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

(Received for publication, December 3, 1996, and in revised form, January 23, 1997)

Ahmed T. Beggah , Philippe Jaunin and Käthi Geering Dagger

From the Institute of Pharmacology and Toxicology, University of Lausanne, rue du Bugnon 27, CH-1005 Lausanne, Switzerland

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES


ABSTRACT

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 beta 1 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.


INTRODUCTION

Na,K-ATPase is a ubiquitous plasma membrane transporter that is responsible for the maintenance of the potassium and sodium homeostasis in animal cells. The minimal functional enzyme unit is composed of two subunits. The large alpha  subunit has 10 putative transmembrane spans and carries out the catalytic and transport functions of the enzyme. The smaller beta  subunit is a type II glycoprotein with a short cytoplasmic domain, one transmembrane segment, and a large C-terminal ectodomain that comprises several N-glycosylation sites and three highly conserved disulfide bridges. The beta  subunit plays an essential role in the structural and functional maturation of the alpha  subunit. Only alpha  subunits that assemble with beta  subunits in the ER1 are stably expressed, acquire their functional properties, and are transported to the plasma membrane (for review see Ref. 1). Upon assembly, the beta  subunit itself also undergoes a structural maturation that is reflected by an increased resistance against proteolysis (2).

The molecular mechanisms that guide the important process of assembly of the beta  subunit with the alpha  subunit are still poorly understood. The structural requirements for alpha -beta assembly have only been partially characterized (3-7). Several regions in the beta  subunit participate in the interaction with the alpha  subunit. It is possible to distinguish, on the one hand, structural interaction sites including the ecto- and the transmembrane domains of the beta  subunit that mediate the maturation of the alpha  subunit and, on the other hand, functional interaction sites such as the cytoplasmic N terminus that participate in the modulation of the transport activity of the functional enzyme (2).

Little is known how the beta  subunit acquires a conformation that is compatible with assembly, e.g. how it folds into a correct tertiary structure that exposes the structurally defined surfaces assuring the specific interaction with the alpha  subunit. Molecular chaperones such as BiP (binding protein) are associated with the newly synthesized beta  subunit (8) suggesting that they might be important for the correct folding and/or in the ER retention of unassembled beta  subunits.

Because the beta  subunit, as many other membrane and secretory proteins, acquires N-linked sugars and forms intramolecular disulfide bonds during its synthesis in the ER, it appeared interesting to us to use this protein as a model protein to compare the role of these co-translational modifications in protein folding, assembly, and in the functional expression. The role of N-glycosylation and disulfide bonds in the folding of proteins has been extensively studied (for review, see Refs. 9 and 10) but rarely compared in the same protein. Their importance in the folding process varies. When N-glycosylation is prevented by site-directed mutagenesis, many but not all glycoproteins misfold, aggregate, and degrade in the ER (11). Disulfide bonds do or don't directly affect the folding process, but, in most cases, their formation stabilizes the folded form. In this study, we used the Xenopus oocyte expression system to study 1) the structural properties of mutant beta  subunits deficient in glycosylation or disulfide bonds, 2) their interaction with BiP, and 3) their ability to associate with alpha  subunits and to be transported to the plasma membrane as functional alpha ·beta complexes. Our results show that both co-translational modifications play an important but qualitatively distinct role in the correct folding of the beta  subunit. Indeed, the interaction of the beta  subunit with molecular chaperones, its stability, and its association efficiency are differently affected after abolition of glycosylation sites or cysteine bonds.


EXPERIMENTAL PROCEDURES

Site-directed Mutagenesis

Site-directed mutagenesis was performed according to the polymerase chain reaction method of Nelson and Long (12). A linearized pSD5 vector (13) containing a beta 1cDNA (pSD5beta 1) of Xenopus Na,K-ATPase (14) was used as a template for the preparation of cysteine mutants (see Fig. 1). First, single mutants in which Cys149, Cys175, or Cys276 were substituted by a serine residue were prepared. The DNAs of the single mutants served as templates to prepare double mutants in which Cys126/Cys149 (CC1), Cys159/Cys175 (CC2), or Cys213/Cys276 (CC3), respectively, were substituted with serine residues. The mutated DNA fragments of the CC1 and CC2 mutants were introduced into the wild type pSD5beta 1 by using unique BamHI and HindIII restriction sites and that of the CC3 mutant by using HindIII and SmaI (present in the vector).


Fig. 1. Site-directed mutagenesis of the beta 1 subunit of Xenopus Na,K-ATPase affecting cysteine residues implicated in disulfide bond formation and asparagine residues implicated in N-glycosylation. A, linear model of the beta 1 subunit of Xenopus Na,K-ATPase indicating the cytoplasmic N terminus, the transmembrane (M) span, and the ectodomain with the six disulfide bond-forming cysteine residues and the three N-glycosylation sites. wt, wild type. B, mutants with cysteines replaced by serine residues. C, mutants with glycosylation consensus asparagines replaced by glutamine residues.
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A linearized pGEM2 vector or a pSD5 vector containing a beta 1cDNA (pGEM2beta 1, pSD5beta 1) of Xenopus Na,K-ATPase was used as a template for the preparation of the N2 or the N1 and the N3 glycosylation mutants, respectively (see Fig. 1). In single mutants Asn158, Asn193, or Asn265, respectively, were substituted by a glutamine residue. The mutated DNA fragments of the N1 and N2 mutants were introduced into the wild type pSD5beta 1 by using BamHI and PstI restriction sites and that of the N3 mutant by using HindIII and SmaI. For the construction of the double mutant N2,3, the mutated BamHI-PstI fragment of the pGEMbeta 1 N193Q mutant was introduced into the pSD5beta 1 N265Q (N3) mutant. For the preparation of the triple mutant N1,2,3, the mutated BamHI-HindIII fragment of the N158Q (N1) mutant was introduced into the pSD5beta 1 N193Q/N265Q (N2,3) mutant. All polymerase chain reaction-generated fragments were sequenced by dideoxy sequencing (15). All mutant beta 1 subunits were tested for their translation efficiency in a reticulocyte lysate (6).

Expression in Xenopus Oocytes and Immunoprecipitation of alpha  and beta  Subunits of Na,K-ATPase

cRNAs encoding Xenopus alpha 1 (14), beta 1 (14), and beta  mutants were obtained by in vitro transcription (16). Stage V and VI oocytes were obtained from Xenopus females (Noerdhoek, Republic of South Africa) as described (6). Routinely, 5-7 ng of alpha  and/or 0.2-0.6 ng of beta cRNA were injected into oocytes. Oocytes were incubated in modified Barth's medium containing 0.6-2.5 mCi/ml [35S]methionine (Amersham Corp.) for the times indicated in the figure legends and eventually subjected to different chase periods in the presence of 10 mM cold methionine. Microsomes were prepared as described (2), and the alpha  and beta  subunits of Na,K-ATPase or BiP were immunoprecipitated under denaturing or nondenaturing conditions as described (6, 8) by using alpha  antibodies produced against the N terminus of the Xenopus alpha 1 subunit (17), beta  antibodies against the ectodomain of the Xenopus beta 1 subunit (17), or BiP antibodies produced against Xenopus BiP (8). In some instances, immunoprecipitates were subjected to endoglycosidase (Endo H) (Calbiochem) treatment as described (18). Dissociated immunocomplexes were separated by SDS-PAGE in the absence or presence of 2% beta -mercaptoethanol and the labeled proteins revealed by fluorography. Quantifications and determinations of the molecular mass of the immunoprecipitated bands were performed with an analytic program for electrophoretic images (Bio-1D) from Vilbert Lourmat (Marne La Vallée, France).

Proteolysis of beta  Subunits

Wild type and mutant beta cRNAs were injected into oocytes together with 3 µCi/oocyte of [35S]methionine. Oocytes were incubated for 5 h before homogenization by 12 gentle strokes with a glass Teflon homogenizer in a solution containing in mM, 250 sucrose, 50 potassium acetate, 5 MgCl2, 1 dithiothreitol, 50 Tris-HCl, pH 7.5. After addition of 10 mM CaCl2, samples were incubated in the absence or presence of 200 mg/ml proteinase K (Merck) and in the absence or presence of 1% Triton X-100 for 1 h at 4 °C. Proteolysis was stopped with 5 mM phenylmethylsulfonyl fluoride and 3.7% hot SDS before immunoprecipitation of the samples.

[3H]Ouabain Binding to Intact Oocytes

[3H]Ouabain binding was essentially done as described (6). After cRNA injection, oocytes were incubated for 2-4 days. Oocytes were then loaded with Na+ by incubation for 2 h in a solution containing in mM, 110 NaCl, 10 Tris-HCl, pH 7.5. After recovery for 30 min in a solution containing in mM, 90 NaCl, 2 CaCl2, 5 BaCl2, 20 tetraethylammonium chloride, 5 MOPS, pH 7.4, the oocytes were transferred to a solution containing in mM, 50 NaCl, 50 N-methyl-D-glucamine-Hepes, 2 CaCl2, 5 BaCl2, 20 tetraethylammonium chloride, 5 MOPS, pH 7.4, and 1 µM [21,22-3H]ouabain (Amersham Corp.) was added. In some instances, increasing concentrations of KCl were added to study the competition of ouabain binding. In this case, the osmolality of the solution was maintained by adapting the concentration of N-methyl-D-glucamine-Hepes. After 12 min at 19 °C, oocytes were washed with a solution containing in mM, 90 NaCl, 2 CaCl2, 5 BaCl2, 5 MOPS, pH 7.4, before dissolution in 0.5% SDS and counting. Nonspecific ouabain binding determined in the presence of a 1000-fold excess of cold ouabain amounted to about 3-7% of the total binding.

86Rb+ Uptake Measurements

To study the K+ activation of the Na,K-ATPase-mediated 86Rb+ uptake, oocytes were injected with Xenopus wild type or mutant beta 1cRNA plus alpha cRNA coding for alpha 1 subunits of Bufo marinus Na,K-ATPase that is more ouabain resistant than the Xenopus alpha 1 subunit (19). After loading of the oocytes with Na+ (see above) and recovery in a solution containing in mM, 90 NaCl, 1 CaCl2, 5 BaCl2, 10 Hepes, pH 7.4, oocytes were transferred to a solution containing varying concentrations of KCl, 90 mM TMA-Cl or 90 mM NaCl, 2 mM CaCl2, 5 mM BaCl2, 1 mM MgCl2, 10 mM Hepes, pH 7.4, and 1 µM ouabain to inhibit the endogenous, oocyte Na,K-ATPase (19). After addition of 5 µCi/ml 86Rb+ (Amersham Corp.), oocytes were incubated for 12 min at room temperature before washing in a solution containing in mM, 90 TMA-Cl, 1 CaCl2, 1 MgCl2, 10 KCl, Hepes, pH 7.4. To determine the ouabain sensitivity of the Na,K-ATPase-mediated 86Rb+ uptake, oocytes were injected with Xenopus wild type or mutant beta 1cRNA plus Xenopus alpha 1cRNA, and 86Rb+ uptake measurements were performed in the presence of 90 mM NaCl and varying concentrations of ouabain. The parameters of the Hill equation V = Vmax/[1+(K1/2/CK)nH were fitted to the data of the 86Rb+ uptake (V) induced by various concentrations of K+ (CK) and yielded least-square estimates of the maximal uptake (Vmax), the half-maximal activation constant (K1/2), and the Hill coefficient (nH).


RESULTS

Effects of Abolition of Disulfide Bonds in the beta  Subunit of Na,K-ATPase on Its Initial Folding and Its Interaction with BiP

To assess the role in the initial folding of the three disulfide bonds present in the beta  subunit of oligomeric P-type ATPase, we introduced mutations in each S-S bond-forming cysteine pair (CC1, CC2, CC3, see Fig. 1) of Xenopus beta 1 subunits. The degree of misfolding of the disulfide bond-deficient mutants was probed by various tests after expression and metabolic labeling in Xenopus oocytes. In a first test, the structural characteristics of the immunoprecipitated, wild type beta  subunits and of the cysteine mutants were compared by performing SDS-PAGE under nonreducing conditions (Fig. 2). The abolition of each one of the disulfide bonds appears to have a distinct effect on the structure of the mutants since their migration in the absence of beta -mercaptoethanol differed slightly from that of the wild type and among each other (Fig. 2A, lanes 5-8). In comparison, the migration in the presence of beta -mercaptoethanol of wild type and mutant beta  subunits was generally slower and identical (Fig. 2A, lanes 1-4). Overexposure of a gel from a similar experiment (Fig. 2B) revealed in addition that a small fraction of individual Xenopus beta 1 subunits, which are retained in the ER (17), occur as disulfide-bonded multimers. However, aggregate formation seen with nonreduced samples is quantitatively and qualitatively similar to wild type (Fig. 2B, lane 5) and disulfide bond-deficient (lanes 6-8) beta  subunits. This result indicates that the lack of one cysteine bond does not enhance the tendency of individual beta  subunits to form inappropriate, intermolecular disulfide bonds and beta  aggregates.


Fig. 2. Migration of Xenopus beta 1 cysteine mutants on SDS-polyacrylamide gels under nonreducing conditions. Oocytes were injected with 0.3 ng of wild type Xenopus beta 1 (A and B, lanes 1 and 5) or mutant beta 1 (A and B, lanes 2-4, and 6-8) cRNA and metabolically labeled for 24 h before preparation of microsomes. The beta  subunits were immunoprecipitated, treated (A and B, lanes 1-4) or not treated (A and B, lanes 5-8) with beta -mercaptoethanol (beta -mercapto.), and resolved by SDS-PAGE and fluorography as described under "Experimental Procedures." A, the immunoprecipitated beta  subunits were deglycosylated with Endo H to better resolve differences in the migration on SDS-polyacrylamide gels. The gel shown in B was overexposed to reveal the small fraction of disulfide-bonded aggregates. Mutants are described in Fig. 1. st, standard proteins of known molecular mass.
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To further characterize the structural effect of the abolition of disulfide bonds, homogenates of oocytes expressing wild type or mutant beta  subunits were subjected to proteolysis in the absence or presence of Triton X-100. Proteinase K did not attack the wild type beta  subunit (Fig. 3A, lanes 1 and 2) but digested the three mutants by about 40% (lanes 5, 8, and 11). Probably due to partial cleavage of the cytoplasmic N terminus (2), protease digestion also produced a slight shift in the molecular mass of the residual mutant proteins (lanes 5, 8, and 11) and to a lesser extent in that of the wild type beta  subunits (lane 2). Finally, in the presence of Triton X-100, which opens closed membrane vesicles and exposes the ectodomain of the beta  subunit, only the wild type (lane 3) but not the mutant beta  subunits (lanes 6, 9 and 12) is partially resistant to proteinase K digestion. Thus, abolition of each one of the disulfide bonds produces a structural effect on the beta  subunit that is reflected by an increased protease sensitivity.


Fig. 3. Sensitivity of beta  cysteine mutants to degradation by proteinase K and cellular proteases and their interaction with BiP. A, digestion of cysteine mutants with proteinase K. Oocytes were injected with 0.2 ng of wild type (lanes 1-3) or 0.3 ng of mutant (lanes 4-12) beta cRNA and 3 µCi/oocyte of [35S]methionine. After 5 h of incubation, homogenates were prepared as described under "Experimental Procedures," and aliquots were incubated for 1 h at 4 °C in the absence (lanes 1, 4, 7 and 10) or presence (lanes 2, 3, 5, 6, 8, 9, 11, and 12) of 200 mg/ml proteinase K and in the absence (lanes 1, 2, 4, 5, 7, 8, 10, and 11) or presence (lanes 3, 6, 9, and 12) of 1% Triton X-100. After addition of 5 mM phenylmethylsulfonyl fluoride and 3.7% (final concentration) hot SDS, the beta  subunits were immunoprecipitated under denaturing conditions and revealed by SDS-PAGE and fluorography. B, cellular degradation of cysteine mutants. Oocytes were injected with 0.2 ng of wild type (lanes 1-3) or 0.3 ng of mutant (lanes 4-12) beta cRNA, metabolically labeled for 24 h, and subjected to a 24- or a 72-h chase. After the pulse and the chase periods, microsomes were prepared, and the beta  subunits were immunoprecipitated under denaturing conditions. C, interaction of cysteine mutants with BiP. Aliquots of samples prepared as described in B were immunoprecipitated with an anti-BiP serum under nondenaturing conditions. A-C show one out of three similar experiments.
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We have previously shown that Xenopus beta 1 when expressed in Xenopus oocytes without an alpha  subunit is slowly degraded in or close to the ER (17). Despite the increased protease sensitivity, the disulfide bond-deficient mutants showed a similar rate of cellular degradation to the wild type beta  subunits (Fig. 3B). Furthermore, the wild type as well as the newly synthesized CC1, CC2, and CC3 mutants interacted with BiP and the chaperone interaction correlated with the degradation rate of the beta  subunits (compare Fig. 3, C with B).

Thus altogether, these data indicate that abolition of only one disulfide bond in the beta  subunit leads to a significant change in its tertiary structure which, however, does not influence the cellular half-life of the proteins or their interaction with BiP.

Association of beta  Cysteine Mutants with alpha  Subunits and Formation of Functional alpha ·beta Complexes

To assess whether the cysteine mutants can adopt an assembly-competent conformation, we co-expressed the mutants with alpha  subunits in oocytes and performed immunoprecipitations with an alpha -antibody under nondenaturing conditions that preserve alpha -beta interactions. As expected, wild type beta  subunits co-precipitated with alpha  subunits either in their core-glycosylated form after a pulse (Fig. 4A, lane 3) or in their fully glycosylated form after a chase period (lane 4) reflecting the transport of the alpha ·beta complexes from the ER to the plasma membrane (18). Furthermore, alpha  subunits that were expressed together with beta  subunits became stabilized (lanes 3 and 4) in contrast to alpha  subunits that were synthesized alone in the oocyte (lanes 1 and 2). The CC1, CC2, and CC3 mutants associated, although inefficiently, with co-expressed alpha  subunits during a pulse period (lanes 5, 7 and 9), but they did not visibly stabilize them (lanes 6, 8 and 10). As shown for the CC1 mutant, the cysteine mutants also associated transiently with the endogenous, oocyte alpha  subunits (lanes 11 and 12) that represent a stable pool of unassembled alpha  subunits (15). Results obtained on the conformation and on the ability to assemble with alpha  subunits were similar for mutants affected in only one of the cysteine residues forming the disulfide bonds (data not shown).


Fig. 4. Assembly of beta  cysteine mutants with the alpha  subunit of Na,K-ATPase. Oocytes were injected with 5 ng of Xenopus alpha 1cRNA alone (lanes 1 and 2), with 5 ng of alpha cRNA plus 0.3 ng of Xenopus wild type (lanes 3 and 4), or mutant (lanes 5-10) beta cRNA or with 0.3 ng of the CC1 mutant beta cRNA alone (lanes 11 and 12). The oocytes were metabolically labeled for 10 h and subjected to a 3-day chase before preparation of microsomes and immunoprecipitation. A, nondenaturing immunoprecipitations with an anti-alpha serum. B, denaturing immunoprecipitations with an anti-beta serum. fg, fully glycosylated; cg, core-glycosylated beta  subunit; st, standard proteins of known molecular mass. * represents an artifactual band (probably actin, 15) that co-migrates with fully glycosylated beta  subunits in front of the heavy chains of immunoglobulins and that is frequently observed in nondenaturing immunoprecipitations.
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During the chase period, the CC2 and CC3 mutants co-expressed with alpha  subunits were degraded in a pre-Golgi compartment in their core-glycosylated form, similar to when they are expressed alone (compare Fig. 4B, lanes 7-10 to lanes 11 and 12 and to Fig. 3B). On the other hand, a small portion of the CC1 mutants became fully glycosylated when they were co-expressed with alpha  subunits (Fig. 4B, lane 6) but not when they were expressed alone (lane 12). This result suggested that in contrast to CC2 and CC3 mutants, the CC1 mutant is still able, although inefficiently, to form alpha ·beta complexes that can leave the ER. To verify this observation, we tested the expression of functional pumps at the cell surface by measuring [3H]ouabain binding and Na,K-ATPase-mediated 86Rb+ uptake in oocytes co-expressing alpha  and wild type beta  or one of the beta  mutants. Oocytes expressing wild type alpha ·beta complexes produced about three or four times more functional Na+,K+ pumps at the cell surface than oocytes expressing alpha  subunits alone (Fig. 5, A and B). On the other hand, a similar number of Na+,K+ pumps to alpha -expressing oocytes was found in oocytes co-expressing alpha  subunits and the CC2 or the CC3 mutant. In agreement with the biochemical data shown in Fig. 4, the CC1 mutant, however, produced a small but significant increase in the number of functional pumps at the cell surface. This increase was only observed when the CC1 mutant was co-expressed with alpha  subunits (Fig. 5).


Fig. 5. Functional expression of Na+,K+ pump complexes containing beta  cysteine mutants. A fraction of oocytes of the experiment described in Fig. 4 was incubated for 4 days before the expression and the transport activity of Na+,K+ pumps at the cell surface were studied by [3H]ouabain binding (A) and by 86Rb+ uptake (B) measurements, respectively. For experimental details see "Experimental Procedures." Shown are means ± S.E. Numbers of oocytes tested are indicated in the bars. Unpaired Student t test: compared with oocytes expressing alpha  subunits alone, the number of functional pumps expressed at the cell surface were only statistically significantly different in oocytes expressing alpha ·wild type beta 1 or alpha ·CC1 mutant beta  complexes. One out of three similar experiments is shown.
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In conclusion, despite the considerable perturbation of the structural integrity of the ectodomain of the beta  subunit by the abolition of only one disulfide bond, all the mutants are capable to associate transiently with alpha  subunits. However, only the CC1 mutant affected in the most N-terminal disulfide bond is partially able to form stable and functional alpha ·beta complexes. This result indicates that the C-terminal domain containing the last two disulfide bonds is particularly important to ensure the acquisition of a conformation compatible with an efficient association of the beta subunit with the alpha  subunit.

Effects of Abolition of Glycosylation Sites in the beta  Subunit of Na,K-ATPase on Its Initial Folding and Its Interaction with BiP

All beta  isoforms of the Na,K-ATPase as well as the beta  subunits of H,K-ATPases are subjected to N-glycosylation during their synthesis. To assess the role of the sugar moieties in the initial folding of beta  subunits, we prepared mutants in which one (N1, N2, or N3), two (N2, 3) or three (N1,2,3) of the potential glycosylation sites in the beta 1 subunit of Xenopus Na,K-ATPase were abolished by replacement of the consensus asparagine residues with glutamine (see Fig. 1). Expression of these mutants in Xenopus oocytes revealed that all three potential glycosylation sites are used. Single, double, and triple mutants exhibited a progressively decreasing molecular mass (Fig. 6). For so far undefined reasons, but perhaps due to differential trimming, the core-glycosylated, single mutants N1, N2, and N3, which each lacks one of the glycosylation sites, exhibited slightly different molecular masses (compare lanes 3, 5, and 7). However, after deglycosylation with Endo H, all mutants as well as the wild type beta  subunit showed the same molecular mass that was identical to that of the triple mutant lacking all glycosylation sites (compare lanes 2, 4, 6, 8, and 10, to lanes 11 and 12) or to that of the beta  subunit synthesized in a reticulocyte lysate (data not shown).


Fig. 6. State of glycosylation of Xenopus beta 1 mutants affected in one, two, or three consensus N-glycosylation sites. Oocytes were injected with 0.2 ng of wild type Xenopus beta 1 (lanes 1 and 2) or 0.6 ng of mutant (lanes 3-12) beta cRNA and metabolically labeled for 24 h. Microsomes were prepared, and the beta  subunit was immunoprecipitated under denaturing conditions. Immunoprecipitates were not treated (lanes 1, 3, 5, 7, 9, and 11) or treated (lanes 2, 4, 6, 8, 10, and 12) with Endo H before separation by SDS-PAGE. Mutants are described in Fig. 1. cg, core-glycosylated; ng, nonglycosylated beta  subunits.
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To assess the effect of glycosylation on the structural stability of the newly synthesized beta  subunit, we subjected the triple N1,2,3 mutant synthesized in oocytes to proteolysis in the presence or absence of Triton X-100. In contrast to the wild type beta  subunit (Fig. 7A, lanes 1-3), the triple glycosylation mutant was completely degraded by proteinase K in the presence of Triton X-100 (lanes 4-6) indicating that the sugar chains either influence the folding of the beta  subunit or directly protect it from proteolytic attack.


Fig. 7. Sensitivity of beta -glycosylation mutants to degradation by proteinase K and cellular proteases and their interaction with BiP. A, digestion of glycosylation mutants with proteinase K. Oocytes were injected with 0.2 ng of wild type (lanes 1-3) or 0.6 ng of mutant (lanes 4-6) beta cRNA and 3 µCi/oocyte of [35S]methionine. After 5 h of incubation, homogenates were prepared as described under "Experimental Procedures," and aliquots were incubated for 1 h at 4 °C in the absence (lanes 1 and 4) or presence (lanes 2, 3, 5 and 6) of 200 mg/ml proteinase K and in the absence (lanes 1, 2, 4, and 5) or presence (lanes 3 and 6) of 1% Triton X-100. After addition of 5 mM phenylmethylsulfonyl fluoride and 3.7% (final concentration) hot SDS, the beta  subunits were immunoprecipitated under denaturing conditions and revealed by SDS-PAGE and fluorography. B, cellular degradation of glycosylation mutants. Oocytes were injected with 0.2 ng of wild type (lanes 1 and 2) or 0.3 ng of mutant (lanes 3-10) beta cRNA, metabolically labeled for 24 h, and subjected to a 72-h chase. After the pulse and the chase periods, microsomes were prepared, and the beta  subunits were immunoprecipitated under denaturing conditions. C and D, cellular degradation of the nonglycosylated beta  subunit and its interaction with BiP. Oocytes were injected with 0.2 ng of wild type (lanes 1 and 2) or 0.6 ng of N1,2,3 mutant (lanes 3-6) beta cRNA, metabolically labeled for 24 h, and subjected to a 24-, 48-, or 72-h chase before preparation of microsomes. Immunoprecipitations were performed with anti-beta serum under denaturing conditions (C) or with anti-BiP serum under nondenaturing conditions (D). E, migration of beta  subunits under nonreducing conditions. Aliquots of samples shown in C were not treated with beta -mercaptoethanol before migration on SDS-polyacrylamide gels. A-E show one out of two or three similar experiments.
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With the exception of the N1,2,3 mutant, all glycosylation mutants expressed without alpha  subunits in the oocyte were degraded during a chase period of 3 days similar to the wild type beta  subunit (Fig. 7B). Interestingly, the nonglycosylated N1,2,3 mutant remained stable over the whole chase period (Fig. 7C). During its stable expression in the ER, the N1,2,3 mutant remained associated with BiP (Fig. 7D). This result provides further support for the previously raised hypothesis (8) that interaction with BiP might be important for the ER retention of partially misfolded or overexpressed Na,K-ATPase beta  subunits until they are recognized by a pre-Golgi degradation system. On the other hand, a comparison of the gel migration of nonreduced samples (Fig. 7E) reveals no increase in the formation of disulfide-bonded aggregates by nonglycosylated beta  subunits that could explain their high stability.

Association of beta  Glycosylation Mutants with alpha  Subunits and Formation of Functional alpha ·beta Complexes

The ability of the glycosylation mutants to form stable alpha ·beta complexes was assessed by performing immunoprecipitations under nondenaturing conditions on microsomes of pulse/chase-labeled oocytes expressing alpha  and beta  mutants. After the pulse period, all glycosylation mutants co-precipitated with an alpha  antibody indicating that they were able to associate with the alpha  subunit (Fig. 8A). The beta  mutants with one or two residual glycosylation sites were in their core-glycosylated (Endo H-sensitive) form (Fig. 8, A and B). After a chase of 3 days, the beta  mutants remained associated with the alpha  subunit (Fig. 8C), and with the exception of the N1,2,3 mutant that is nonglycosylated, the other mutants with residual glycosylation were now mainly processed to the fully glycosylated (Endo H-resistant) form (Fig. 8, C and D) indicating that the alpha ·beta complexes had been transported through a distal Golgi compartment. In comparison to the residually glycosylated beta  mutants that stabilized the alpha  subunit similarly to the wild type beta  subunit (Fig. 8C, compare lanes 2-5 to lane 1), the nonglycosylated beta  mutant only achieved about 50% (average of two experiments) stabilization of the alpha subunit (Fig. 8C, compare lane 6 to lane 1). Thus, these data indicate that the acquisition of an assembly-competent conformation of the beta  subunit depends on the presence of the sugars. However, the presence of only one sugar chain is necessary and sufficient to ensure an efficient association with the alpha  subunit.


Fig. 8. Assembly of beta -glycosylation mutants with the alpha  subunit of Na,K-ATPase. Oocytes were injected with 5 ng of Xenopus alpha 1cRNA alone (lanes 7) or with 5 ng of alpha cRNA plus 0.3 ng of Xenopus wild type beta 1 (lanes 1) or 0.6 ng of mutant (lanes 2-6) beta cRNA. The oocytes were metabolically labeled for 26 h and subjected to a 3-day chase before preparation of microsomes and immunoprecipitation of alpha and beta  subunits. Shown are nondenaturing immunoprecipitations with an anti-alpha serum after a 26-h pulse (A) or a 3-day chase (C) and denaturing immunoprecipitations with an anti-beta serum after a 26-h pulse (B) or a 3-day chase (D). fg, fully glycosylated; cg, core-glycosylated beta  subunits. One out of two similar experiments is shown.
[View Larger Version of this Image (61K GIF file)]


The cell surface expression of alpha ·beta complexes containing the N1,2,3 mutant was estimated by [3H]ouabain binding on intact oocytes combined with pulse/chase experiments. Denaturing immunoprecipitations of the labeled, wild type beta  subunit expressed without the alpha  subunit showed that a small fraction of the beta  subunit population became fully glycosylated during the chase (Fig. 9A, lanes 3 and 4) due to its association with endogenous, oocyte alpha  subunits (15). The fraction of fully glycosylated beta  subunits was more important when the beta  subunit was expressed together with alpha  subunits (lanes 7 and 8). In contrast to the wild type beta  subunit, the nonglycosylated beta  subunit showed, as expected, no change in its molecular mass after the chase when it was expressed alone (lanes 5 and 6) or together with alpha  subunits (lanes 9 and 10). The N1,2,3 mutant co-precipitated with the alpha subunit during the pulse period similarly to the wild type beta  subunit but, as shown above, it was not able to stabilize the alpha  subunit to a similar extent during the chase period (Fig. 9B, compare lanes 5 and 6 to lanes 3 and 4).


Fig. 9. Cell surface expression of Na+,K+ pump complexes containing nonglycosylated beta 1 mutants. Oocytes were not injected (ni) or injected with 0.3 ng of Xenopus wild type beta 1 or 0.6 ng of N1,2,3 mutant beta cRNA alone or together with 5 ng of Xenopus alpha 1cRNA. A, glycosylation processing of beta  subunits. The oocytes were metabolically labeled for 6 h and subjected to a 48-h chase before preparation of microsomes and immunoprecipitations with an anti-beta serum under denaturing conditions. B, assembly competence of beta  subunits. Aliquots from samples described in A were immunoprecipitated with an anti-alpha serum under nondenaturing conditions. C, expression of alpha ·beta complexes at the cell surface. A fraction of noninjected or cRNA-injected oocytes was incubated for 2 days before measurements of [3H]ouabain binding to intact oocytes. Shown are means ± S.E. Unpaired Student t test: compared with noninjected oocytes, the number of pumps expressed at the cell surface was statistically significantly different in oocytes expressing beta  subunits alone or alpha ·beta complexes. One out of two similar experiments is shown.
[View Larger Version of this Image (46K GIF file)]


Expressed alone, the wild type beta  subunit and the N1,2,3 mutant increased the expression of the number of Na+,K+ pumps at the cell surface about 2-fold compared with noninjected oocytes due to association with the endogenous alpha  subunit (Fig. 9C). When co-expressed with alpha  subunits, the wild type beta  subunit allowed for about a 4-fold increase and the N1,2,3 mutant for about a 3.5-fold increase in the number of Na+,K+ pumps (Fig. 9C). A comparison between the stabilization of the alpha  subunit by the wild type or mutant beta  subunit and the expression of Na+,K+ pumps at the cell surface consistently showed that the expression of alpha ·N1,2,3 mutant complexes was more important than expected from the ability of the beta  mutant to stabilize the alpha  subunit. While the expression of alpha ·N1.2,3 mutant complexes, assessed by ouabain binding, was only about 20% less than that of alpha ·wild type beta  complexes (Fig. 9C), the stabilization of alpha ·N1,2,3 complexes was about 50% lower (average of two experiments) than that of alpha ·wild type beta complexes (Fig. 9B, lanes 3-6). This discrepancy might be explained by a regulated expression of Na+,K+ 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 alpha ·N1,2,3 complexes might be that the mutant complexes have a different ouabain sensitivity than the alpha ·wild type beta  complexes. This latter possibility incited us to study the effect of the sugars in the beta  subunit on several functional properties of the cell surface-expressed alpha ·beta complexes. The apparent K+ affinity (K1/2) of Bufo Na,K-ATPase, measured as the half-maximal K+ activation of the Na+,K+ pump-mediated 86Rb+ flux, was about 300 µM in the absence of extracellular Na+ and 1 mM in the presence of extracellular Na+ for wild type as well as for mutant, nonglycosylated Na+,K+ pumps (Fig. 10, A and B) These values are similar to those previously measured by electrophysiological means (21). Furthermore, the affinity for ouabain of the Xenopus Na+,K+ pumps containing the nonglycosylated mutant beta  subunit was similar to that of the glycosylated wild type pumps. The inhibition constant (Ki) measured as the half-maximal concentration needed to inhibit the Na,K-ATPase-mediated 86Rb+ uptake was close to 50 nM for both types of pumps (Fig. 10C). Finally, no significant differences could be observed between the wild type and the nonglycosylated Xenopus pumps in the K+ concentrations needed to compete with ouabain binding (Fig. 10D).


Fig. 10. Functional properties of Na,K-ATPase alpha ·beta complexes containing nonglycosylated beta  subunits. A and B, K+ activation of Na,K-ATPase-mediated 86Rb+ uptake in the absence (A) or presence (B) of extracellular Na+. Oocytes were injected with 9 ng of Bufo alpha 1cRNA plus 0.2 ng of Xenopus wild type (open circle ) or 0.5 ng of N1,2,3 mutant (bullet ) beta cRNA. After incubation for 3 days, 86Rb+ uptake was measured in the presence of varying concentrations of K+ as described under "Experimental Procedures." All measurements were done in the presence of 1 µM ouabain to inhibit the endogenous, oocyte Na+,K+ pumps. Each data point represents the mean ± S.E. 86Rb+ uptake of 20-30 individual oocytes. One out of two similar experiments is shown. Maximal 86Rb+ uptake in oocytes expressing wild type or mutant Na+,K+ pumps was 87 and 79 pmol/oocyte/min, respectively, in A and 307 and 311 pmol/oocyte/min, respectively, in B. C, ouabain inhibition of Na,K-ATPase-mediated 86Rb+ uptake. Oocytes were injected with 5 ng of Xenopus alpha 1cRNA plus 0.3 ng of Xenopus wild type (open circle ) or 0.4 ng of N1,2,3 mutant (bullet ) beta cRNA. After incubation for 3 days, 86Rb+ uptake was measured in the presence of varying concentrations of ouabain in the presence of 5 mM K+. Each data point represents the mean ± S.E. 86Rb+ uptake of 15-25 individual oocytes. The curve is fitted to the data of five experiments. Calculations of the Hill coefficient (see "Experimental Procedures") predicts one site of ouabain binding. 86Rb+ uptake mediated by endogenous, oocyte pumps was determined in noninjected oocytes and represented about 15% of the Vmax measured in cRNA-injected oocytes. The endogenous, oocyte Na+,K+ pumps showed a sensitivity to ouabain similar to the exogenous Na+,K+ pumps. Maximal 86Rb+ uptake in oocytes expressing wild type or mutant Na+,K+ pumps was 148 and 137 pmol/oocyte/min, respectively. D, competition of ouabain binding by K+. Oocytes were injected with 5 ng of Xenopus alpha 1cRNA plus 0.3 ng of Xenopus wild type (open circle ) or 0.5 ng of N1,2,3 mutant (bullet ) beta cRNA. After incubation for 3 days, [3H]ouabain binding was measured as described under "Experimental Procedures" in the presence of increasing concentrations of K+ and in the presence of 50 mM external Na+. Each data point represents the mean ± S.E. [3H]ouabain binding of 20-25 individual oocytes. Maximal ouabain binding to oocytes expressing wild type or mutant Na+,K+ pumps was 86 and 79 fmol/oocyte, respectively.
[View Larger Version of this Image (26K GIF file)]


Thus, these data demonstrate that the presence of at least one sugar moiety is necessary for the correct folding of the beta 1 subunit of Na,K-ATPase permitting its efficient assembly with the alpha  subunit. On the other hand, our functional tests did not permit us to reveal a role of the sugars in the functional properties of the mature enzyme expressed at the cell surface.


DISCUSSION

The present study reveals the importance of co-translational modifications for the initial folding of the beta  subunit of Na,K-ATPase. We show that disulfide bond formation and to a lesser extent core glycosylation during the synthesis of the beta  subunit are needed to render it competent for the assembly with the catalytic alpha  subunit.

Six cysteine residues in the ectodomain of the beta  subunits of the Na,K-ATPase and the H,K-ATPase, the positions of which are highly conserved, form disulfide bonds in a sequential pattern (22-24). Cleavage of these disulfide bonds by reducing agents leads to structural and functional changes in purified enzyme preparations (25-28) suggesting that they are important to maintain the beta  subunit in a conformation that is compatible with a correct association with the alpha  subunit. In this study, we show that prevention of the formation of only one of the three disulfide bonds in the beta  subunit of Na,K-ATPase indeed is sufficient to abolish its ability to correctly assemble with alpha  subunits and as a consequence to impede the maturation and efficient expression of functional pumps.

Acquisition of disulfide bridges is catalyzed by protein disulfide isomerase in the ER, and it is thought to limit the number of folding states a protein can adopt during or after synthesis, to guide the folding toward a final state and ultimately to contribute to the stability of the correctly folded state (for reviews see Refs. 10 and 29). Thus, the observed inability of an efficient assembly of the beta  subunit of Na,K-ATPase or of oligomeric, viral proteins (for review see Ref. 9) in which cysteine residues involved in disulfide bond formation are changed by site-directed mutagenesis is probably due to destabilization and increased misfolding of the newly synthesized proteins. However, in the cysteine-deficient beta  subunit, misfolding is not accompanied, as in many other proteins, by the formation of intermolecular disulfide-bonded aggregates or by a more pronounced interaction with the molecular chaperone BiP (for review, see Ref. 10). Possibly, the lack of only one cysteine residue in beta  subunits is not enough for the formation of covalent aggregates or for a global misfolding but leads to local structural perturbations that impede the acquisition of a completely folded association domain. Such partial misfolding might explain why cysteine bond-deficient beta  subunits can transiently but not permanently associate with alpha  subunits.

Our data indicate that the relative importance of the three disulfide bonds in the beta  subunit on the acquisition of an assembly-competent form is different. Indeed, while beta  mutants, affected in the formation of disulfide bonds Cys159-Cys175 (numbering refers to the beta 1 subunit of Xenopus laevis) or Cys213-Cys276, completely lose their ability to assemble permanently with the alpha  subunit, the beta  mutant affected in the formation of the most N-terminal disulfide bond Cys126-Cys149 permits the formation of a small number of functional alpha ·beta complexes at the cell surface. Thus, the domain comprised between the two most C-terminal disulfide bonds is able to adopt a structure that partially permits correct subunit interaction. Probably this domain in the beta  subunit is more compact as suggested by its lower sensitivity to reduction by beta -mercaptoethanol that first breaks the least stable disulfide bond Cys126-Cys149 in purified enzyme preparations (26). On the basis of these results, it has been speculated that the most N-terminal disulfide bond might function as a kind of hinge that allows establishment of close contact between the hydrophobic, membrane-anchored domain and the hydrophilic C-terminal part of the beta  subunit that contains the sugar chains.

Our data showing the structural importance of the two most C-terminal disulfide bonds in the beta  subunit are in agreement with similar studies performed with cysteine mutants of Torpedo beta  subunits. However, our results obtained with the beta  mutant lacking the first N-terminal disulfide bond differ from those of Noguchi et al. (30) who reported that Torpedo beta  subunits lacking this disulfide bond are able to assemble and stabilize alpha  subunits but cannot form functional Na+,K+ pumps. Although we cannot completely rule out that species differences are responsible for the discrepancies in the results, it is more likely that they are due to differences in the experimental approach. Indeed, Noguchi et al. (30) performed continuous pulse labeling protocols to analyze alpha -beta interaction. In contrast, in this study, we performed pulse/chase protocols that are the only means to distinguish between a short and a long term association of beta  subunits. Furthermore, it is likely that, in contrast to the highly sensitive 86Rb+ uptake measurements used in our study, the Na,K-ATPase assay used by Noguchi et al. (30) was not sensitive enough to detect a small fraction of functional pumps formed with mutant beta  subunits and/or that sodium thiocyanate used during sample preparation led to dissociation and inactivation of the loosely assembled alpha ·beta mutant complexes.

As with most cell surface proteins, all beta  isoforms of Na,K- or H,K-ATPase are glycoproteins and possess 3-7 N-linked sugar chains (for review see Ref. 31). The functional role of the glyco-moieties is poorly understood. In this study, we confirm previous reports (32-34) that nonglycosylated beta  subunits are able to assemble with alpha  subunits and permit the expression of functional Na+,K+ pumps at the cell surface. Nevertheless, our data show that in the beta  subunit of Na,K-ATPase, as in many other glycoproteins (for review see Ref. 11), inhibition of glycosylation using tunicamycin (35) or elimination of the consensus sequences for N-linked glycosylation by site-directed mutagenesis leads to folding problems of the newly synthesized protein. These are reflected by an increased sensitivity of the beta  subunit against proteolysis and a decreased efficiency in the assembly with alpha  subunits. As reported for vesicular stomatitis virus G-glycoprotein or influenza hemagglutinin, the effects of elimination of carbohydrate sites in the beta  subunit is additive (for references, see Ref. 9). No single oligosaccharide is essential for the correct folding, but all sugars must be eliminated to significantly affect the assembly competence.

Addition of N-linked sugar chains occurs as soon as the nascent polypeptide emerges in the ER lumen and thus occurs, in most cases, before folding and assembly. The addition of the large, hydrophilic carbohydrate chains facilitates the ongoing folding process of the nascent chain by favoring the recruitment of discrete polypeptide domains to the surface of the protein (for review and references see Ref. 36). In addition to local effects on folding, it has been proposed that core sugars have a global effect on the solubility of newly synthesized proteins that counteracts the tendency to form irreversible aggregates (37).

Although the degradation rates of wild type and sugar-deficient glycoproteins have not rigorously been compared, proteins in which the sugar moieties have been eliminated can be degraded in the ER (38). The nonglycosylated beta  subunit, however, is very resistant to ER degradation. The high stability of the nonglycosylated mutant compared with that of the wild type, glycosylated beta  subunit is surprising, since the presence rather than the absence of sugars would be expected to protect from proteolytic attack. In contrast to the prediction mentioned above, the nonglycosylated beta  subunit does not form disulfide-bonded aggregates that could explain its resistance to cellular degradation. On the other hand, it is permanently associated with BiP, perhaps in aggregates formed by hydrophobic interactions that could impede the recognition of the nonglycosylated beta  subunit by the pre-Golgi degradation pathway. Alternatively, the beta  subunit of Na,K-ATPase might be an example of a protein whose quality control and degradation depends on glycosylation. A similar hypothesis has recently been raised for carboxypeptidase Y whose unglycosylated species is stably retained in the ER (39). An important player in the quality control of glycoproteins is calnexin that binds to newly synthesized monoglucosylated N-linked oligosaccharides. A role for glycoprotein trimming and/or calnexin interaction is suggested by a recent study that reports that a mutant, secretory alpha 1-antitrypsin Z is degraded by the proteasome by first digesting the cytoplasmic tail of calnexin molecules that are associated with alpha 1-antitrypsin Z (40).

In contrast to disulfide bonds, complex-type sugar chains are often not important for overall stability and function of mature glycoproteins (for references see Ref. 11). In agreement, enzymatic deglycosylation of the beta  subunit in purified Na,K-ATPase has little effect on the enzyme activity or ouabain binding (41). Our studies using beta  subunits in which the glycosylation sites had been abolished by site-directed mutagenesis also do not reveal significant effects of the sugars in the beta 1 subunit on either the K+ activation or the ouabain binding of functional Na+,K+ pumps expressed at the cell surface. The question remains open whether complex-type oligosaccharides in the beta 1 subunit of Na,K-ATPase might represent specific recognition sites for interaction with external ligands, as suggested for many glycoproteins (42).


FOOTNOTES

*   This work was supported by the Swiss National Fund for Scientific Research Grants No 31-33676.92 and 31-42954.95.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Dagger    To whom correspondence should be addressed: Institute of Pharmacology and Toxicology, University of Lausanne, rue du Bugnon 27, CH-1005 Lausanne, Switzerland. Tel.: 41 21 692 53 50; Fax: 41 21 692 53 55.
1   The abbreviations used are: ER, endoplasmic reticulum; PAGE, polyacrylamide gel electrophoresis; BiP, binding protein; Endo H, endoglycosidase H; MOPS, 4-morpholinepropanesulfonic acid.

ACKNOWLEDGEMENTS

We thank Jean-Daniel Horisberger and Dmitri Firsov for a critical reading of the manuscript.


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