against Triton X-100-mediated dissociation, and (ii) ␤2 subunits must carry N-glycans to combine with ␣1 but not with ␣2. The interacting surfaces are mainly exposed to the extracellular side because coexpression of a truncated ␤1 subunit comprising the ectodomain results in assembly with ␣1 and ␣2, but not with ␣3; the ␤2 ectodomain combines with ␣2 only. A chimera consisting of 81% and 19% of the ␣1 N terminus and ␣2 C terminus, respectively, behaves like ␣2 and coprecipitates with the ␤2 ectodomain. In contrast, the reciprocal chimera does not coprecipitate with the ␤2 ectodomain. These results provide evidence for a selective interaction of Na,K-ATPase ␣ and ␤ subunits.The Na,K-ATPase is a heterodimer consisting of a 110-kDa ␣ subunit that is noncovalently linked with a glycoprotein ␤ subunit (protein core, 33-35 kDa). The ␣ subunit is the catalytic active part and bears the site for ATP hydrolysis, the binding sites for Na ϩ , K ϩ , and cardiac glycosides (1). The ␣ subunit spans the membrane several times. The ␤ subunit exposes a short N-terminal tail to the cytoplasm and the majority of its mass at the extracellular surface. It confers conformational stability to the ␣ subunit during or after assembly in the endoplasmic reticulum and is needed for routing of the enzyme to the plasma membrane (2-5).Three ␣ isoforms (␣1, ␣2, and ␣3) and two ␤ isoforms (␤1 and ␤2) have been identified in mammals and birds, each encoded by a separate gene (6). The ␣ isoforms show more than 80% homology even for very diverse species. The two ␤ isoforms have only about 40% amino acid sequence identity within the same species. Sequences encoding a mammalian ␤3 isoform have been deposited in the GenBank database. Both ␣ and ␤ isoforms display complex patterns of tissue-specific and developmental expression (7,8).All six possible ␣͞␤ isozymes are formed from the three ␣ and two ␤ isoforms of birds (9) and mammals (this work) in vitro, supporting the assumption that different isozymes exist in vivo (10, 11). Hybrid Na,K-pumps are readily formed from ␣1 and ␤1 subunits of virtually all species investigated (for a review, see ref. 12). The ␤1 isoform can even be replaced by the ␤ subunit of the H,K-pump to yield a functional Na,Kpump (13).Protein domains involved in ␣1͞␤1 interactions have only partially been identified. Deletion of the cytoplasmic domain and part of the transmembrane domain of chicken ␤1 does not disturb assembly as long as the truncated mutants are inserted into the membrane (14). Chimeric ␤1 subunits containing the N terminus and the membrane spanning domain...

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Section: Resultsmentioning

confidence: 99%

Isoform-specific interactions of Na,K-ATPase subunits are mediated via extracellular domains and carbohydrates

Schmalzing

Ruhl

Gloor

1997

Proc. Natl. Acad. Sci. U.S.A.

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“…Bma x ofoocytes which have been permeabilized selectively with digitonin is approximately equal to that of intact cells (Schmalzing et al, 1989). Additional permeabilization of inner membranes with the nonselective detergent SDS discloses intracellular Na+/K § pumps in oocytes.…”

Section: Effect Of Phorbol Esters On Ouabain Bindingmentioning

confidence: 99%

“…In some experiments, oocytes were pretreated with PMA as above, washed in EGTA, and permeabilized with 10/zM digitonin as described by Schmalzing, KrOner & Passow, (1989). 3Houa-bain binding was quantified by incubating the cells for 4 hr at 25~ with 1-40 nM 3H-ouabain in the NaCI-Tris medium containing (in mM): 110 NaCI, 2 MgC[,, I EGTA, I ATP, 10 Tris/HCI (pH 7.0) with or without 0.02% SDS.…”

Section: Measurements Of Ouabain Bindingmentioning

confidence: 99%

Activation of protein kinase C by phorbol ester induces downregulation of the Na+/K+-ATPase in oocytes ofXenopus laevis

et al. 1990

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Full-grown prophase-arrested oocytes of Xenopus laevis were treated with 50 nM phorbol 12-myristate 13-acetate (PMA), an activator of protein kinase C, or with 50 nM 4 alpha-phorbol 12,13-didecanoate (4 alpha PDD) that does not activate protein kinase C. The effect on membrane currents and capacitance, inulin uptake and ouabain binding, and on membrane morphology were analyzed. (i) During application of PMA, current generated by the Na+/K+ pump decreases; in addition, Cl- and K+ channels become inhibited. This general decrease in membrane conductance reaches steady state after about 60 min. 4 alpha PDD was ineffective. (ii) Ouabain binding experiments demonstrate that PMA (K1/2 = 7 nM), but not 4 alpha PPD, induces a reduction of the number of pump molecules in the surface membrane. Permeabilization of oocytes by digitonin plus 0.02% SDS renders all binding sites present prior to PMA treatment again accessible for ouabain. The KD value for ouabain binding is not influenced. 4 alpha PDD was ineffective. (iii) Exposure of oocytes to PMA reduces membrane capacitance and stimulates uptake of inulin suggesting an increase in endocytosis. Electron micrographs show that PMA reduces the number and length of microvilli, leading finally to a smooth membrane surface with a reduced surface area. From these results we conclude that stimulation of protein kinase C leads to downregulation of the sodium pump. A major portion of this inhibition is brought about by reduction in area of surface membrane with a concomitant internalization of pump molecules. In addition to this mode of downregulation, a direct effect of stimulation of protein kinase C on the pump molecule cannot be excluded.

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“…9B, lanes 3-6). This discrepancy might be explained by a regulated expression of Na ϩ ,K ϩ pumps in Xenopus oocytes that only permits the expression of a limited number of pumps at the cell surface and that would establish an intracellular pool of pumps (20). Another possibility to explain the differences between the expression and the stabilization of the ␣⅐N1,2,3 complexes might be that the mutant complexes have a different ouabain sensitivity than the ␣⅐wild type ␤ complexes.…”

Section: Fig 5 Functional Expression Of Namentioning

confidence: 99%

Role of Glycosylation and Disulfide Bond Formation in the β Subunit in the Folding and Functional Expression of Na,K-ATPase

Beggah

Jaunin

Geering

1997

Journal of Biological Chemistry

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Initial folding is a prerequisite for subunit assembly in oligomeric proteins. In this study, we have compared the role of co-translational modifications in the acquisition of an assembly-competent conformation of the beta subunit, the assembly of which is required for the structural and functional maturation of the catalytic Na,K-ATPase alpha subunit. Cysteine or asparagine residues implicated in disulfide bond formation or N-glycosylation, respectively, in the Xenopus beta1 subunit were eliminated by site-directed mutagenesis, and the assembly efficiency of the mutants and the functional expression of Na+,K+ pumps were studied after expression in Xenopus oocytes. Our results show that lack of each one of the two most C-terminal disulfide bonds indeed permits short term but completely abolishes long term assembly of the beta subunit. On the other hand, lack of the most N-terminal disulfide bonds allows the expression of a small number of functional Na+,K+ pumps at the cell surface. Elimination of all three but not of one or two glycosylation sites produces beta subunits that remain stably expressed in the endoplasmic reticulum, in association with binding protein but not as irreversible aggregates. The assembly efficiency of nonglycosylated beta subunits is decreased but a reduced number of functional Na+,K+ pumps is expressed at the cell surface. The lack of sugars does not influence the apparent K+ or ouabain affinity of the Na+,K+ pumps. Thus, these data show that disulfide bond formation and N-glycosylation may play important but qualitatively distinct roles in the initial folding of oligomeric protein subunits. Moreover, the results suggest that an endoplasmic reticulum degradation pathway exists, which is glycosylation-dependent.

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Evidence for intracellular sodium pumps in permeabilized Xenopus laevis oocytes

Cited by 23 publications

References 13 publications

Isoform-specific interactions of Na,K-ATPase subunits are mediated via extracellular domains and carbohydrates

Isoform-specific interactions of Na,K-ATPase subunits are mediated via extracellular domains and carbohydrates

Activation of protein kinase C by phorbol ester induces downregulation of the Na+/K+-ATPase in oocytes ofXenopus laevis

Role of Glycosylation and Disulfide Bond Formation in the β Subunit in the Folding and Functional Expression of Na,K-ATPase

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