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

The β-subunit of the Na,K-ATPase is required to deliver functional αβ–heterodimers to the plasma membrane (PM) of baculovirus-infected insect cells. We have investigated the molecular determinants in the β-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 α-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 β-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 β-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 β-subunits and some disulfide bridge substitution mutants assemble with the α-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. The β-subunit of the Na,K-ATPase is required to deliver functional αβ–heterodimers to the plasma membrane (PM) of baculovirus-infected insect cells. We have investigated the molecular determinants in the β-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 α-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 β-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 β-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 β-subunits and some disulfide bridge substitution mutants assemble with the α-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. The Na,K-ATPase is an ion-transporting plasma membrane (PM) 1The abbreviations used are: PM, plasma membrane; ER, endoplasmic reticulum; G, Golgi apparatus; TPCK, N-p-tosyl-l-phenylalanine chloromethyl ketone; PBS, phosphate-buffered saline.1The abbreviations used are: PM, plasma membrane; ER, endoplasmic reticulum; G, Golgi apparatus; TPCK, N-p-tosyl-l-phenylalanine chloromethyl ketone; PBS, phosphate-buffered saline. protein that exchanges three intracellular Na+ ions for two extracellular K+ ions in an ATP-dependent fashion. The Na,K-ATPase belongs to the P-type ATPase family of membrane enzymes, which are characterized by their ability to harness the energy of ATP hydrolysis to pump ions across the membrane against electrochemical gradients through the formation of a phosphoenzyme intermediate. The Na,K-ATPase and closely related H,K-ATPase are the only members of this family composed of two subunits, α and β (1Lutsenko S. Kaplan J.H. Biochemistry. 1995; 34: 15607-15613Crossref PubMed Scopus (417) Google Scholar). The α-subunit is the P-type defining subunit of 1016 amino acid residues containing an ATP hydrolysis domain and 10 transmembrane segments capable of ion occlusion (for a review, see Ref. 2Kaplan J.H. Annu. Rev. Biochem. 2002; 71: 511-535Crossref PubMed Scopus (885) Google Scholar). Although the β-subunit is essential for pump activity (3Hasler U. Wang X. Crambert G. Beguin P. Jaisser F. Horisberger J.D. Geering K. J. Biol. Chem. 1998; 273: 30826-30835Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar, 4Jaisser F. Jaunin P. Geering K. Rossier B.C. Horisberger J.D. J. Gen. Physiol. 1994; 103: 605-623Crossref PubMed Scopus (131) Google Scholar, 5Jaunin P. Jaisser F. Beggah A.T. Takeyasu K. Mangeat P. Rossier B.C. Horisberger J.D. Geering K. J. Cell Biol. 1993; 123: 1751-1759Crossref PubMed Scopus (74) Google Scholar, 6Lutsenko S. Kaplan J.H. Biochemistry. 1993; 32: 6737-6743Crossref PubMed Scopus (125) Google Scholar), a primary role currently attributed to the β-subunit is that of a molecular chaperone to aid in the correct membrane insertion, stability, and trafficking of the α-subunit to the PM (for a review, see Ref. 7Geering K. J. Bioenerg. Biomembr. 2001; 33: 425-438Crossref PubMed Scopus (269) Google Scholar). The β1-subunit is a glycoprotein of 303 amino acid residues consisting of a cytoplasmic amino-terminal domain of about 40 residues, a single membrane-spanning segment, and a larger extracellular carboxyl-terminal domain comprising about 240 amino acid residues. The extracellular domain has three N-linked glycosylation sites and three disulfide bonds that are conserved among all of the β-subunit isoforms have investigated the role of the of the β-subunit in an to the β-subunit with the α-subunit and to the functioning of the all of the of the β-subunit have important for the of functional but is of functional pump is to the of the β-subunit to assemble with the heterodimer to the PM, or Na,K-ATPase The for the and β-subunits to a prerequisite for the targeting of the pump to the PM has the of the β-subunit for assembly and for trafficking have not or have attributed to the extracellular domain of the The to functional pump the in the and has to the or of a PM targeting or an for the heterodimer to is not to the assembly and trafficking in of the assembly and delivery is essential for the of of the β-subunit the of functional The a by which the and activity of Na,K-ATPase and the molecular of the β-subunit required for The has for of Na,K-ATPase, no Na,K-ATPase or β-subunits are in the or insect for of membrane have and sufficient of protein produced for Kaplan J.H. J. Physiol. 2001; PubMed Google Scholar). the the of the site and catalytic activity of the Na,K-ATPase, the N-linked the disulfide bridges and the extracellular by and the amino-terminal and in the and activity of that the Na,K-ATPase is to the PM, that enzymatic is not a prerequisite for trafficking to the We that N-linked glycosylation of the β-subunit is not for delivery to the or activity of the Analysis of disulfide mutants reveals that correct formation of the and disulfide bridges is essential for the exit of the heterodimer from the but not for the assembly of the and reveals that no trafficking is in the extracellular domain of the β-subunit and that the extracellular carboxyl-terminal domain of the β-subunit is for enzymatic and were through or a β-subunit mutants required the of an to for Western the the for the a carboxyl-terminal was through The was to the of the β-subunit to a for the containing α or β were by in the or to the The was used to β was site of containing in site α site of the containing β in site Kaplan J.H. J. Physiol. 2001; PubMed Google Scholar, Kaplan J.H. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, Kaplan J.H. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). which the were with and of was for by was and used for to the bridge substitution in the β-subunit and the D369A α-subunit mutation were by of from mutants were by and the presence of the on the of were with containing to the and to for was removed and used to of in for Cell was removed by for was and used for Cell and were and in containing or were from were in the presence of with that of from to were by and and and and by was and were on and of protein was with a protein Western to The and β-subunits were The β-subunit is a the by with and and J. were Kaplan J.H. J. Physiol. 2001; PubMed Google Scholar). were in 10 and by were removed by 10 ER, Golgi, and PM were by on a and and in a for ER, Golgi, and PM were from to in and for was and were in containing TPCK, and and on or for was by the of a Kaplan J.H. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). ATPase activity of membranes was for with of membrane protein in the Kaplan J.H. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). Na,K-ATPase activity was the in the of in the presence and of in of of ATPase for Na,K-ATPase from to of of on and Western protein of ER, Golgi, and PM were in or in the presence of and to were with in for or and with used used or used for in with were three for 10 in and for with used in with were three for 10 in were used for were to with and were in of 10 TPCK, and with was by the membrane through a and for was removed by of was a Western to The was to a and of was to a of with and with for or The of used Kaplan J.H. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google was to the of were increased to for in was and with for were for and was removed by were three for with in of the of was to the of were and was to for Western We have used baculovirus-infected insect to heterodimer assembly of the and trafficking of the heterodimer to the PM, and ATPase activity of Na,K-ATPase in which the enzymatic activity of the pump has by the mutation of the essential site in the α-subunit or in which the β-subunit has through of substitution of disulfide or of carboxyl-terminal of the glycoprotein of the Na,K-ATPase in insect an for the not Na,K-ATPase of Na,K-ATPase are of the are capable of and trafficking of the Kaplan J.H. J. Physiol. 2001; PubMed Google Scholar, Kaplan J.H. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). have that of both the and β-subunits is for targeting the to the PM Kaplan J.H. J. Physiol. 2001; PubMed Google Scholar). the α-subunit is in the of the α is in the the β-subunit is in the of β is to the PM the and β-subunits are both subunits are to the PM The of a carboxyl-terminal to the β-subunit not the or activity of the Na,K-ATPase, the β-subunit to increased and that the β-subunit in insect in the ER, and the β-subunit Western of the of a molecular to which the from to and The of in the Golgi of not in insect cells. the β-subunits from the and PM of insect are in from the in the and The for both the and β-subunits are in a single and all both subunits, but the of the and β-subunits are β-subunits are not a of β-subunits in the PM is β-subunits not in a heterodimer. protein to which is a in for β-subunits both β-subunits that by the against the α-subunit and β-subunits that are for this to the functional activity of the Na,K-ATPase was important for PM targeting of the the in the α-subunit essential for enzymatic with an We the D369A α-subunit with the β-subunit in cells. were by and the ER, G, and PM on a Kaplan J.H. J. Physiol. 2001; PubMed Google Scholar). mutation in trafficking J. Biol. Chem. 1993; Full Text PDF PubMed Google Scholar, S. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar). that both the and β-subunits of the are to the PM of baculovirus-infected with a compartmental distribution the Na,K-ATPase activity with the a D369A has three N-linked glycosylation sites that all three of the sites are G. Kaplan J.H. U. S. PubMed Scopus Google Scholar, A.T. Jaunin P. Geering K. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, PubMed Scopus Google Scholar). to the role that in the and activity of the Na,K-ATPase, the in of the three glycosylation sites with to the β The β-subunit was with the in baculovirus-infected and membrane were the β-subunit a of for a β-subunit to the molecular of β-subunit in the of insect the molecular in the β are not in β are to degradation by sites by the in has for β-subunits in glycosylation was Rossier B.C. Geering K. J. Biol. PubMed Scopus Google Scholar). Analysis of Western blots from membrane for the β-subunit reveals delivery of both subunits from the to the PM distribution is in and Kaplan J.H. J. Physiol. 2001; PubMed Google Scholar). distribution of the ATPase activity of the was to the ATPase for protein are the ER, G, and PM and the heterodimer containing the the distribution of ATPase activity from were and in the ER, G, and PM, The activity distribution with the distribution of α-subunit in Western ATPase of the heterodimer in the PM from to of in different which is with protein is that the of N-linked not the PM or functional activity of the disulfide bridges in the extracellular domain of the β-subunit are conserved β isoforms of both the Na,K-ATPase and the bridges have in enzymatic of the disulfide bonds by the through the of K+ occlusion S. Kaplan J.H. Biochemistry. 1993; 32: 6737-6743Crossref PubMed Scopus (125) Google Scholar). the have in PM of the pump of the in to of protein containing disulfide bridges A.T. Jaunin P. Geering K. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, S. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). is not from the disulfide bridges are important in the heterodimer assembly the trafficking or both processes. the role of the disulfide three point mutants in which both residues to of the disulfide bridges in the β-subunit were with or We the single point which the disulfide bridge a single The bridge mutants were with the α-subunit in and to and of the disulfide bridge through mutation of both residues not targeting of the Na,K-ATPase to the PM, from for both the α-subunit and β-subunit the membrane The β-subunit Western of all bridge substitution produced not in the protein in which a through the of is and blots of and to the presence of or all three glycosylation The molecular of the which to the β-subunit is of with the β-subunit with and blots of and that substitution of the bridges the of glycosylation on the disulfide formation and glycosylation has A.T. Jaunin P. Geering K. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). of the β-subunit by an the α-subunit that the assembly of the bridge β-subunit the heterodimer in the The the of the of disulfide bridge mutants and and and and is a of the on of the mutants to of ATPase activity of the heterodimer containing the β-subunit was and in the ER, G, and PM, which is the distribution The of Na,K-ATPase in the PM from to of and were a of protein from different trafficking was the disulfide bridge was through the of only both residues. The mutation a in for the α-subunit and a of the β-subunit and of are to the PM to an ATPase activity of of in the PM the of the α-subunit and of the the distribution of Na,K-ATPase activity ER, G, and PM with the β-subunit was and which is to that a of the that is is in the ER. the β-subunit is the α-subunit and the α-subunit is the catalytic only the distribution of the α-subunit in to the distribution of ATPase The of ATPase with that two of the β-subunit a and functional which is to the PM in the of the and a which is in the ER. of the β-subunit by that the heterodimer in the this β-subunit only or both and The and β-subunits are both to the the or disulfide bridges are through substitution of both residues and that the formation of the and disulfide bonds an important role in exit from the ER. that the and bridge substitution mutants are capable of assembly with the α-subunit and Although the and disulfide mutants are in the ER, the of heterodimer in the PM is sufficient to but of Na,K-ATPase activity of ATPase activity was in the or that the to the PM a of functional have with the β-subunit that the disulfide bridges an role in the of heterodimer capable of the ER. the produced by the extracellular domain and by the disulfide bonds are for the of a PM in the heterodimer. the bridges or of the domain and of the protein in the with to a of mutants in which the carboxyl-terminal the of of the disulfide was of the disulfide substitution mutants is to of that of segments the are important for a PM targeting their in the of the β-subunit to the of of the disulfide bonds residues and by the in for was with a to the carboxyl-terminal was or to the and The was the for the The was to the β-subunit to the presence of the or activity of the The of the was the and The β-subunit and the α-subunit are and of the that is by the and with the α-subunit in the the of is of and in with in and and ATPase activity was in membrane for the β-subunit and α The β-subunit not N-glycosylation sites and a through the of with the both subunits were in the ER, some protein in the and PM and of heterodimer assembly by not the β-subunit and is not in used for Na,K-ATPase or H,K-ATPase to not ATPase activity in membrane for the and α The all three extracellular of with α in of in sufficient of are to the PM to the α-subunit to to the PM is to the of the β-subunit the α-subunit The β-subunit is capable of assembly with the by with no ATPase activity is in membrane with had that the transmembrane and amino-terminal of the β-subunit no for assembly and J. Biol. Chem. 1993; Full Text PDF PubMed Google Scholar). We the truncated to this membrane insertion, some extracellular residues were and the carboxyl-terminal was to for Western of β-subunit and α-subunit that both subunits are to the PM and of β-subunit by the ability of the β-subunit to assemble a heterodimer with the α-subunit of β-subunit a in the heterodimer. ATPase activity was in membrane with the the have baculovirus-infected insect to the assembly and delivery to the PM of Na,K-ATPase Although the β-subunit is to the PM in the of the the is not the of the the α-subunit is in the Kaplan J.H. J. Physiol. 2001; PubMed Google Scholar). a role for the β-subunit in the trafficking the the of assembly of heterodimer in the and the delivery of the heterodimer or subunits from the to the PM. We the of extracellular of the β-subunit in the and functioning of the that assembly of to exit from the ER, assembly is not sufficient for PM the and disulfide bridge substitution are capable of assembly with the α-subunit but in the ER. Trafficking of mutation of the enzymatic site of in ATPase in the to and intracellular of the pump J. Biol. Chem. 1993; Full Text PDF PubMed Google Scholar, S. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar). in the essential of the Na,K-ATPase was with the of the pump the in not S. K. PubMed Scopus Google Scholar). that the presence of catalytic activity important for the and delivery of P-type the to catalytic of Na,K-ATPase has in membrane from J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar), and trafficking of Na,K-ATPase in has J.H. Biochemistry. PubMed Scopus Google Scholar), that the Na,K-ATPase was to the PM in J.H. Biochemistry. PubMed Scopus Google have the of catalytic mutants among P-type and in the ability to of ATPase in the of pump subunits a in the ability to J.H. Biochemistry. PubMed Scopus Google in that the is to the in in delivery is not have the baculovirus-infected insect and the distribution the ER, G, and PM of Na,K-ATPase containing the α D369A We that the α D369A Na,K-ATPase, is to the PM. that functional activity is not a for PM delivery and the D369A α-subunit of the Na,K-ATPase, the a and of the β1-subunit in has three N-linked glycosylation sites in extracellular the that of all N-linked by the three residues the of with no effect on Na,K-ATPase assembly or PM delivery in insect cells. The distribution of protein among the membrane is in with the from in and in which of glycosylation not or activity of Na,K-ATPase composed of the from Rossier B.C. Geering K. J. Biol. PubMed Scopus Google Scholar, K. S. PubMed Scopus Google Scholar, Rossier B.C. Geering K. Biol. Google Scholar, J. Biol. Chem. Full Text PDF Google Scholar). Although had from with to mutants in that was for the β-subunit to an A.T. Jaunin P. Geering K. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar), have that the β-subunit is capable of delivery to the PM, and enzymatic is not for or of the Na,K-ATPase for subunit G. K. U. S. PubMed Scopus Google Scholar), but to to the of the has that to is by of glycosylation of β Rossier B.C. Geering K. J. Biol. PubMed Scopus Google Scholar), but the degradation in is J. Biol. Chem. Full Text PDF Google Scholar), and intracellular of β-subunit mutants glycosylation have in A.T. Jaunin P. Geering K. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). of susceptibility of the β-subunit in membranes from which is not with the β-subunit in of and by that the N-linked glycosylation of the β1-subunit is of for the or functioning of the Na,K-ATPase but that the glycosylation to the β1-subunit from degradation by and a role in the of the of in and of the all Na,K-ATPase and H,K-ATPase β-subunits conserved extracellular residues that to three disulfide has some or all of essential for S. Kaplan J.H. Biochemistry. 1993; 32: 6737-6743Crossref PubMed Scopus (125) Google Scholar, A.T. Jaunin P. Geering K. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, S. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar, S. 1994; PubMed Scopus Google Scholar). of bridges through has that the formation of the and bridges a the mutation of the bridge A.T. Jaunin P. Geering K. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, S. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar, S. 1994; PubMed Scopus Google Scholar). the the ability of β-subunits to assemble with and the α-subunit and S. 1994; PubMed Scopus Google that the β-subunit of the with the bridge removed assemble with the α-subunit but produced ATPase the bridge substitution mutants were able to assemble with the Beggah A.T. Jaunin P. Geering K. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google that β-subunits with the bridge assemble with the α-subunit and a in the of the and that or disulfide bridges were able to assemble with the The have in the insect the of of the disulfide bonds in the assembly and trafficking of the Na,K-ATPase heterodimer. of β-subunits with an the α-subunit from of of the disulfide bridge substitution mutants that β-subunits are capable of heterodimer assembly and and and substitution of the and disulfide bridge in and to some the the ability of the β-subunits and of the heterodimer to to the PM. that the disulfide the two carboxyl-terminal are important for the and the the of the mutation the disulfide bridge the ability to The of the disulfide through the single mutation of the but of both a heterodimer that is to the PM. is to that the presence of disulfide formation the and some and that this formation to an or the β-subunit in a intermediate. The of correct disulfide formation of the and the bridges in the β-subunit extracellular domain residues are not in the extracellular domain of The of of the disulfide mutants in of two the of disulfide formation to a of the β-subunit extracellular or a or that is essential for trafficking to the PM or to of the We that was to of of the the through the of the extracellular We a of mutants in which the β-subunits were truncated to the of of the disulfide that of larger of the of heterodimer to the PM and PM of Western blots in to conclude that the of the that is a of the β-subunits We that this is through the of the β-subunit with chaperone is that different in are to the of We are currently the role of molecular in the assembly the of trafficking or on the of subunits, which PM of amino acid targeting have in the and of amino are to targeting of through the with an or (for see F. G. Cell 2001; PubMed Scopus Google and Annu. Rev. Cell Biol. PubMed Scopus Google Scholar). in which the β-subunit carboxyl-terminal residues were removed J. Biol. Chem. 1993; Full Text PDF PubMed Google Scholar, A.T. Beguin P. Jaunin P. Geering K. Biochemistry. 1993; 32: PubMed Scopus Google Scholar, in The The to the that the 10 amino acid residues of the β-subunit a PM targeting to the of this β-subunit A.T. Beguin P. Jaunin P. Geering K. Biochemistry. 1993; 32: PubMed Scopus Google Scholar). that residues not an essential role in delivery of the β-subunit to the PM. Elimination of a PM to the PM delivery of the that β-subunits with carboxyl-terminal larger 10 residues not PM delivery. We that β-subunits with larger and to the PM the which the extracellular delivery of the heterodimer to the PM. has that Na,K-ATPase with β-subunits the amino-terminal domain are to the PM (3Hasler U. Wang X. Crambert G. Beguin P. Jaisser F. Horisberger J.D. Geering K. J. Biol. Chem. 1998; 273: 30826-30835Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar, J. Biol. Chem. Full Text PDF PubMed Google Scholar). in which the β-subunit transmembrane domain was with a transmembrane from protein were to the PM, to the that the extracellular domain was in J. Biol. Chem. 1993; Full Text PDF PubMed Google Scholar). and this of to that no PM targeting in the of has to the of the and β-subunits that are in the assembly of the Although the amino-terminal K. Beggah P. S. S. Jaunin P. J. Cell Biol. PubMed Scopus (125) Google and transmembrane Biochemistry. PubMed Scopus Google Scholar, U. Crambert G. Horisberger J.D. Geering K. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google have in with the the β-subunit extracellular domain has and sufficient for assembly with the α-subunit J. Biol. Chem. 1993; Full Text PDF PubMed Google Scholar, Takeyasu K. J. Physiol. 1994; PubMed Google Scholar, J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). the extracellular domain of β1-subunit residues have to with the extracellular of the α-subunit through J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). residues to the from which is to of the to which is to the of the disulfide bridge in the β1-subunit have that the β-subunit which only of the residues in the is capable of assembly with the α-subunit by that the cytoplasmic and transmembrane are the extracellular domain is not for heterodimer We that both the transmembrane and extracellular of the and β-subunits to the subunits and to the of the heterodimer. of some of through not the heterodimer to but the of the the β-subunit of the Na,K-ATPase is the of the two subunits, is that the β-subunit to the functional of the Na,K-ATPase in a is by the different with a of and with different β-subunit is to from single of in a single no of a in the β-subunit that PM the of with the α-subunit that the heterodimer from of the not the extracellular domain had assembly of and β-subunits is required for delivery of functional Na,K-ATPase to the PM, assembly is not sufficient to that the heterodimer to the PM. in in the β-subunit in an in trafficking is not an and some PM delivery We F. for and for