©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Topology of the Na,K-ATPase
EVIDENCE FOR EXTERNALIZATION OF A LABILE TRANSMEMBRANE STRUCTURE DURING HEATING (*)

Elena Arystarkhova , Don L. Gibbons , Kathleen J. Sweadner (§)

From the (1) Laboratory of Membrane Biology, 149-6118, Massachusetts General Hospital, Charlestown, Massachusetts 02129

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The topological organization of the Na,K-ATPase subunit is controversial. Detection of extracellular proteolytic cleavage sites would help define the topology, and so attempts were made to find conditions and proteases that would permit digestion of Na,K-ATPase in sealed right-side-out vesicles from renal medulla. The subunit is predominantly extracellular and could mask the surface of the subunit. Most of the tested proteases cleaved , and some digested it extensively. However, without further disruption of structure, there was still no digestion of the subunit. Reduction (at 50 °C) of disulfide bonds that might stabilize the subunit fragments, or heating alone at 55 °C, permitted tryptic digestion of at a site close to the C terminus, while simultaneously increasing digestion of . A 90-kDa N-terminal fragment of was recovered, but the C-terminal fragment was further digested. Heating and reduction resulted in the extracellular exposure of a protein kinase A phosphorylation site, Ser-938, and the C terminus, both of which have been proposed to be located on the intracellular surface. At the same time, access to a distant protein kinase C phosphorylation site was not increased. The data suggest that the harsh treatment simultaneously resulted in alteration of the subunit and the extrusion of a segment of that normally spans the membrane, without causing complete denaturation or opening the sealed vesicles. Preincubation with Rb+was protective, consistent with prior evidence that it stabilizes the protein segments in the C-terminal third of . We conclude that this portion of the subunit contains a transmembrane structure with unique lability to heating.


INTRODUCTION

The Na,K-ATPase() catalyzes the active transport of ions across the plasma membrane. It has a catalytic subunit, , with a molecular mass of 110 kDa, and a smaller glycoprotein subunit, , with an apoprotein molecular mass of 32 kDa. A clear role for the subunit has not been determined, but there is evidence for its importance in - assembly during biogenesis (1) , in cell-cell communication (2) , and in modulating the affinity for K+(3, 4, 5) . Both subunits have been cloned (6) , and topological models have been based on deduced primary structure, hydropathy plot predictions, selective proteolysis, chemical modification, and electron microscopy (7, 8, 9, 10) . According to current models, the N and C termini of the subunit are located inside the cell, and there is a large central intracellular portion, anchored by 6-10 transmembrane segments. The models predict that very little of is exposed on the extracellular surface, mostly in the C-terminal third where the structure is much debated. In contrast, the majority of the subunit is exposed outside the cell (11) . Its extracellular domain has three asparagine-linked carbohydrate groups and three disulfide bridges (12, 13) . Only a small portion, including the N terminus, is intracellular, connected by one transmembrane span.

To study the spatial organization of the Na,K-ATPase and the sidedness of events of membrane transport, sealed vesicles have been extensively employed (11, 14, 15, 16, 17, 18, 19, 20, 21) . Right-side-out vesicles, i.e. those in which Na,K-ATPase has the same orientation as in the cell, are formed during homogenization of renal medulla by the resealing of involuted basolateral membrane fragments. Vesicles are typically 80-90% sealed, as determined by measuring how much latent ATPase activity can be detected by assay with and without mild detergent treatment to open them. Chemical modification of right-side-out vesicles revealed a 3:1 relative distribution of reactive amino groups on the subunit between the intra- and extracellular surfaces (11) , which predicts a respectable number of potential tryptic cleavage sites. Attempts to get tryptic cleavage of the subunit at the extracellular surface have been unsuccessful, however (14, 15, 17, 18) . To explain the discrepancy, one hypothesis is that the subunit masks the subunit.

In this paper we report that exposure of a certain stretch of the subunit at the extracellular surface can be achieved in right-side-out vesicles, and the data implicate the close interaction of the and subunits. Independent work by Karlish et al. (22, 23) has produced evidence for the exposure of sites at the extracellular surface of as a consequence of heating. Evidence for substantial reorganization of the C-terminal domain is described here within the framework of topological models of Na,K-ATPase structure.


EXPERIMENTAL PROCEDURES

Materials

Antiserum K1 (24) was raised against the purified subunit of rat kidney Na,K-ATPase. Antipeptide directed antisera 757 (25) and 845 (26) were the gift of Dr. William J. Ball, Jr., University of Cincinnati. Polyclonal antiserum FP1 was provided by Dr. Alicia McDonough, University of Southern California; polyclonal antibody UBI 1 was purchased from United Biotechnology Inc. (Lake Placid, NY); these two antibodies were prepared by the same procedure (27) . Monoclonal antibody McK1 was raised against rat kidney Na,K-ATPase (16) . Peptide-directed antibody ETYY (28) was the gift of Dr. Jack Kyte, University of California at San Diego. All proteases and secondary antibodies used for staining blots were obtained from Sigma.

General Procedures

Purification of Na,K-ATPase from pig or rat kidney outer medulla microsomes, determination of protein concentration, ATPase activity assay, and [H]ouabain binding were performed as described elsewhere (29) .

Right-side-out Vesicles

Right-side-out vesicles from rat kidney outer medulla were prepared by modification of published methods (11) . Briefly, freshly prepared crude microsomes were loaded onto step gradients consisting of 20 and 5% metrizamide (Sigma, grade I) in 0.25 M sucrose, 25 m M MOPS, pH 7.4, 1 m M Tris-EDTA. Centrifugation was at 27,000 rpm for 4 h at 4 °C in the SW 50.1 rotor of a Beckman ultracentrifuge. The vesicle fraction was collected from the interface between the two metrizamide solutions and stored without further manipulation at -70 °C after the addition of dimethyl sulfoxide to 5% as a cryoprotectant. The extent of vesicle sealing was assessed in a latency test by measuring the hydrolysis of ATP before and after permeabilization, which was with 0.016% sodium dodecyl sulfate in the presence of 3 m M ATP to protect activity. For the preparations used here, activity was stimulated 5.5-7.7-fold, indicating that vesicles were 82-88% sealed. With cryoprotection, vesicle sealing was changed very little by storage for weeks or months at -70 °C.

Proteolytic Digestion

Right-side-out vesicles (0.5-0.6 mg/ml) were incubated with different proteases (in all cases, 1:3 w/w with respect to vesicle protein) for periods of time from 1 min to 1 h as indicated, in medium containing 250 m M sucrose, 25 m M MOPS, pH 7.4, 1 m M Tris EDTA. For some experiments, different ligands were added as specified in the text and tables; for others, digestion was preceded by treatment with reducing agent (dithiothreitol or -mercaptoethanol) at different temperatures, or by heating at 55 °C for 30 min (the latter condition according to Ref. 23). Proteolysis was performed at 37 °C, and the reaction was stopped either with the addition of acidified electrophoresis sample buffer (125 m M Tris, 4% SDS, 20% glycerol, and 5% -mercaptoethanol, pH < 2.0) or by addition of 3 m M diisopropylfluorophosphate (Sigma), a covalent inhibitor of serine proteases. As shown previously, acidified sample buffer is more effective at preventing digestion by trypsin during gel electrophoresis than addition of soybean trypsin inhibitor (30) , and it was successful with other proteases as well. The acidification of the sample lasts only until it penetrates the gel, but it appears to cause irreversible denaturation of the protease.

Gel Electrophoresis

For most experiments, gel electrophoresis was performed according to Laemmli (31) , using 7.5%, 10%, or gradient 5-15% polyacrylamide for separating gels. To resolve short peptides, the Tricine-SDS buffer system of Schagger and von Jagow (32) was employed with 16% polyacrylamide gels. Electrophoretic transfer was performed in a buffer containing 20 m M Tris, 150 m M glycine, and 20% methanol, at 100 mA overnight at 4 °C. Nitrocellulose was quenched with Tris-buffered saline, 0.5% Tween 20, and stained with antibodies as described previously (16) , with visualization with ECL (enhanced chemiluminescence) (Amersham). For autoradiography of 32P, nitrocellulose blots, stained with 0.1% Amido Black, were exposed to Kodak XAR-5 film. Densitometry was accomplished with a Molecular Dynamics laser densitometer.

The renal medulla preparation used here contains the 1 and 1 isoforms of the Na,K-ATPase. Without digestion, both subunits migrate on SDS gels at anomalous positions. The subunit migrates as if it had a molecular mass of 95-99 kDa, slightly faster than the 112-kDa mass deduced from the cDNA. This is thought to be due to peculiarities in the interaction of very hydrophobic domains with the detergent (30) . The subunit, with its protein molecular mass of 32 kDa, migrates as a fuzzy band of roughly 60 kDa because of its carbohydrate groups. The extra band of 70 kDa which stained with an antiserum against 1 (K1) seen in Fig. 1 was absent from Na,K-ATPase purified from renal medulla microsomes by SDS extraction (data not shown), and it was not investigated further as it was not digested.


Figure 1: Digestion of vesicles under mild conditions. Right-side-out vesicles (36 µg) were equilibrated with either 15 m M NaCl ( lanes 1-4) or 15 m M KCl ( lanes 5-8) overnight at 4 °C. Lanes 1 and 5 were controls with no trypsin. Samples were digested with trypsin at 37 °C, and aliquots were taken at 1 min ( lanes 2 and 6), 10 min ( lanes 3 and 7), and 30 min ( lanes 4 and 8) of proteolysis and loaded on a 5-15% gradient gel. Blots were stained first with antibody K1 to detect ( A) and reprobed with a polyclonal antibody, UBI 1 ( B). The positions of undigested subunit, subunit, and the 50-kDa fragment of the subunit are denoted with arrows. The undigested band at approximately 70 kDa, indicated by an asterisk, has been seen consistently in vesicle and microsome preparations when the K1 antiserum is used, but appears not to be digested or to affect the results. Its identity is uncertain; it does not stain with Na,K-ATPase 1-specific monoclonal antibody McK1 (data not shown). Double asterisks indicate the position of trypsin, at 27 kDa, which is autodigested during the incubation. We do not know why ECL reagents detect trypsin, but its identity was confirmed on gels loaded with trypsin alone (not shown).



Competition Enzyme-linked Immunosorbent Assay

Heated (55 °C, 30 min) or unheated vesicles were used directly or permeabilized by treatment with 0.016% SDS in the presence of 3 m M ATP for 15 min at room temperature. Various concentrations of vesicles were preincubated with fixed concentrations of antibody in a solution containing 250 m M sucrose, 25 m M MOPS, 1 m M EDTA, 0.1% bovine serum albumin for 1 h at 37 °C. Vesicle-bound antibody was removed by centrifugation in an Airfuge for 15 min. Unbound antibody was measured on microtiter plates (Nunc Maxi-Sorp immunoplates) coated with rat kidney microsomes (0.7 µg/well in phosphate-buffered saline) and quenched with 1% bovine serum albumin in phosphate-buffered saline. Detection was performed with biotinylated goat anti-rabbit or anti-mouse IgG (Life Technologies, Inc.), and color was developed with horseradish peroxidase-conjugated streptavidin and o-phenylenediamine as a substrate. The wells were read on a EIA plate reader (Bio-TEK Instruments, Inc.).

Phosphorylation by Protein Kinase A

Pretreated or untreated right-side-out vesicles (10 µg) or purified Na,K-ATPase from rat kidney (1 µg) were preincubated in a buffer containing 20 m M HEPES, pH 7.4, 1 m M EGTA, 5-10 m M MgClin a total volume 10-20 µl. After 15 min, 0.05% Triton X-100 (if specified) and 100 ng of protein kinase A catalytic subunit (Sigma) were added. After 5 min, the reaction was started by addition of 200 pmol of [ -32P]ATP (DuPont NEN) (specific activity 10,000 cpm/pmol). Phosphorylation was allowed to proceed for 15 min at 30 °C and was stopped with an equal volume of electrophoresis sample buffer containing 125 m M Tris-Cl, pH 6.8, 4% SDS, 20% glycerol, and 5% -mercaptoethanol. When trypsinolysis of phosphorylated Na,K-ATPase was performed, vesicles were first heated at 55 °C for 30 min, next phosphorylated by protein kinase A catalytic subunit without Triton as described above, and then trypsin was added at 1:3 ratio with respect to vesicle protein. Proteolysis was for 1 h at 30 °C and was stopped with acidified electrophoresis sample buffer.

Phosphorylation by Protein Kinase C

An aliquot of right-side-out vesicles containing 3 m M ATP (sodium salt) was first treated with 0.016% SDS for 15 min at room temperature for permeabilization. Then opened and sealed vesicles were subjected to treatment with 200 m M dithiothreitol (DTT) at 50 °C for 15 min or heated at 55 °C for 30 min. For optimal phosphorylation by protein kinase C (PKC), samples (10 µg) were preincubated in the presence of 10 m M Tris phosphate, pH 7.4, 5 m M magnesium acetate, 500 µ M CaCl, 100 n M phorbol 12-myristate 13-acetate (Sigma), and 50 ng of protein kinase C (Calbiochem). The reaction was started with 200 pmol of [-32P]ATP (specific activity 1,000 cpm/pmol) and stopped after 15 min by the addition of an equal volume of electrophoresis sample buffer.


RESULTS

Trypsinolysis in the Presence of Different Ligands

Ion-induced conformation changes are well-known to alter the digestion of cytoplasmic loops of the subunit (33) . To evaluate whether such changes would also affect the extracellular portion of either subunit, trypsinolysis of right-side-out vesicles was performed after overnight incubation in either 15 m M Na+or 15 m M K+, to induce the E1 or E2 conformation, respectively (Fig. 1). Digestion from the extracellular surface was unaffected by the cations. Staining with a polyclonal antibody against (K1) revealed no digestion even at 30 min (Fig. 1 A). In contrast, the subunit was readily cleaved at 1 min (Fig. 1 B; the blot shown in A was reprobed with a second antibody, and the stain is the difference between the two images). Prior work (34) has established that is preferentially cleaved at Arg-142, dividing the 302-amino acid protein into roughly equal pieces. Only the C-terminal half contains carbohydrate groups, however, and, accordingly, staining of the tryptic digest with an antibody raised against a fusion protein containing amino acids 150-302 (UBI 1) revealed a large, smudgy band with an apparent molecular mass of 50 kDa. The N-terminal 16-kDa fragment, which contains the cytoplasmic and transmembrane domains of the subunit, was detected with a peptide-directed antiserum (757) raised against the first 12 amino acids of the subunit. This fragment also appeared after 1 min of trypsinolysis, and as a doublet still containing the N-terminal epitope at longer times. This is seen in Fig. 2, which shows digestion in the absence of added ligands. In both figures, there was reduction of total staining by the UBI 1 antibody of the 60-kDa subunit and its 50-kDa fragment, which could be explained by digestion of epitopes very close to the C terminus.() Thus, while exposure of the subunit could not be detected, a site in the middle of the subunit was easily accessible at the extracellular surface, as well as a site near the C terminus.


Figure 2: Digestion of the extracellular domain of the subunit in right-side-out vesicles. Vesicles (33 µg) were digested with trypsin without external ligands present. Lanes 1 and 5 contained no trypsin. Aliquots were taken at 1 min ( lanes 2 and 6), 10 min ( lanes 3 and 7), and 30 min ( lanes 4 and 8) of proteolysis. Samples containing 2.5% -mercaptoethanol ( 1-4) or without reducing agent (5-8) were loaded on 5-15% gradient gels. Blots were stained with UBI 1 antibody to detect the C-terminal half ( A) and reprobed with antibody 757 to detect fragments containing the N terminus ( B). The subunit and its 50-kDa and 16-kDa fragments are denoted by arrows. The asterisk indicates the position of trypsin. C shows the sequence of the subunit within the first disulfide bridge and in the close vicinity. Vertical arrows indicate proposed sites of initial tryptic cleavage, whereas arrowheads denote additional positions for tryptic attack at 30 min of proteolysis.



Similar tryptic digestion patterns were observed when other physiological ligand combinations were used. The tested conditions are listed in . The pretreatments did not cause the opening of more than a small fraction of the sealed vesicles, as determined by measuring the latency of ATP hydrolysis. Regardless of conditions, the subunit was resistant to digestion. Cleavage of the subunit was restricted to formation of the N-terminal 16-kDa and C-terminal 50-kDa fragments at early times, with some additional cleavage at the C-terminal end of each fragment.

Fig. 2 also shows the pattern of subunit fragments detected when gels were run under reducing ( lanes 1-4) and nonreducing ( lanes 5-8) conditions. In A, the blot was probed with the antibody against amino acids 150-302, while in B it was reprobed with antibody against amino acids 1-12. It appears that the initial digestion occurred within a stretch of protein stabilized by a disulfide bond, which prevented the separa-tion of the fragments when -mercaptoethanol was omitted. These results are in agreement with previous reports (19, 34, 35) . By 30 min, however, another cleavage occurred outside the region connected by the disulfide, permitting the sepa-ration of the fragments. The delayed appearance of frag-ments on nonreducing gels is probably because cleavage at the initial points (Arg-135, Arg-142, or Lys-146, i.e. within the disulfide bond between Cys-125 and Cys-148) causes the protein to lose its tight globular structure, allowing trypsin access to Arg-149 or Arg-151 (Fig. 2 C).

The results verify the accessibility of the subunit to limited tryptic digestion, as well as the inaccessibility of the extracellular surface of the subunit, and establish that there is little effect of ligands which alter Na,K-ATPase conformation.

Digestion of Vesicles with Various Proteases

A variety of other specific and nonspecific proteases was tried for digestion of either subunit at the extracellular surface; summarizes the results. The subunit was not cleaved with any of them. This indicates how inaccessible its surface is and also serves as an internal control establishing that proteolysis did not occur during gel electrophoresis. Digestion patterns of the subunit were different with different proteases. No digestion was detected with papain, which was surprising because it had been reported previously to cleave the subunit at the extracellular surface (15, 36) . Like trypsin, chymotrypsin and elastase digested some, but not all, of the . Subtilisin, ficin, proteinase K, and bromelain caused extensive digestion, leaving only 15-20% of intact. A 16-kDa N-terminal fragment like that generated by trypsin was detected after digestion by chymotrypsin, elastase, subtilisin, and ficin, indicating that this marks a generally accessible site on the surface of the protein. A new N-terminal fragment with an apparent molecular mass of 40 kDa was detected after digestion by chymotrypsin, ficin, and proteinase K, marking a second exposed region. After digestion by bromelain, no major fragments were seen, indicating extensive digestion at secondary sites.

Proteolysis of Vesicles after Heat and Reduction

The most complete trypsinolysis of the subunit described in the literature was achieved after first treating it with 300 m M -mercaptoethanol to disrupt tertiary structure by reduction of its three disulfide bonds (37) . We hypothesized that such a reorganization of the extracellular domain of could result in exposure of hidden stretches of the subunit. Preincubation of vesicles with 100 m M DTT at 37 °C did not inhibit the activity of the Na,K-ATPase and did not make the vesicles leaky, as measured by the latency of ATP hydrolysis. Trypsinolysis after this treatment resulted in typical cleavage of the subunit into 16-kDa and 50-kDa fragments and no digestion of the subunit. Increasing the concentration of DTT to 200 m M and varying the duration of treatment and the temperature over the range from 4-37 °C did not promote digestion of . Although at least partial reduction of 's disulfide bonds should occur under some of these conditions, as shown for purified enzyme (38) , the digestion pattern in vesicles was unchanged. Heating of the vesicles to 50 °C during incubation with 200 m M DTT or 250 m M -mercaptoethanol, however, led to significant changes in the subsequent tryptic digestion of both subunits, accompanied by loss of most of the ATPase activity. Table III demonstrates the correlation between the disappearance of the 16-kDa fragment of the subunit and the exposure of the tryptic site on the subunit.

Fig. 3 illustrates the result of trypsinolysis of rat kidney vesicles after incubation with 200 m M DTT at 37 °C ( lanes 1-4) and 50 °C ( lanes 5-8) for 15 min. A distinct doublet of the subunit was seen in lanes 6-8 but not in lanes 2-4, thus demonstrating access to a new cleavage site. As calculated from several experiments, the fragment of the subunit was 8-9 kDa smaller than the intact protein. Similar fragments were generated by digestion with chymotrypsin. Site-specific antibodies were used to determine whether the cleavage point was near the N or C terminus (Fig. 4). The doublet was stained by the antibody against the N terminus ( A, lanes 8 and 9), while staining of the subunit by the antibody against the C terminus was reduced ( B). When DTT was omitted and pretreatment consisted only of heating at 50 °C, no digestion of was seen ( lanes 5 and 6). Proteolysis 8-9 kDa from the C terminus would place the cleavage site approximately between Lys-907 and Asn-937.


Figure 3: Dual effect of DTT and high temperature on digestion of the subunit. Right-side-out vesicles were preincubated with 200 m M DTT for 15 min at 37 °C ( lanes 1-4) or at 50 °C ( lanes 5-8). Trypsinolysis was then performed at 37 °C for different periods of time: 1 min ( lanes 2 and 6), 10 min ( lanes 3 and 7), and 30 min ( lanes 4 and 8). Lanes 1 and 5 were without trypsin. Samples were electrophoresed on 5-15% gradient gels. Blots were stained with the K1 antiserum. Arrows denote the positions of the intact subunit and the fragment resulting from digestion. The unidentified 70-kDa band again appeared as in Fig. 1, but it was not digested.



Recently, Goldshleger and Karlish (22, 23) reported that cleavage of the subunit occurred if pig kidney vesicles were heated at 55 °C for 30 min prior to trypsinolysis. Proteolysis resulted in a 91-kDa fragment, and digestion was near the C terminus, as found here. When we used those conditions, as shown in Fig. 4 ( lanes 11-12), a larger fraction of the subunit in rat kidney vesicles was digested than after heating to 50 °C in DTT, but the resulting fragment was similar in size. According to densitometric analysis of antibody-stained blots, as little as 15-20% of intact protein was left undigested, whereas 60-70% of the subunit remained uncleaved in DTT/50 °C-treated samples.


Figure 4: Location of cleavage sites in and . Vesicles (9 µg) were digested with trypsin or chymotrypsin (1 h, 37 °C) with or without prior treatment as indicated at the bottom of the figure. Samples were analyzed on a 7.5% polyacrylamide gel ( A and B) or on a 16% polyacrylamide Tricine gel ( C). Blots were stained with McK1 antibody, which detects the N terminus of rat 1, amino acids 14-22 (16) ( A); peptide-directed antiserum 845, which detects the C terminus of , amino acids 1003-1013 ( B); and peptide-directed antibody 757, which detects the N terminus of ( C). The digestion fragments are marked with arrows. The asterisk denotes the position of chymotrypsin, which was evidently autodigested in the presence of DTT ( lane 9). As with trypsin, the basis for ECL detection of chymotrypsin is not understood. Autodigestion of trypsin prevented its visualization in this experiment (see Fig. 1).



To detect fragments of the subunit, 16.5% polyacrylamide Tricine gels were used. Fig. 4 C illustrates staining with the antibody against amino acids 1-12. Digestion patterns in control vesicles and those only heated at 50 °C were identical: undigested and the 16-kDa fragment could be detected, and with chymotrypsin, the 40-kDa N-terminal fragment was also seen. In contrast, proteolysis of the glycoprotein was altered in conditions where digestion of the subunit was observed. If DTT treatment at 50 °C preceded digestion, about twice as much remained intact with both trypsin and chymotrypsin. This may be due to enhanced autodigestion of the proteases in the presence of the reducing agent. For the fraction of that was digested, however, the yield of the 16-kDa N-terminal fragment was greatly reduced, and instead there was an N-terminal fragment of approximately 6 kDa. After heating the vesicles to 55 °C, the subunit became entirely sensitive to digestion by both proteases (Fig. 4 C). There was a major N-terminal fragment of 6-7 kDa in trypsin, suggesting exposure of a cleavage site just above the membrane; Goldshleger et al. (22) reported detection of a 10-kDa fragment of pig subunit in similar conditions. We detected three similar fragments with chymotrypsin, suggesting the exposure of multiple sites. Two identical blots of the fragments generated by trypsin treatment were stained with an antibody (FP1) against amino acids 150-302 ( lanes 1-6) and an antibody (757) against amino acids 1-12 ( lanes 7-12). It can be seen that the staining of the 50-kDa fragment was greatly reduced and no major subfragments were seen, indicating that there was further digestion that removed the epitopes for this antibody (Fig. 5).


Figure 5: subunit in DTT-treated vesicles is more resistant to proteologysis. Vesicles (9 µg were heated as indicated in the presence or absence of 200 mM DTT prior to proteoloysis. After 1 h of digestion (37 °C), samples were separated on a Tricine gel, and blots were stained with the polyclonal antibody FP1, which detects the C-terminal half of (lanes 1-6), or with peptide-directed antibody 757, which detects its N terminus (lanes 7-12). The subunit and its fragments are denoted with arrows.



Taken together, the data of Figs. 3, 4, and 5 and the loss of ATPase activity suggest that harsh treatment caused rearrangements of the structure of the Na,K-ATPase. The most quantitative digestion of correlated with the most thorough digestion of .

Protection of the C-terminal Domain of by Rb+

K+(and its congener Rb+) is known to stabilize the Na,K-ATPase against denaturation by heat (39, 40) , to stabilize the disulfide bonds in the subunit against reduction (41) , and to stabilize a 19-kDa segment at the C terminus of to digestion by trypsin (42) . Goldshleger et al. (22) have reported that Rb+also protects against proteolysis of sites on pig at the extracellular surface, which is logical since the cleavage point in that is exposed by 50 °C/DTT or 55 °C lies within the 19-kDa fragment (8-9 kDa from the C terminus). Fig. 6 shows that 10 m M Rb+fully protected the C-terminal domain of the rat subunit from trypsinolysis in vesicles that had been heated at 55 °C for 30 min. Moreover, digestion of the subunit under the same conditions followed the pattern of the control sample, in which a single cleavage generating an N-terminal 16-kDa fragment was the final product of proteolysis. Choline chloride at 10 m M did not provide protection for either or subunits (data not shown). Table IV confirms that the presence of Rb+during heating of pig kidney vesicles to 55 °C also protected [H]ouabain binding in parallel with ATPase activity.


Figure 6: Rb+ ions protect a native configuration of the C-terminal domain of and the N-terminal domain of . Vesicles were trypsinized (1 h, 37 °C) in the presence or absence of 10MM RbCl with or without prior hearting as indicated. Samples were separated on a 7.5% polyacrylamide gel (A) or a Tricine gel (B). Blots were stained with the monoclonal antibody of McK1, which detects the N terminus of (A), or site-specific antibody 757, which detects the N terminus of (B). The positions of , , and the fragments of digetsion are denoted with arrows. Digestion products after heating in Rb+ were indistinguishable from those in the control.

Exposure of Kinase Phosphorylation Sites

Does the extracellular cleavage site on become accessible because of loosening of compact structure, or is there a more fundamental reorganization of the C-terminal domain caused by the harsh treatment? The recent identification of Ser-938 (rat numbering) as the site of phosphorylation by protein kinase A (43, 44) provides a way to address this, since the cleavage site is predicted to be near this residue, and exposure of one site would be predicted to be accompanied by exposure of the other. Evidence that Ser-938 is normally exposed on the intracellular surface is that expression of mutagenized protein in transfected cells blunted the modest inhibition of Na,K-ATPase activity elicited by stimulation of protein kinase A (43) . Phosphorylation of Na,K-ATPase subunit by protein kinase A has a peculiarity, however. Once the cell is disrupted and membranes are isolated, phosphorylation requires the presence of a detergent like Triton X-100 (45) , even when there are no intact vesicles. The requirement for detergent is not understood, but suggests that Ser-938 is close to the membrane or is affected by conformation changes brought about by interaction of transmembrane segments with their lipid or detergent environment. The detergent alone can inhibit Na,K-ATPase activity (46) .

Prior to examining the phosphorylation of right-side-out kidney vesicles, we used purified Na,K-ATPase before and after heating and reduction. Fig. 7 A confirms that Na,K-ATPase was normally phosphorylated only in the presence of Triton X-100 (compare lanes 9 and 10). In contrast, samples that were pretreated with 200 m M DTT at 50 °C for 15 min were phosphorylated even without detergent, although addition of Triton enhanced it. Heating to 55 °C for 30 min resulted in the highest level of 32P incorporation, and addition of Triton had little additional effect.() The presence of Rb+during both pretreatment and phosphorylation reduced phosphorylation in all cases, including in the control with Triton, where a 25% reduction was observed. The effect of Rb+was most pronounced, however, in the absence of Triton. Fig. 7 B shows antibody staining of the same blot to demonstrate that variation in subunit recovery did not account for the differences seen. Under the same conditions, treatment with Triton X-100 caused essentially complete loss of ATPase activity, while the DTT/50 °C and 55 °C pretreatments caused 80-90% loss (data not shown). Preincubation with 1 m M ouabain before heating, like Rb+, prevented the exposure of the phosphorylation site in medium without Triton (also not shown).


Figure 7: subunit of Na,K-ATPase treated with heat and reduction can be phosphorylated by protein kinase A catalytic subunit without detergent. Rat kidney microsomes (1 µg) were phosphorylated by protein kinase A in the presence or absence of Triton X-100 or 10 m M RbCl, with or without prior treatment, as indicated. A shows an autoradiogram of 32P-labeled subunit after SDS-gel electrophoresis and electrophoretic transfer of the samples. The same blot was restained with the antibody McK1 ( B). Although the original blot was still radioactive, the time required to expose x-ray film for visualization of ECL signal is too short (5-20 min) for autoradiographic detection of the 32P (which required 16 h). The harsh treatments were able to substitute for Triton X-100.



The critical question is whether the phosphorylation site remains hidden at the intracellular surface in right-side-out vesicles. Only a lack of phosphorylation would be consistent with the retention of both normal topology and intact vesicles. Since trypsin did not obtain access to the many tryptic cleavage sites on on the inside of the vesicles after heating and reduction, and since protein kinase A is much larger than trypsin, the harsh treatments were unlikely to have made the vesicles leaky to the kinase. Fig. 8 A shows, nonetheless, that phosphorylation of rat kidney vesicles by kinase in the extravesicular space occurred after heating and reduction even in the absence of detergent. In control vesicles, phosphorylation was seen only after addition of Triton X-100. By densitometry, 49% of the Na,K-ATPase in vesicles treated at 50 °C with DTT became phosphorylated, and 59% of that treated at 55 °C. These proportions are too large to have been derived from the 15% open vesicles in the preparation. Fig. 8 B shows the constant amount of subunit present per lane. The incomplete level of phosphorylation after heating and reduction corresponds with the incomplete cleavage of to the 90-kDa fragment seen in other experiments. The data suggest that in a significant proportion of the Na,K-ATPase units, a radical conformation change has resulted in the movement of the kinase and tryptic sites to the outside.


Figure 8: Protein kinase A phosphorylation of subunit in right-side-out vesicles reveals a structural reorganization of the C-terminal domain. Phosphyorylation of rat kidney vesicles (10 mg) by protein kinase A catalytic subunit was performed int he presence or absence of Triton X-100 with or without prior treatment as indicated. A blot is an autoradiogram of 32P-labeled subunit; straining of the same blot with McK1 antibody is presented in B. After harsh treatments, the phosphorylation site became accessible from the outside.



Phosphorylation by protein kinase A catalytic subunit was performed in rat kidney vesicles after heating but prior to digestion by trypsin, to see if the smaller cleaved fragment(s) could be detected (Fig. 9). In the same experiment, we employed an antibody (anti-ETYY) capable of detecting fragments containing the subunit C terminus. In Fig. 9 A, staining by an antibody (McK1) against the N terminus of illustrates the production of the 90-kDa fragment. In Fig. 9 B, it is seen that in the heated and undigested sample was phosphorylated by protein kinase A catalytic subunit. In the digested sample, the cleaved 90-kDa fragment was not phosphorylated, as predicted if the cleavage site is on the N-terminal side of Ser-938. Uncleaved was also not phosphorylated, as predicted if only those enzyme units that have undergone a radical conformation change are susceptible to both trypsin and the kinase. The data suggest that the pretreatment used was not sufficient to achieve 100% conversion of subunits to the altered conformation. In Fig. 9 C, staining with antibody against the C terminus of illustrated that only very small amounts of small cleavage products could be detected, at 22 kDa and 15.2 kDa. Both of these may have been derived from enzyme in open vesicles, and since they were not phosphorylated by protein kinase A in the absence of Triton X-100, they were more likely to be derived from undenatured enzyme than the heat-modified enzyme. Their predicted N termini would be Asn-833 (42) and Trp-889 (23) (rat numbering). Autoradiography of the same gel to detect 32P (Fig. 9 D) showed that there was a small amount of a phosphorylated fragment of approximately 17.5 kDa which did not coincide with either of the fragments stained by antibody against the C terminus; we cannot be certain whether this fragment was derived from Na,K-ATPase or from a phosphorylated contaminant of 45 kDa also seen in Fig. 9D. There was a smudge of phosphorylated fragments of molecular mass around 8.5 kDa, also not stained by the antibody, and also in low yield (Fig. 9, C and D). The results indicate that the phosphorylated portion removed from to generate the major trypsin-stable 90-kDa fragment was further digested, and that neither phosphate- nor ETYY-containing peptides were recovered in useful amounts.


Figure 9: The fragment containing the phosphorylation site and the C terminus can be further digested. Heated or untreated rat kidney vesicles were phosphorylated by protein kinase A catalytic subunit without the addition of Triton X-100. After phosphorylation, portions were digested with trypsin, and all samples were electrophoresed on a 7.5% polyacrylamide gel ( A and B) or a 16% polyacrylamide Tricine gel ( C and D). Blots were first exposed to x-ray film for autoradiography of the 32P ( B and D). The positions of the intact subunit and its 90-kDa fragment are indicated with arrows ( B): only the intact, undigested contained 32P. The asterisk in D shows the position of the smallest fragment containing 32P. The yield of 32P-labeled fragments was very low, suggesting losses due to further digestion. The blots were then stained with monoclonal antibody McK1 ( A) to detect the intact subunit and its 90-kDa fragment ( A) or with anti-ETYY to detect intact and any C-terminal fragment ( C). The yield of C-terminal fragment was negligible and did not coincide with any 32P, indicating that the epitope was cleaved off.



An alternative hypothesis, that the affected Na,K-ATPase units could be denatured and entirely exposed at the extracellular surface, is not viable because of the resistance of the 90-kDa major fragment to extravesicular trypsin. Further evidence that normal topology was maintained for the rest of the subunit was obtained by using another enzyme, protein kinase C, as a probe for the exposure of an intracellular site closer to the N terminus (47, 57) . Fig. 10 shows that protein kinase C does phosphorylate a fraction of the Na,K-ATPase in right-side-out vesicles ( lane 1), presumably because of unsealed vesicles in the preparation. Treatment with 0.016% SDS to open the vesicles significantly increased the level of subsequent phosphorylation ( lane 4). Pretreatment with heating and reduction decreased the overall level of phosphorylation by 30% in SDS-opened vesicles ( lanes 5 and 6), indicating either some denaturation of the kinase target site or a DTT effect on the kinase. It can be seen clearly, however, that the heating did not increase phosphorylation, compared to the samples treated with DTT but not heated, in vesicles that had not been opened with SDS (compare lane 2 with lane 3). Similar results were obtained when vesicles were heated at 55 °C (data not shown). The failure to detect, in lanes 2 and 3, phosphorylation comparable to that in lanes 5 and 6, allowed us to conclude that the integrity of the lipid bilayer was retained, and that the protein kinase C phosphorylation site, unlike the protein kinase A site, was not exposed at the extravesicular surface.


Figure 10: Exposure of the protein kinase C phosphorylation site is not increased in sealed vesicles. Phosphorylation of vesicles by protein kinase C without or without permeabilization with SDS was performed without or without prior incubation with heat and 200 mM DTT, as indicated. A shows an autoradiogram of 32P-labeled protein. Arrows indicate the position of the phosphorylated subunit and autophosphorylated protein kinase C. Staining of the same blot with McK1 antibody is presented in B. A basal level of phosphorylation was seen due to open vesicles, but no increase was caused by the harsh treatment.

Exposure of the Subunit C Terminus

As shown above (Fig. 9 C), the absence of anti-ETYY staining of fragments suggested that the C terminus had been digested from the extracellular surface after heating and reduction. The extravesicular exposure of this site was tested by a competition enzyme-linked immunosorbent assay, in which antibody that binds to membrane-bound antigen in solution is removed by centrifugation, and residual antibody is tested for binding to antigen that has been denatured by adsorption to a polystyrene plate. The anti-ETYY antibody is known to bind only to denatured enzyme (28) . We hypothesized that heating and reduction would denature the C terminus enough to permit it to bind, and we verified this using rat kidney microsomes (data not shown). The experiment was then performed with sealed right-side-out vesicles, with and without permeabilization (Fig. 11). Without prior heating, anti-ETYY bound very little to the vesicles (high concentrations of vesicles were required to reduce binding), and permeabilization had little effect. The concentration of SDS used here was sufficient to open vesicles and unmask the epitope for antibody McK1 near the N terminus (10, 16) , but it did not affect the epitope for anti-ETYY. After heat treatment of the vesicles, however, antibody bound well, even to vesicles that had not been opened by detergent treatment. The conclusion is that the C terminus, like the protein kinase A phosphorylation site, became exposed at the extracellular surface.


Figure 11: Heating exposes the C terminus of at the extravesicular surface. Anti-ETYY antibody (1:1,000 dilution) was preincubated in solution with various amounts of sealed rat kidney vesicles ( open circles and squares) or SDS-treated vesicles ( closed circles and squares, with ( squares) or without ( circles) prior heating at 55 °C. After removing the antigen-antibody complex by centrifugation, the residual soluble antibody was assayed by conventional enzyme-linked immunosorbent assay using rat kidney microsomes as the antigen bound to the plate. Vesicles competed effectively for antibody only after they had been heated, and sealed right-side-out vesicles were almost as effective as those opened with SDS.




DISCUSSION

Implications for Na,K-ATPase Subunit Structure

Previous attempts to demonstrate proteolysis of the Na,K-ATPase subunit at the extracellular surface have not been successful, but cleavage at sites that are predicted to be extracellular has been observed when digestion is performed with enzyme that is accessible from both sides (Arg-880 between H7 and H8 (9, 37) ; Arg-974 between H9 and H10 (19, 37) ). This led us to seek new conditions that would permit digestion from the outside. Although the original rationale for heating and reduction was to disrupt the subunit to expose the natural surface of the subunit, we observed a discrete step of subunit denaturation. What makes the result significant is that the denaturation event affected the exposure of only a portion of the subunit near its C terminus.

The predicted structure of the C-terminal third of the Na,K-ATPase has been controversial, and the 10-span model in Fig. 12 A is provisional. Attempts to predict transmembrane -helices have been complicated by low hydropathy values and by an unconvincing alignment of predicted segments from different members of the ATPase gene family (48) . Attempts to determine topology by the detection of epitopes, or the insertion of exogenous epitopes, have resulted in models (10, 29, 49, 50, 51) that conflict in some instances with models resulting from proteolytic digestion studies (9, 34, 37, 52) . Not all of the predicted membrane spans in this region can act as signal anchor sequences or stop-transfer signals during biosynthesis in vitro (53, 54, 55) . There is also controversy about whether H9 and H10 span the bilayer (19, 52, 56) . In sum, the physical evidence for 6 -helical membrane spans in this region is not compelling.

Heating and reduction, of course, have the potential to bring about radical structural change. The extensive digestion of the subunit after treatment is consistent with its substantial unfolding. To what extent the subunit was denatured is not known, but ATP hydrolytic activity and ouabain binding were destroyed, whereas the membrane barrier was not. A small portion of the subunit, normally hidden, was exposed at the extracellular face. A minimum of three sites within this region became accessible: the cleavage site generating the 90-kDa N-terminal fragment, the protein kinase A phosphorylation site, and a cleavage site near the C terminus. Strictly speaking, we do not know whether these sites are normally hidden by compact folding at the extracellular surface, by burying them within the transmembrane portion, or by exposing them on the intracellular surface. For this reason, the structure is represented as a black box in Fig. 12 B.

Location of the First Cleavage Site

The exact site of cleavage is not yet known, since we were unable to recover a C-terminal fragment to determine its sequence. From the difference in electrophoretic mobility between intact and the N-terminal fragment, we calculated that the cleavage site was 8-9 kDa from the C terminus. The closest predicted sites are Lys-933, Arg-935, or Arg-936. The fact that the phosphorylation site (Ser-938) was removed by proteolysis rules out any cleavage sites closer to the C terminus. The next nearest sites are at Arg-906 and Arg-907, but cleavage here would be predicted to reduce the size of by 12,000 kDa. Phosphorylated fragments of approximately the expected size, 8.5 kDa, were seen on Tricine gels, but they were present in very low yield. Since they were not stained by an antibody against the C-terminal amino acids ETYY, we cannot be certain that they were not derived from an unrelated protein.

In the model shown in Fig. 12 A, the sites at 906-907 are predicted to be on the extracellular surface, whereas those at 933-936 are predicted to be on the cytoplasmic surface. If cleavage is really at 933-936, and if the model is correct, the exposure of this region to proteolysis from the extracellular side of sealed right-side-out vesicles implies that it moved (``popped out'') as an early step of denaturation.


Figure 12: Folding models for the Na,K-ATPase. A shows a conventional 10-helix model for the subunit, and a 1-helix model for the subunit. The N and C termini are indicated, and the P marks the location of phosphorylation by protein kinase A. The oval marks the segment beginning at Trp-887, which has been identified as the location of the epitope for antibody IICand also as a site important for the assembly of with . Two hot spots for proteolysis of the subunit are marked with arrows; the subunit is resistant to digestion. B shows an alternative model for . The last 3 predicted spans are replaced by an unspecified structure, buried within the protein core of the subunit. The site of IICbinding and - assembly ( oval) is still oriented to the extracellular surface, and the phosphorylation site to the cytoplasm, but the C terminus of is suggested to be buried. C shows the structure obtained after heating and reduction. The phosphorylation site is exposed on the extracellular side of the membrane, as well as the C terminus. New proteolytic digestion sites ( arrows) on result in the cleavage of both the phosphorylation site and the C terminus from the extracellular surface. Additional cleavage sites on the subunit are also exposed. The association between and is assumed to be disrupted. The harsh conditions may have also altered structure at the intracellular surface, but this is not shown for simplicity.



Goldshleger and Karlish (23) digested preparations of pig kidney vesicles after heating to 55 °C; they reported detection of 4 fragments (7.4-14.5 kDa) containing ETYY. The yield of the peptides was not reported. The peptides were sequenced, and the largest was the result of cleavage at Trp-889 (rat numbering). Although initially interpreted in terms of an 8-span model (22) , cleavages were later shown to occur between H7 and H8, H8 and H9, and H9 and H10, implying that H8 and H9 popped out of the membrane to the extracellular surface.() Our failure to detect ETYY-containing phosphorylated peptides could be explained if the rat Na,K-ATPase is more labile than pig Na,K-ATPase and undergoes more complete digestion in similar conditions.

Location of the Protein Kinase A Phosphorylation Site

The protein kinase A phosphorylation site at Ser-938 became accessible as a result of the treatment, and it is adjacent to the predicted cleavage site (43, 44, 57) . Two different groups have inserted epitopes at this location to determine its topology, but reached opposite conclusions (49, 51) . Some consideration should be given to the possibility that Ser-938 isn't a physiological kinase regulatory site, since phosphorylation cannot be observed in vitro without the addition of Triton X-100, and Triton X-100 has inhibitory effects on ATPase activity. If the site were actually on the extracellular side, but buried, it would be plausible for both heating and Triton X-100 to cause similar changes in its exposure. In this event, the harsh treatments used here would have performed as originally predicted: exposing sites that were already extracellular, but masked. The weight of opinion, however, is that the phosphorylation site normally lies on the inside, leading to the conclusion that in these conditions it popped out.

Location of the C Terminus

If only a hairpin pair of transmembrane helices (H8 and H9) containing the protein kinase A phosphorylation site and the new tryptic cleavage site was extruded during heating and reduction, the C terminus of the subunit would be predicted to remain protected on the intracellular surface. The paucity of any digestion fragments staining for the epitope at the very C terminus (ETYY), however, implies that the C terminus became accessible to trypsin in the extravesicular space. The accessibility of the C terminus was confirmed directly by showing that heated vesicles acquired the ability to bind anti-ETYY antibodies in solution, and that opening the vesicles caused very little additional exposure of sites. Thus, all of the data support the heat-treated structure modeled in Fig. 12 C.

The normal location of the C terminus has been controversial. The weight of evidence suggests that it is normally inaccessible from either side. Two groups have produced antibodies against the peptide sequences of the C terminus that failed to recognize native enzyme (26, 28) , providing no support for binding to exposed sites on either surface. Another antibody against the C terminus was initially thought to bind to the extracellular surface based on fluorescence-activated cell sorting, but the epitope was readily removed by trypsin (58) , suggesting in retrospect that the fluorescence was due to a small fraction of denatured enzyme or to antibody contaminants binding to unrelated surface proteins. Two additional antibodies against the C terminus were thought to bind on the cytoplasmic surface because binding to right-side-out kidney vesicles was seen only when they had been opened by treatment with SDS (17) . However, those vesicles showed a maximum of 6.7-fold increase in ATPase activity, while the antibody binding increased more than 1000-fold. The conclusion that the site is on the intracellular surface is less likely than that the site was buried and exposed by detergent. Further evidence for the normal inaccessibility of the C terminus is that access of the sequence ETYY to carboxypeptidase Y was restricted in trypsin-digested membranes which have intact Rb+occlusion capacity, but greatly increased in digested membranes treated with Ca2+, which destroys Rb+occlusion capacity (9) . It was concluded that digestion occurred only in the inactivated sample. Whether the Ca2+treatment caused externalization of a large portion of the subunit near the C terminus as shown here is not known. Covalent labeling studies with pyridoxal phosphate (21) or lactoperoxidase followed by carboxypeptidase digestion (59, 60) are consistent with an intracellular location, but are not quantitative enough to prove that all of the C termini are normally exposed, rather than buried. Other evidence that the C terminus must be on (or close to) the cytoplasmic surface is that a cDNA fusion product between the and subunits could be expressed as active enzyme (61) . The flexibility of the linker may have allowed repositioning of the Na,K-ATPase C terminus, but it seems unlikely that it could have allowed it to move from the outside to the inside without disruption of enzyme activity.

The data presented here throw no light on the normal location of the C terminus. The observation that there was no loss of ETYY binding (on blots) to chains that were neither cleaved to 90 kDa nor phosphorylated suggests, however, that the exposure of the primary cleavage site, the phosphorylation site, and the C terminus was a single concerted event.

Implications for Na,K-ATPase Subunit Structure

The identification of fragments of the untreated subunit revealed that it has two proteolytic hot spots, one yielding an N-terminal fragment of 16 kDa described before (34, 35) , and the other yielding an N-terminal fragment of about 40 kDa. The location of the second site can be predicted to be in the vicinity of residue 238, if each amino acid has an average molecular mass of 110 and each of the three carbohydrate groups changes the electrophoretic mobility by the equivalent of 9 kDa. The 40-kDa fragment would thus contain 75-80% of the protein and two carbohydrate groups. In both cases, the cleavage sites would lie within disulfide bridges. Consequently, the cleavages would not be expected to have a major impact on Na,K-ATPase tertiary structure.

When enzyme was digested after heating and reduction or heating at 55 °C, an antibody against the first 12 amino acids of the subunit still stained the ~6-kDa trypsin digestion product. Trypsin cleaves off 4 amino acids from the N terminus only when it has access to the intracellular surface (18, 34) . In the presence of Mg2+/P, or in the presence of Ca2+, both of which promote more extensive digestion of and , the first 26 amino acids are removed (9, 19) , which would remove the binding site for this antibody. The site's continued presence here implies that the N terminus of is still inaccessible. Goldshleger et al. (22) reported that their 10-kDa N-terminal fragment of pig subunit began at the N-terminal alanine, also demonstrating that trypsin had not entered the vesicles. The 6-kDa fragment seen after tryptic digestion here should be long enough to span the membrane if cleavage was at Arg-70. The difference in apparent molecular mass (6 kDa versus 7.7 kDa predicted) could be due to anomalous mobility of hydrophobic fragments on the gel.

Since the subunit is stabilized by three disulfide bonds, one would expect its cleavage to be more complete after reduction than after heating alone. In the absence of reduction, digestion after treatment at 50 °C for 15 min resulted in cleavage of at one site like the control, while after 55 °C for 30 min, it was digested extensively. Addition of reducing agent in the milder conditions promoted more extensive digestion of at least some of the subunit. The most salient observation was that pretreatment at 55 °C did a good job of making sensitive to proteolysis without DTT.

Implications for - Interaction

There is strong evidence that the subunit associates specifically with portions of the C-terminal third of the subunit (62, 63, 64) . Chimeras of Na,K-ATPase with Ca2+-ATPase have narrowed down an essential region to 26 extracellular amino acids between the 7th and 8th spans of the 10-span model, from Asn-891 to Ala-916 (63) . We have observed that an antibody (IIC) that maps to the same region binds to native enzyme only under certain circumstances, suggesting that the region is subject to conformational changes (10) . An antibody to the H,K-ATPase, mAb 146-14, has been shown to interact with both the subunit and the subunit at the extracellular surface near H7, in the region implicated in - interaction in the Na,K-ATPase (50) . The region of the subunit most important for assembly appears to be within the N-terminal half (4, 5) and has been narrowed down to 96 amino acids just on the C-terminal side of the membrane span by construction of chimeras (65) . Other sites are also implicated (66, 67) , but may have their effect through influence on conformation. Within the required region, only amino acids 68-77 have a high degree of conservation between species and isoforms, a property expected of the assembly site because of the interchangeability of subunits.

These observations are consistent with the present report that heating and reduction disrupt - interaction sufficiently to expose new tryptic cleavage sites at or near the interaction site. The ~6-kDa N-terminal tryptic fragment of the subunit should result from cleavage at RVAP, just above the membrane span and within the most likely site of assembly (amino acids 68-77). The subunit cleavage site that leaves a 90-kDa N-terminal fragment should be just on the C-terminal side of the assembly site, in a region that appears to be stabilized by the complex with , but extruded when - interactions are disrupted. The observation that conditions that expose one subunit to digestion also expose the other subunit supports the hypothesis that and interact closely at these sites.

The effect of Rb+in preserving activity and ouabain binding and in protecting against digestion is consistent with the hypothesis that the Rb+-occluded form of the enzyme is a conformation with maximum compactness and stability (19, 22, 42) . We can also point out an inverse correlation between Rb+(K+) binding and the accessibility of the protein kinase A catalytic subunit phosphorylation site in unheated, but Triton-treated enzyme: optimal phosphorylation is seen in the absence of monovalent cations and in the presence of Mg2+/P, and next best in the presence of Na+, whereas phosphorylation is lower in the presence of K+and is markedly inhibited by ouabain (44) . The conditions that are best for phosphorylation thus are those that are worst for Rb+occlusion.

The conclusion that emerges is that both K+(Rb+) and ouabain stabilize a conformation in which the protein kinase A catalytic subunit site is buried and other sites within the 19-kDa C-terminal portion are protected from proteolysis. The stabilizing effect of Rb+against a variety of insults (heat, reduction, and proteases), combined with the fact that both subunits are protected, implies that and subunits go through condensed and expanded conformations in a concerted way. Within this framework it is logical to propose that, after heating, the transmembrane spans of the subunit that do not directly participate in interaction with the subunit remain stably associated with the membrane, while those that do interact with unfold into the extracellular space.


Table I Tryptic cleavage of and resistance of is unaffected by Na,K-ATPase ligands

Rat kidney vesicles were presoaked in media containing various ligands for the indicated periods of time. The latency of the vesicles was determined by the stimulation of Na,K-ATPase activity by mild detergent treatment. The latency of untreated vesicles (kept on ice after thawing) in a medium containing no external ligands was taken as 100%.





Table II

Digestion of right-side-out vesicles with various proteases reveals two ``hot spots'' in the structure of subunit

Rat kidney vesicles were pre-equilibrated for 1 h at 4 °C in different media prior to proteolysis. Digestion was performed for 30 min at 37 °C. Blots were stained with peptide-directed antibody 757 to detect any fragments containing the N terminus of the subunit.





Table III

Effect of heating and reduction on proteolysis of and subunits of Na,K-ATPase in right-side-out vesicles

Rat kidney vesicles were treated with various concentrations of DTT for the indicated periods of time prior to trypsinolysis. No external ligands were added to the incubation media. Detection of digestion fragments was performed with K1 antiserum ( subunit) or peptide-directed antibody 757 ( subunit).





Table IV

Protective effect of Rb+ions against thermal denaturation

Pig kidney Na,K-ATPase (specific activity 20 µmol of Pmg of protein-1 min-1) was heated at 55 °C in the presence or absence of 10 m M RbCl. [H]Ouabain binding was measured at equilibrium (37 °C, 1 h, in the presence of 5 m M Mg2+and 5 m M P), and was determined to be 6.5 nmol/mg of protein in the control sample. The results represent the average of two independent experiments.




FOOTNOTES

*
This work was supported by National Institutes of Health Grant HL 36271. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence and reprint requests should be addressed. Tel.: 617-726-8579; Fax: 617-726-7526.

The abbreviations used are: Na,K-ATPase, Na+- and K+-stimulated adenosine triphosphatase; DTT, dithiothreitol; MOPS, 4-morpholinepropanesulfonic acid.

Indeed, in further experiments, we have been able to demonstrate that most of the antigenic determinants responsible for binding of FP1 antibody (the equivalent of the UBI 1 antibody used here) are localized within the C-terminal quarter (amino acids 230-302) of the subunit. More direct confirmation was obtained in experiments using the peptide-directed antibody 940 against amino acids 294-302 (gift of W. J. Ball, Jr.), which indicated that the extreme C terminus of the subunit can be easily clipped off when rat kidney right-side-out vesicles are proteolyzed even under mild conditions. In similar experiments performed with pig kidney vesicles, we did not see any loss of staining of the subunit or its 50-kDa fragment with monoclonal antibody M17-P5-F11 (gift of W. J. Ball, Jr.), which recognizes amino acids 234-236 in . Data not shown.

We have observed, however, that heating alone is not sufficient to expose the protein kinase A phosphorylation site in purified pig kidney Na,K-ATPase (data not shown), despite essentially complete loss of activity (Table IV). We confirmed that these conditions do expose a cleavage site in pig kidney right-side-out vesicles (23). Subtle species differences thus complicate the results.

S. J. D. Karlish, personal communication.


ACKNOWLEDGEMENTS

We thank W. J. Ball, Jr., Alicia McDonough, and J. Kyte for their generosity with antibodies and S. J. D. Karlish for early and stimulating sharing of research results.


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