Beta 1- and beta 3-subunits can associate with presynthesized alpha-subunits of Xenopus oocyte Na,K-ATPase.

Oligomerization of newly synthesized alpha- and beta-subunits is a prerequisite for the structural and functional maturation of Na,K-ATPase. In this study, we have tested the competence of presynthesized alpha- and beta-subunits to assemble into functional enzyme complexes. Antisense oligonucleotides complementary to alpha-mRNA were used to inhibit alpha-subunit synthesis in Xenopus oocytes leaving a presynthesized trypsin-sensitive alpha-subunit pool. beta-Subunits expressed in these oocytes from injected cRNA assembled with the preexisting alpha-subunits, rendered them trypsin-resistant, and permitted the expression of more ouabain binding sites at the plasma membrane. Similarly, presynthesized beta 1- or beta 3-subunits produced in Xenopus oocytes by injection of beta-cRNA and later of specific antisense oligonucleotides were stabilized and transported out of the endoplasmic reticulum when alpha-cRNA was injected into oocytes. These data indicate that alpha- and beta-subunits can insert into endoplasmic reticulum membranes independent of each other in an assembly-competent form and retain their ability for oligomerization after synthesis.     

The gamma subunit is a specific component of the Na,K-ATPase and modulates its transport function.

The role of small, hydrophobic peptides that are associated with ion pumps or channels is still poorly understood. By using the Xenopus oocyte as an expression system, we have characterized the structural and functional properties of the gamma peptide which co-purifies with Na,K-ATPase. Immuno-radiolabeling of epitope-tagged gamma subunits in intact oocytes and protease protection assays show that the gamma peptide is a type I membrane protein lacking a signal sequence and exposing the N-terminus to the extracytoplasmic side. Co-expression of the rat or Xenopus gamma subunit with various proteins in the oocyte reveals that it specifically associates only with isozymes of Na,K-ATPase. The gamma peptide does not influence the formation and cell surface expression of functional Na,K-ATPase alpha-beta complexes. On the other hand, the gamma peptide itself needs association with Na,K-ATPase in order to be stably expressed in the oocyte and to be transported efficiently to the plasma membrane. Gamma subunits do not associate with individual alpha or beta subunits but only interact with assembled, transport-competent alpha-beta complexes. Finally, electrophysiological measurements indicate that the gamma peptide modulates the K+ activation of Na,K pumps. These data document for the first time the membrane topology, the specificity of association and a potential functional role for the gamma subunit of Na,K-ATPase.     

The H,K-ATPase beta-subunit can act as a surrogate for the beta-subunit of Na,K-pumps.

Na,K-ATPase and H,K-ATPase are the only members of the P-type ATPases in which a glycosylated beta-subunit is part of the purified active enzyme. In this study, we have followed the synthesis and the posttranslational processing of the beta-subunit of H,K-ATPase (beta HK) in Xenopus oocytes injected with beta HK cRNA and have tested whether it can act as a surrogate for the beta-subunit of Na,K-ATPase (beta NaK) to support the functional expression of Na,K-pumps. In Xenopus oocytes, beta HK is processed from an Endo H-sensitive 51-kDa coreglycosylated form to an Endo H-resistant 71-kDa fully glycosylated form. Similar to beta NaK, beta HK can stabilize and increase the trypsin resistance of alpha-subunits of Na,K-ATPase (alpha NaK). Finally, expression of beta HK together with alpha NaK leads to an increased number of ouabain binding sites at the plasma membrane accompanied by an increased Rb+ uptake and Na,K-pump current. Our data suggest that beta HK, similar to beta NaK, can assemble to alpha NaK, support the structural maturation and the intracellular transport of catalytic alpha NaK, and ultimately form active alpha NaK-beta HK complexes with Na,K-pump transport properties.     

Oligomerization of the Na,K-ATPase in cell membranes

The higher order oligomeric state of the Na,K-ATPase alphabeta heterodimer in cell membranes is the subject of controversy. We have utilized the baculovirus-infected insect cell system to express Na,K-ATPase with alpha-subunits bearing either His(6) or FLAG epitopes at the carboxyl terminus. Each of these constructs produced functional Na,K-ATPase alphabeta heterodimers that were delivered to the plasma membrane (PM). Cells were simultaneously co-infected with viruses encoding alpha-His/beta and alpha-FLAG/beta Na,K-ATPases. Co-immunoprecipitation of the His-tagged alpha-subunit in the endoplasmic reticulum (ER) and PM fractions of co-infected cells by the anti-FLAG antibody demonstrates that protein-protein associations exist between these heterodimers. This suggests the Na,K-ATPase is present in cell membranes in an oligomeric state of at least (alphabeta)(2) composition. Deletion of 256 amino acid residues from the central cytoplasmic loop of the alpha-subunit results in the deletion alpha-4,5-loop-less (alpha-4,5LL), which associates with beta but is confined to the ER. Co-immunoprecipitation demonstrates that when this inactive alpha-4,5LL/beta heterodimer is co-expressed with wild-type alphabeta, oligomers of wild-type alphabeta and alpha-4,5LL/beta form in the ER, but the alpha-4,5LL mutant remains retained in the ER, and the wild-type protein is still delivered to the PM. We conclude that the Na,K-ATPase is present as oligomers of the monomeric alphabeta heterodimer in native cell membranes.     

Betam,a structural member of the X,K-ATPase beta subunit family,resides in the ER and does not associate with any known X,K-ATPase alpha subunit

betam, a muscle-specific protein, is structurally closely related to the X,K-ATPase beta subunits, but its intrinsic function is not known. In this study, we have expressed betam in Xenopus oocytes and have investigated its biosynthesis and processing as well as its putative role as a chaperone of X,K-ATPase alpha subunits, as a regulator of sarcoplasmic reticulum Ca(2+)-ATPase (SERCA), or as a Ca(2+)-sensing protein. Our results show that betam is stably expressed in the endoplasmic reticulum (ER) in its core glycosylated, partially trimmed form. Both full-length betam, initiated at Met(1), and short betam species, initiated at Met(89), are detected in in vitro translations as well as in Xenopus oocytes. betam cannot associate with and stabilize Na,K-ATPase (NK), or gastric and nongastric H,K-ATPase (HK) alpha isoforms. betam neither assembles stably with SERCA nor is its trypsin sensitivity or electrophoretic mobility influenced by Ca(2+). A mutant, in which the distinctive Glu-rich regions in the betam N-terminus are deleted, remains stably expressed in the ER and can associate with, but not stabilize X,K-ATPase alpha subunits. On the other hand, a chimera in which the ectodomain of betam is replaced with that of beta1 NK associates efficiently with alpha NK isoforms and produces functional Na,K-pumps at the plasma membrane. In conclusion, our results indicate that betam exhibits a cellular location and functional role clearly distinct from the typical X,K-ATPase beta subunits.     

Phosphorylation of Na,K-ATPase alpha-subunits in microsomes and in homogenates of Xenopus oocytes resulting from the stimulation of protein kinase A and protein kinase C.

The phosphorylation of the alpha-subunit of Na+/K(+)-transporting ATPase (Na,K-ATPase) by cAMP-dependent protein kinase (PKA) and protein kinase C (PKC) was characterized in purified enzyme preparations of Bufo marinus kidney and duck salt gland and in microsomes of Xenopus oocytes. In addition, we have examined cAMP and phorbol esters, which are stimulators of PKA and PKC, respectively, for their ability to provoke the phosphorylation of alpha-subunits of Na,K-ATPase in homogenates of Xenopus oocytes. In the enzyme from the duct salt gland, phosphorylation by PKA and PKC occurs on serine and threonine residues, whereas in the enzyme from B. marinus kidney and Xenopus oocytes, phosphorylation by PKA occurs only on serine residues. Phosphopeptide analysis indicates that a site phosphorylated by PKA resides in a 12-kDa fragment comprising the C terminus of the polypeptide. Studies of phosphorylation performed on homogenates of Xenopus oocytes show that not only endogenous oocyte Na,K-ATPase but also exogenous Xenopus Na,K-ATPase expressed in the oocyte by microinjection of cRNA can be phosphorylated in response to stimulation of oocyte PKA and PKC. In conclusion, these data are consistent with the possibility that the alpha-subunit of Na,K-ATPase can serve as a substrate for PKA and PKC in vivo.     

Characterization of Na,K-ATPase and H,K-ATPase enzymes with glycosylation-deficient beta-subunit variants by voltage-clamp fluorometry in Xenopus oocytes

The role of N-linked glycosylation of beta-subunits in the functional properties of the oligomeric P-type ATPases Na,K- and H,K-ATPase has been examined by expressing glycosylation-deficient Asn-to-Gln beta-variants in Xenopus oocytes. For both ATPases, the absence of the huge N-linked oligosaccharide moiety on the beta-subunit does not affect alpha/beta coassembly, plasma membrane delivery or functional activity of the holoenzyme. Whereas this is in line with several previous glycosylation studies on Na,K-ATPase, this is the first report showing that the cell surface delivery and enzymatic activity of the gastric H,K-ATPase is unaffected by the lack of N-linked glycosylation. Sulfhydryl-specific labeling of introduced cysteine reporter sites with the environmentally sensitive fluorophore tetramethylrhodamine-6-maleimide (TMRM) upon expression in Xenopus oocytes enabled us to further investigate potential effects of the N-glycans on more subtle enzymatic properties, like the distribution between E 1P/E 2P states of the catalytic cycle and the kinetics of the E 1P/E 2P conformational transition under presteady state conditions. For both Na,K-ATPase and H,K-ATPase, we observed differences in neither the voltage-dependent E 1P/E 2P ratio nor the kinetics of the E 1P/E 2P transition between holoenzymes comprising glycosylated and glycosylation-deficient beta-subunits. We conclude that the N-linked glycans on these essential accessory subunits of oligomeric P-type ATPases are dispensable for proper folding, membrane stabilization of the alpha-subunit and transport function itself. Glycosylation is rather important for other cellular functions not relevant in the oocyte expression system, such as intercellular interactions or basolateral versus apical targeting in polarized cells, as demonstrated in other expression systems.     

Expression, localization, and function of an N-terminal half fragment of the rat Na,K-ATPase beta-subunit in HeLa cells.

The N-terminal half of the beta-subunit of rat brain Na,K-ATPase was expressed in HeLa cells transfected with the plasmid pSV2TKneo beta N containing the truncated beta-subunit cDNA to study the assembly and transport of alpha-beta complex. Immunoprecipitation from extracts of metabolically labeled transformed cells demonstrated that the truncated beta-subunit polypeptide (beta N) was neither transported to the plasma membrane nor assembled into an alpha-beta complex with the endogenous alpha-subunit. Cell fractionation experiments showed that the beta N truncated subunit remained unassembled within rough microsomes, suggesting that it never exited from the endoplasmic reticulum (ER). The assembly of the endogenous alpha-and beta-subunits in the beta N-expressing cells was significantly inhibited compared with control cells or with the transformants that did not express the beta N. These results suggest that the N-terminal portion of the beta-subunit interferes with the normal assembly of the endogenous complex which normally takes place in the ER.     

The functional role of the beta-subunit in the maturation and intracellular transport of Na,K-ATPase.

The minimal functional enzyme unit of Na,K-ATPase consists of an alpha-beta complex. The alpha-subunit bears all functional domains of the enzyme and so far a regulatory role for the beta-subunit in the catalytic cycle has not been established. On the other hand, increasing experimental evidence suggests that the beta-subunit is an indispensable element for the structural and functional maturation of the enzyme as well as its intracellular transport to the plasma membrane. This brief review summarizes the experimental data supporting the hypothesis that assembly of the beta-subunit is needed for the alpha-subunit to acquire the correct, stable configuration necessary for the acquisition of functional properties and its exit from the ER.     

The alpha and beta subunits of the Na,K-ATPase can assemble at the plasma membrane into functional enzyme

Synthesis and assembly of most oligomeric plasma membrane proteins occurs in the ER. However, the role the ER plays in oligomerization is unknown. We have previously demonstrated that unassociated alpha and beta subunits of the Na,K-ATPase are targeted to the plasma membrane when individually expressed in baculovirus-infected Sf-9 cells. This unique property allows us to determine if assembly of these two polypeptides is restricted to the ER, or if it can also occur at the plasma membrane. To investigate the assembly of the Na,K-ATPase we have taken advantage of the ability of baculovirus-infected cells to fuse. Lowering the extracellular pH of the infected cells triggers an endogenously expressed viral protein to initiate plasma membrane fusion. When individual Sf-9 cells expressing either the Na,K-ATPase alpha or beta subunits are plated together and subjected to a mild acidic shock, they form large syncytia. In the newly continuous plasma membrane the separate alpha and beta polypeptides associate and assemble into functional Na,K-ATPase molecules. However, a hybrid ATPase molecule consisting of a Na,K-ATPase alpha subunit and a H,K- ATPase beta subunit, which efficiently assembles in the ER of coinfected cells, does not assemble at the plasma membrane of fused cells. When cells expressing the Na,K-ATPase alpha subunit are fused to cells coexpressing the Na,K-ATPase beta subunit and the H,K-ATPase beta subunit, the Na,K-ATPase alpha subunit selectively assembles with the Na,K-ATPase beta subunit. However, when cells are coinfected and expressing all three polypeptides, the Na,K-ATPase alpha subunit assembles with both beta subunits in the ER, in what appears to be a random fashion. These experiments demonstrate that assembly between some polypeptides is restricted to the ER, and suggests that the ability of the Na,K-ATPase alpha and beta subunits to leave the ER and assemble at the plasma membrane may represent a novel mechanism of regulation of activity.     

The steroid-binding subunit of the Na/K-ATPase as a progesterone receptor on the amphibian oocyte plasma membrane

Progesterone acts at a plasma membrane receptor on the Rana oocyte to initiate meiosis. A cascade of lipid messengers occurs within seconds, followed by sequential changes in membrane phospholipid composition. We now show that progesterone binding to the plasma membrane increases continuously over the first 4 h. Subsequently, about 60% of the total plasma membrane and > 90% of membrane-bound progesterone, ouabain binding sites, and Na/K-ATPase activity are internalized. Until the completion of membrane internalization, oocytes must be continuously exposed to nanomolar concentrations of exogenous progesterone for meiosis to continue. The membrane-bound progesterone remains unchanged, whereas microinjected [(3)H]progesterone is rapidly metabolized. We find that progesterone and the plant steroid ouabain compete for one of two ouabain binding sites on the oocyte surface. Ouabain blocks progesterone action and inhibits subsequent meiosis if added at any time during the first 4-5 h. Western blots of SDS/PAGE extracts of isolated oocyte plasma membranes contain a -110 kDa band which binds an antibody to the steroid-binding c-terminal domain in rat and human PR. The number of binding sites and K(d) for progesterone binding to the plasma membrane is comparable to those for low-affinity ouabain binding to the alpha-subunit of the Na/K-ATPase (112 kDa). Our results suggest that progesterone binding to the ouabain binding site on the N-terminal region of the alpha-subunit of Na/K-ATPase may modulate early plasma membrane events over the first 4-6 h. Progesterone may thus act in part through the plasma membrane Na/K-ATPase signaling system.     

Na,K-ATPase mutations in familial hemiplegic migraine lead to functional inactivation

The Na,K-ATPase is an ion-translocating transmembrane protein that actively maintains the electrochemical gradients for Na+ and K+ across the plasma membrane. The functional protein is a heterodimer comprising a catalytic alpha-subunit (four isoforms) and an ancillary beta-subunit (three isoforms). Mutations in the alpha2-subunit have recently been implicated in familial hemiplegic migraine type 2, but almost no thorough studies of the functional consequences of these mutations have been provided. We investigated the functional properties of the mutations L764P and W887R in the human Na,K-ATPase alpha2-subunit upon heterologous expression in Xenopus oocytes. No Na,K-ATPase-specific pump currents could be detected in cells expressing these mutants. The binding of radiolabelled [3H]ouabain to intact cells suggested that this could be due to a lack of plasma membrane expression. However, plasma membrane isolation showed that the mutated pumps are well expressed at the plasma membrane. 86Rb+-flux and ATPase activity measurements demonstrated that the mutants are inactive. Therefore, the primary disease-causing mechanism is loss-of-function of the Na,K-ATPase alpha2-isoform.     

beta-subunit assembly is essential for the correct packing and the stable membrane insertion of the H,K-ATPase alpha-subunit

The alpha-subunits of H,K-ATPase (HKAalpha) and Na,K-ATPase require a beta-subunit for maturation. We investigated the role of the beta-subunit in the membrane insertion and stability of the HKAalpha expressed in Xenopus oocytes. Individual membrane segments M1, M2, M3, M4, and M9 linked to a glycosylation reporter act as signal anchor (SA) motifs, and M10 acts as a partial stop transfer motif. In combined HKAalpha constructs, M2 acts as an efficient stop transfer sequence, and M3 acts as a SA sequence. However, M5 and M9 have only partial SA function, and M7 has no SA function. Consistent with the membrane insertion properties of segments in combined alpha constructs, M1-3 alpha-proteins are resistant to cellular degradation, and M1-5 up to M1-10 alpha-proteins are not resistant to cellular degradation. However, co-expression with beta-subunits increases the membrane insertion of M9 in a M1-9 alpha-protein and completely protects M1-10 alpha-proteins against cellular degradation. Our results indicate that HKAalpha N-terminal (M1-M4) membrane insertion and stabilization are mediated by intrinsic molecular characteristics; however, the C-terminal (M5-M10) membrane insertion and thus the stabilization of the entire alpha-subunit depend on intramolecular and intermolecular beta-subunit interactions that are similar but not identical to data obtained for the Na,K-ATPase alpha-subunit.     

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

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.     

Intersubunit interactions in human X,K-ATPases: role of membrane domains M9 and M10 in the assembly process and association efficiency of human, nongastric H,K-ATPase alpha subunits (ATP1al1) with known beta subunits

Na,K- and H,K-ATPase (X,K-ATPase) alpha subunits need association with a beta subunit for their maturation, but the authentic beta subunit of nongastric H,K-ATPase alpha subunits has not been identified. To better define alpha-beta interactions in these ATPases, we coexpressed human, nongastric H,K-ATPase alpha (AL1) and Na,K-ATPase alpha1 (alpha1NK) as well as AL1-alpha1 and alpha1-AL1 chimeras, which contain exchanged M9 and M10 membrane domains, together with each of the known beta subunits in Xenopus oocytes and followed their resistance to cellular and proteolytic degradation and their ER exit. We show that all beta subunits (gastric betaHK, beta1NK, beta2NK, beta3NK, or Bufo bladder beta) can associate efficiently with alpha1NK, but only gastric betaHK, beta2NK, and Bufo bladder beta can form stably expressed AL1-beta complexes that can leave the ER. The trypsin resistance and the forces of subunit interaction, probed by detergent resistance, are lower for AL1-beta complexes than for alpha1NK-beta complexes. Furthermore, chimeric alpha1-AL1 can be stabilized by beta subunits, but alpha1-AL1-gastric betaHK complexes are retained in the ER. On the other hand, chimeric AL1-alpha1 cannot be stabilized by any beta subunit. In conclusion, these results indicate that (1) none of the known beta subunits is the real partner subunit of AL1 but an as yet unidentified, authentic beta should have structural features resembling gastric betaHK, beta2NK, or Bufo bladder beta and (2) beta-mediated maturation of alpha subunits is a multistep process which depends on the membrane insertion properties of alpha subunits as well as on several discrete events of intersubunit interactions.     

Identification of a phospholemman-like protein from shark rectal glands. Evidence for indirect regulation of Na,K-ATPase by protein kinase c via a novel member of the FXYDY family

The Na,K-ATPase provides the driving force for many ion transport processes through control of Na(+) and K(+) concentration gradients across the plasma membranes of animal cells. It is composed of two subunits, alpha and beta. In many tissues, predominantly in kidney, it is associated with a small ancillary component, the gamma-subunit that plays a modulatory role. A novel 15-kDa protein, sharing considerable homology to the gamma-subunit and to phospholemman (PLM) was identified in purified Na,K-ATPase preparations from rectal glands of the shark Squalus acanthias, but was absent in pig kidney preparations. This PLM-like protein from shark (PLMS) was found to be a substrate for both PKA and PKC. Antibodies to the Na, K-ATPase alpha-subunit coimmunoprecipitated PLMS. Purified PLMS also coimmunoprecipitated with the alpha-subunit of pig kidney Na, K-ATPase, indicating specific association with different alpha-isoforms. Finally, PLMS and the alpha-subunit were expressed in stoichiometric amounts in rectal gland membrane preparations. Incubation of membrane bound Na,K-ATPase with non-solubilizing concentrations of C(12)E(8) resulted in functional dissociation of PLMS from Na,K-ATPase and increased the hydrolytic activity. The same effects were observed after PKC phosphorylation of Na,K-ATPase membrane preparations. Thus, PLMS may function as a modulator of shark Na,K-ATPase in a way resembling the phospholamban regulation of the Ca-ATPase.     

Concepts of the oligomeric structure and function of Na, K-ATPase based on the results of studying ligand binding and of determining the size of the molecular target

This review is devoted to the discussion of the sizes and molecular structure of the minimal functional unit of Na, K-ATPase. Special attention is paid to the data obtained by radiation inactivation method and studies on ligand binding. The model for the stepwise radiation inactivation of Na, K-ATPase is proposed. The conclusion is drawn that Na, K-ATPase has a dimeric structure, the interactions between its alpha-subunits stabilize the quaternary structure of the pump. Functionally, each alpha-subunit in a stabilized structure possesses a full hydrolytic activity.     

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

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 alpha and beta subunits of Na,K-ATPase. Xenopus oocytes injected with alpha and/or beta cRNA were treated with brefeldin A, which permitted the accumulation of individual subunits or alpha-beta complexes in the ER. Only alpha subunits that are associated with beta subunits become resistant to trypsin digestion and cellular degradation. Similarly, assembly with beta subunits is necessary and probably sufficient for the catalytic alpha 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, ouabain-inhibitable fashion. Not only the alpha but also the beta 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 NH2- terminally modified beta subunits revealed a K(+)-dependent interaction of the cytoplasmic NH2 terminus of the beta subunit with the alpha 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.     

Expression of an active Na,K-ATPase with an alpha-subunit lacking all twenty-three native cysteine residues

We have constructed a mutant Na,K-ATPase alpha1-subunit with all native cysteine residues replaced. Using the baculovirus system, this cysteine-less alpha1-subunit and wild-type beta1-subunit were expressed in High Five cells. After 3 days of infection, cells were fractionated, and endoplasmic reticulum, Golgi apparatus, and plasma membranes were isolated. The molecular activity of the cysteine-less mutant in the plasma membranes was close to the wild-type protein (8223 min(-)(1) versus 6655 min(-)(1)). Cation and ATP activation of Na,K-ATPase activities revealed that replacing all 23 cysteines resulted in only a 50% reduction of K(m) for Na(+), a 2-fold increase in K(m) for K(+), and no changes in K(m) for ATP. The distribution of alpha-subunits among the membranes showed a high percentage of cysteine-less protein in the endoplasmic reticulum and Golgi apparatus compared with the wild-type protein. Furthermore, the cellular stability of the alphabeta assembly appeared reduced in the cysteine-less mutant. Cells harvested after more than 3 days of infection showed extensive degradation of the cysteine-less alpha-subunit, which is not observed with the wild-type enzyme. Thus the Na,K-ATPase contains no cysteine residues that are critical for function, but the folding and/or assembly pathway of this enzyme is affected by total cysteine substitution.