Characterization of Disulfide Cross-links between Fragments of Proteolyzed Na,K-ATPase
IMPLICATIONS FOR SPATIAL ORGANIZATION OF TRANS-MEMBRANE HELICES*

Eran OrDagger , Rivka Goldshleger, and Steven J. D. Karlish§

From the Department of Biological Chemistry, Weizmann Institute of Science, Rehovot, 76100 Israel

    ABSTRACT
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Abstract
Introduction
References

This study characterizes disulfide cross-links between fragments of a well defined tryptic preparation of Na,K-ATPase, 19-kDa membranes solubilized with C12E10 in conditions preserving an intact complex of fragments and Rb occlusion (Or, E., Goldshleger, R., Tal, D. M., and Karlish, S. J. D. (1996) Biochemistry 35, 6853-6864). Upon solubilization, cross-links form spontaneously between the beta  subunit, 19- and 11.7-kDa fragments of the alpha  subunit, containing trans-membrane segments M7-M10 and M1/M2, respectively. Treatment with Cu2+-phenanthroline (CuP) improves efficiency of cross-linking. Sequencing and immunoblot analysis have shown that the cross-linked products consist of a mixture of beta -19 kDa dimers (approx 65%) and beta -19 kDa-11.7 kDa trimers (approx 35%). The alpha -beta cross-link has been located within the 19-kDa fragment to a 6.5-kDa chymotryptic fragment containing M8, indicating that beta Cys44 is cross-linked to either Cys911 or Cys930. In addition, an internal cross-link between M9 and M10, Cys964-Cys983, has been found by sequencing tryptic fragments of the cross-linked product. The M1/M2-M7/M10 cross-link has not been identified directly. However, we propose that Cys983 in M10 is cross-linked either to Cys104 in M1 or internally to Cys964 in M9. Based on this study, cross-linking induced by o-phthalaldehyde (Or, E., Goldshleger, R., and Karlish, S. J. D. (1998) Biochemistry 37, 8197-8207), and information from the literature, we propose an approximate spatial organization of trans-membrane segments of the alpha  and beta  subunits.

    INTRODUCTION
Top
Abstract
Introduction
References

Renal Na,K-ATPase consists of a catalytic alpha  subunit (112 kDa), a glycosylated beta  subunit (33 kDa), and a gamma  subunit (approx 6.5 kDa) (3). The alpha  subunit contains the ATP hydrolytic site and the Na+ and K+ transport sites. The beta  subunit is required for correct post-translational processing and stabilizes the alpha  subunit (4). The gamma  subunit may be a tissue-specific regulator (5). Recent studies on P-type pumps have focused on residues involved in cation or ATP binding, or conformational transitions (3, 6). Cation occlusion sites are located within trans-membrane segments (8), and site-directed mutagenesis suggests that side chains within M4, M5, M6, and M8 ligate cations (6, 9-11). In addition, a variety of studies have defined the topological organization of the catalytic subunits, comprising 10 trans-membrane segments (see Refs. 3 and 7 and references therein).

The spatial organization of trans-membrane segments of the catalytic subunits is unknown although such information is essential for defining the structure of the cation transport path. Recently published structures of Ca-ATPase and H-ATPase (12, 13), at 8-Å resolution, show that the 10 trans-membrane segments are alpha -helices and most helices are tilted so that their spatial organization changes at different levels in the membrane. A tentative model of helix packing of Ca-ATPase has been proposed based on constraints of trans-membrane topology, site-directed mutagenesis, and disulfide cross-linking (12, 14, 15). Modeling of Na,K-ATPase has also been attempted based on hydrophobic labeling and prediction of helix orientation with respect to lipid (16). Despite these attempts, direct determination of helix proximity is largely lacking. Recently, covalent cross-linking experiments have begun to provide such specific information (2, 15).

This paper describes experiments utilizing CuP1-catalyzed S-S bridge formation. Older work (17-20) showed that treatment of detergent-solubilized Na,K-ATPase leads primarily to a 1:1 alpha beta cross-linked product. Identification of the cross-linked residues constitutes a formidable task for, whereas the beta  subunit has only one free cysteine, Cys44, located within its single trans-membrane helix (21), the alpha  subunit contains 23 cysteines. The problem could be simplified by using 19-kDa membranes which are obtained by extensive tryptic digestion of Na,K-ATPase. This preparation consists of fragments of the alpha  subunit comprising mainly trans-membrane segments (M1/M2, M3/M4, M5/M6, and M7/M10) connected by the external loops, and a partially cleaved beta  subunit (8, 22). The fragments contain only 10 cysteines located within or next to trans-membrane segments.

A potential problem in cross-linking membrane proteins is the possibility of intermolecular cross-linking via random collisions in the membrane. Detergent solubilization can overcome this problem because the soluble protein can be diluted. Indeed Sarvazyan et al. (23, 24) have reported that treatment of digitonin-solubilized 19-kDa membranes with CuP yielded two cross-linked products, dimers of fragments containing M7/M10 and M1/M2 (22 kDa:11 kDa) and trimers containing these two fragments with the beta  subunit (beta :22 kDa:11 kDa) in 1:1:1 stoichiometries. These studies established that a cysteine residue within Asn831-Tyr1016 of the alpha  subunit (Cys911, Cys930, Cys964, or Cys983) is cross-linked to the beta  subunit (Cys44), but did not identify the cysteine.

19-kDa membranes lack ATP-dependent functions, but retain cation occlusion and ouabain binding (8, 22, 25). Recently we described a procedure for solubilizing 19-kDa membranes with the non-ionic detergent C12E10 which preserves intact the complex of fragments with occluded Rb ions and bound ouabain (1). The intact soluble complex contains one copy of each fragment. After solubilizing 19-kDa membranes in the absence of Rb ions and ouabain, neither Rb occlusion nor the complex of fragments are intact (1). Because cross-linking in the latter condition, as in Ref. 23, might reveal non-native interactions between fragments, one of our objectives has been to characterize cross-linking of the C12E10-solubilized intact complex of fragments containing occluded Rb ions. A second objective has been to identify cross-linked cysteines.

    EXPERIMENTAL PROCEDURES

Na,K-ATPase was prepared from pig kidney red outer medulla (26) and stored at -80 °C. Protein and ATPase activity were determined as described (26). Specific activities were 13-18 units/mg of protein. Before use enzyme was thawed and dialyzed overnight at 4 °C against 1000 volumes of 25 mM histidine, 1 mM EDTA(Tris), pH 7.0. 19-kDa membranes were prepared by tryptic digestion of Na,K-ATPase with trypsin (22), and resuspended at 3 mg/ml in 2 mM RbCl, 25 mM imidazole, 1 mM EDTA, pH 7.5. The specific Rb occlusion capacity was 6 to 6.5 nmol/mg of protein. 19-kDa membranes were solubilized with C12E10 at a ratio of 2.2 (w/w) as described by Or et al. (1). Before cross-linking with CuP the pH was adjusted to 8.0 with RbOH (final concentration, 0.8 mM).

Cross-linking Catalyzed by CuP-- The solubilized preparation (0.4 mg/ml) was treated at 20-22 °C with CuP (final concentration: 0.5/2.5 mM), added in 5 aliquots every 12 min. After 1 h solid urea was added to 2 M and the pH was readjusted to 8.0 with solid Tris base. Free cysteines were blocked by adding iodoacetamide to 40 mM and free Cu2+ was chelated with 10 mM EDTA(Tris). After 30 min at 20-22 °C the mixture was acidified to pH approx  6.0 with acetic acid and protein was precipitated with 4 volumes of methanol/ether (2:1) and stored at -20 °C overnight. When analytical amounts were cross-linked, urea, Tris base, and acetic acid were omitted.

Gel Electrophoresis-- Precipitated protein was collected by centrifugation at 9700 × g for 1 h at 4 °C, dried under a stream of nitrogen, and dissolved in loading buffer. Samples were resolved by Tricine-SDS-PAGE as in Refs. 22 and 27. Either 14-cm short or 23-cm long gels were used. In nonreducing conditions glutathione and mercaptoethanol were omitted from sample and running buffers.

Purification of CuP-catalyzed Cross-linked Products-- Precipitated protein (15 mg) was dissolved in 2 ml of nonreducing sample buffer and resolved on two 1.5-mm thick long 10% Tricine gels. Gels were briefly fixed, stained, and destained and bands of interest were cut out and equilibrated with 660 mM Tris-Cl, pH 8.9, for 1 h. Protein was eluted into 50 mM NH4HCO3, 0.1% SDS using a Bio-Rad Model 422 Electro-Eluter, run at 8 mA/tube overnight. Eluted protein was precipitated with 4 volumes of methanol at -20 °C after adjusting the pH to 7.0 with acetic acid. Pellets were collected by centrifugation and dissolved in 50 mM Tris-Cl, pH 8.0, 0.1% SDS. Samples were analyzed on a minigel to check the amounts and purities of the eluted proteins.

Proteolytic Digestions-- Protein (0.8 mg/ml) in 35 mM Tris-Cl, pH 8.0, 0.07% SDS was digested with trypsin (10% w/w) at 37 °C in the presence of 2 M urea and 10 mM CaCl2. Digestions with chymotrypsin (10% w/w) were done at 37 °C in 25 mM Tris-Cl, 1 mM EDTA, pH 8.0, 0.05% SDS. Proteolytic digestions were stopped with 5 mM phenylmethylsulfonyl fluoride.

Phosphorylation by PKA-- Purified protein (0.1-0.25 mg/ml) was incubated at 30 °C for 30 min in 20 mM Tris-Cl, pH 7.5, 0.02% SDS, 0.1% octyl glucoside, 10 mM MgCl2, 50 µM [gamma -32P]ATP, and 50 ng of catalytic subunit of PKA. Autoradiography was carried out using a Fuji BAS 1000 PhosphorImager after transfer of proteins from the gel to PVDF paper.

Digestion with N-Glycosidase F-- Free or cross-linked beta  subunit (0.1 mg/ml) in 50 mM phosphate buffer, pH 7.5, containing also 0.01% SDS and 0.7% octyl glucoside, was treated with 2500 units of N-glycosidase F at 37 °C for 20 h. The reaction was stopped by adding loading buffer.

Immunoblotting-- Protein samples were separated on short Tricine gels, which were electroblotted onto PVDF (Semi-Phor TE70, Hoefer Scientific Instruments) (28). PVDF sheets were incubated with antisera (22). Immunoblots were stained by diaminobenzidine with metal ion enhancement (29).

Sequencing-- Bands of interest were cut out of the gel, equilibrated in 660 mM Tris-Cl, pH 8.9, for 1 h and transferred to PVDF by either of two procedures. (a) Bands were separated on a second 1.5-mm thick short 10% Tricine gel and electroblotted onto PVDF (22). (b) Bands were cut into small pieces, and protein was eluted into 1.7 ml of 1 M Tris-Cl, 0.1% SDS, pH 8.45, by mixing on a rotating wheel for 20 h. Supernatants were transferred into 3-ml syringes connected to 13-mm Swinnex filter holders (Millipore) and eluted proteins were blotted onto PVDF (Immobillon PSQ Millipore) by centrifugation at 290 × g for 25 min. Sequencing of peptides blotted onto strips of PVDF was done on an Applied Biosystems Model 475A protein sequencer with an on-line Model 120A phenylthiohydantoin analyzer. Each reported sequence was done at least twice.

Materials-- 1,10-Phenanthroline-HCl was from BDH Chemicals, L-1-tosylamido-2-phenylethyl chloromethyl ketone-trypsin (bovine pancreas) was from Worthington, alpha -chymotrypsin was from Merck, N-glycosidase F was from New England Biolabs, and octyl glucoside from Calbiochem. Trypsin inhibitor (type 1-S from soybean), phenylmethylsulfonyl fluoride, iodoacetamide, ouabain, thioglycolate, Tricine, C12E10, and molecular weight markers (SDS17 and VII-L) were from Sigma. Electrophoresis grade reagents for SDS-PAGE and PVDF paper were from Bio-Rad and Millipore, respectively. [gamma -32P]ATP was from Amersham. Antisera recognizing Asn889-Gln903 and Lys1012-Tyr1016 (anti-KETYY) and the catalytic subunit of PKA were gifts from colleagues (see "Acknowledgments"). Antisera were raised against an 11.7-kDa peptide containing M1/M2 (N-terminal Asp68) and a 16-kDa fragment of the beta  subunit (N-terminal Ala5) as described previously (1).

    RESULTS

CuP-catalyzed Cross-linked Peptides-- Fig. 1 presents a representative experiment showing components of C12E10-solubilized 19-kDa membranes in reducing or nonreducing conditions and also cross-linked products after treatment with CuP. In reducing conditions (Red), untreated C12E10-solubilized 19-kDa membranes reveal the standard 19-kDa and smaller (8-12 kDa) fragments of the alpha  subunit (the latter are not well resolved on this 10% gel), an intact beta  subunit or its approx 50-kDa glycosylated and 16-kDa fragments (22). In nonreducing conditions (Non-red) one major new band appeared, 70-80 kDa, and the intensity of the 19-kDa fragment was less than in reducing conditions. The approx 50- and 16-kDa fragments were not seen because they are internally cross-linked between Cys125 and Cys148. After treatment of C12E10-solubilized 19-kDa membranes with CuP (Sol), the intensity of the 70-80-kDa band rose and that of the 19-kDa peptide and beta  subunit decreased further (compare Sol with non-Red), suggesting that this band contains both the 19-kDa peptide and beta  subunit. About 63% of the beta  subunit underwent cross-linking based on the weight ratio of the cross-linked product and remaining beta  subunit extracted from gels (approx 3.1, w/w), and on the composition of the cross-linked product (see Table I). Raising CuP concentration or incubation time did not improve the yield (not shown). CuP treatment of unsolubilized 19-kDa membranes, containing occluded Rb ions, did not produce the 70-80-kDa cross-linked band (Mem). After denaturation of 19-kDa membranes with SDS, CuP treatment did not produce the 70-80-kDa band (SDS), excluding the possibility that the cross-link is the product of a nonspecific association of its components after termination of the reaction. A small amount of a higher molecular weight band (Fig. 1, asterisk) was observed after C12E10 solubilization but the amount was not increased by CuP treatment. This band was also observed with intact 19-kDa membranes. It may represent the product of inter-molecular cross-linking between adjacent complexes, and therefore it was not investigated further. In conditions producing the 70-80-kDa band the remaining 19-kDa fragment ran a little faster than in the other conditions, implying that the peptide is more compact (compare Non-red and Sol with Mem, and SDS). This hints at the possibility of an internal cross-link (see also Table II).


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Fig. 1.   Spontaneous and CuP-catalyzed cross-links detected in C12E10-solubilized 19-kDa membranes. Samples (140 µg) were resolved on a long 10% gel. Red and Non red-solubilized 19-kDa membranes were electrophoresed under reducing or nonreducing conditions, respectively, without prior CuP treatment. Sol, solubilized 19-kDa membranes treated with CuP; Mem, unsolubilized 19-kDa membranes (0.4 mg/ml) in 3.3 mM imidazole, 130 µM EDTA, pH 7.5, plus 5 mM RbCl and 45 mM choline-Cl were treated with CuP. SDS, 19-kDa membranes in 3.3 mM imidazole, 130 µM EDTA, pH 7.5, plus 5 mM RbCl, 10 mM ouabain were denatured with 2% SDS prior to addition of C12E10 and CuP treatment.

                              
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Table I
Sequences found in the major CuP cross-linked product

                              
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Table II
Sequence analysis of cross-linked tryptic fragments

Fig. 2 presents an immunoblot using anti-KETYY to detect cross-links in intact 19-kDa membranes. After CuP treatment in the presence of Rb ions (+Rb) the antibody recognized the 19-kDa fragment but no higher molecular mass species, confirming the absence of cross-links in intact 19-kDa membranes seen in Fig. 1. By contrast, after CuP treatment in the absence of Rb ions (-Rb) the amount of 19-kDa peptide was reduced, and a number of bands appeared (30.8-66 kDa), as well as a smear of material in the upper half of the gel. Using antibodies raised against fragments containing M1/M2, M3/M4, and M5/M6 all these bands were recognized by anti-M1/M2 and some of them also by anti-M3/M4 or anti-M5/M6 (not shown). This heterogeneity suggests that the cross-links might be nonspecific. In fact the same pattern of anti-KETYY recognition was found for 19-kDa membranes in which Rb occlusion had been thermally inactivated (30, 31) (Fig. 2, Th.in). As discussed below these cross-links cannot be assumed to represent proximities between fragments in a native state.


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Fig. 2.   CuP-catalyzed cross-links detected in intact 19-kDa membranes. 19-kDa membranes (0.5 mg/ml) were suspended in 25 mM imidazole, 1 mM EDTA, pH 7.5, without (-Rb, Th.in) or with 30 mM RbCl (+Rb). Membranes were either kept on ice (-Rb) or were incubated at 37 °C for 25 min (+Rb, Th.in). All samples were then treated with CuP as described under "Experimental Procedures," and then dissolved in 2% SDS and treated with 40 mM iodoacetamide and 10 mM EDTA for 30 min at room temperature. Protein was precipitated with 4 volumes of methanol. The samples were resolved on a 10% gel in nonreducing conditions, transferred to PVDF paper, and then probed with the anti-KETYY antibody.

The components of the 70-80-kDa cross-linked product have been identified by sequencing (Table I) and immunoblots (e.g Fig. 3). Immunoblots showed that both spontaneously formed and CuP-induced cross-linked product contain the beta  subunit 19-kDa peptide (M7-M10) and an 11.7-kDa peptide (M1/M2) (not shown). N-terminal sequencing clearly identified four fragments (Table I). Two sequences are derived from the beta  subunit (N terminus Ala5 in 19-kDa membranes, Ref. 22), which is partially cleaved to fragments of 16 kDa (N terminus Ala5) and approx 50 kDa (N terminus, Gly143). The sequence with N terminus Ala5 represents both the 16-kDa fragment and intact beta  subunit, and about 65% is cleaved judging by the yields of 26 and 17 pmol/cycle, respectively. Two sequences are derived from the alpha  subunit: 19 kDa (N terminus Asn831, 17 pmol/cycle) and 11.7 kDa (N terminus Asp68, 9 pmol/cycle). Note that the yields of the components are not stoichiometric. The excess beta  subunit (26 pmol/cycle) over the 19-kDa fragment (17 pmol/cycle) implies that the cross-linked product was not completely resolved from free beta  subunit. The different yields of the 19- and 11.7-kDa fragments could imply that the cross-linked product consists of either a mixture of beta :19-kDa dimers (approx 65%) and beta :19 kDa:11.7 kDa trimers (approx 35%) or a mixture of dimers, beta :19 kDa and beta :11.7 kDa, which are not well resolved on the gel. Deglycosylation of the beta  subunit has allowed us to distinguish between these alternatives. The deglycosylated cross-linked product was clearly resolved into two bands of 62- and 54 kDa (Fig. 3A, Coom). Both bands were recognized by anti-beta antibodies and anti-KETYY but only the upper 62-kDa band was recognized also by anti-M1/M2. Thus the cross-linked product consists of a mixture of beta :19-kDa dimers and beta :19 kDa:11.7-kDa trimers.


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Fig. 3.   Identification of cross-linked peptides following deglycosylation of the beta  subunit. Purified CuP cross-linked product was treated or not with N-glycosidase F (+ or -PNGase, respectively), resolved on a short 10% gel and electroblotted onto PVDF. Coom' lanes were stained with Coomassie (A). Samples treated with PNGase were also probed with antibodies recognizing the 19-kDa fragment (anti-KETYY), the 16-kDa N-terminal fragment of the beta  subunit (anti-16 kDa), or the 11.7-kDa peptide containing M1/M2 (anti-M1/M2) (B).

Identification of Cross-linked Residues and Segments-- The following experiments demonstrate cross-linking of Cys44 of the beta  subunit to a cysteine in M8 (Cys911 or Cys930), and an internal cross-link between Cys964 (M9) and Cys983 (M10). We have not been able to identify the M7/M10-M1/M2 cross-link, probably because the beta :19 kDa:11.7-kDa trimer was formed in too low a yield (see "Discussion").

One approach involved tryptic digestion of the 70-80-kDa product and sequencing of cross-linked fragments. Cross-linked fragments were identified as bands present in non-reducing but absent in reducing conditions (Fig. 4). Several such bands were observed (marked **, *, a and b). The bands marked with an asterisk (*) were not sequenced for they were observed also in digests of the beta  subunit and represent fragments cross-linked by internal S-S bridges (not shown). The two fragments a, 9.3 kDa, and b, 7.7 kDa, were not observed in digests of the beta  subunit and were sequenced (Table II). Fragment a contained three peptides derived only from the alpha  subunit, with N termini Met973 (M10), Asn944, and Ile946 (M9), respectively. The combined average yield of fragments containing M9 is 12.6 pmol/cycle while that of the fragment containing M10 is 11.7 pmol/cycle. These yields are measured against a background of about 1 pmol/cycle, i.e. with a possible error of about 10%. Thus, the experiment demonstrates that M9 and M10 are present in equimolar proportions, suggesting that Cys964 within M9 and Cys983 within M10 are cross-linked to each other. Sequencing of fragment b led to essentially the same result suggesting that it is a truncated version of fragment a (probably cleaved at Arg998 or Arg1003).


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Fig. 4.   Digestion of the CuP-catalyzed cross-linked product with trypsin. The CuP cross-linked product (95 µg) was digested with trypsin for 12 h (see "Experimental Procedures"). Samples (~48 µg/lane) were resolved on a short 1-mm 16.5% T, 6% C gel in either reducing (Red) or non-reducing conditions (Non-red).

The evidence for the internal cross-link Cys964-Cys983 in Table II excludes either residue as a partner of Cys44 of the beta  subunit, unless one makes an unlikely assumption that Cys44 can form S-S bridges with different cysteines in the 19-kDa peptide. Thus, one could predict that Cys44 is cross-linked to either Cys911 or Cys930 in M8. However, direct evidence was not obtained because we detected no cross-linked band containing sequences from both alpha  and beta  subunits. A hypothesis to explain the paradox could be that the broad band (**) in Fig. 4 contains cross-linked fragments of both alpha  and beta  subunits, but the latter are also cross-linked by the internal S-S bridges to other glycosylated fragments of the beta  subunit. Sequencing of this band (**) showed indeed that it consists of a heterogenous mixture of peptides which precludes simple interpretation (not shown).

The hypothesis just proposed was tested as follows (Figs. 5, 6, and Table III). The 70-80-kDa cross-linked product, or the 19-kDa fragment, were first incubated with protein kinase A and [gamma -32P]ATP, in order to phosphorylate Ser936 in the PKA site RRNS (32, 33), and the labeled proteins were then digested with chymotrypsin.2 The digestion products were resolved on a 16.5% gel, blotted onto PVDF paper, which was first autoradiographed (Fig. 5, 32Pi) and then also immunostained with an antibody recognizing residues Asn889-Gln903 in the loop between M7 and M8 (Fig. 5, L7-8). For greater ease of understanding, the scheme in Fig. 5 marks the positions of the Asn889-Gln903 epitope and the phosphorylation site of PKA in relation to the trans-membrane segments, M7-M10. In nonreducing conditions only broad, poorly defined bands of 32Pi-labeled material (*) were observed (Non-red). However, in reducing conditions, a discreet 32Pi-labeled band of 6.5 kDa appeared (Red). The same 6.5-kDa band appeared also in a chymotryptic digest of 32Pi-labeled 19-kDa fragment resolved under reducing conditions (19 kDa). Thus the 6.5-kDa band is a fragment of the alpha  subunit containing Ser936 which is cross-linked to the broad band of material in nonreducing conditions and is released by reduction of an S-S bridge. By contrast to the result with PKA phosphorylation of Ser936, the anti-Asn889-Gln903 antibody recognized an 8.0-kDa chymotryptic fragment, the size of which was not affected by reduction (Fig. 5, L7-8). Thus, this fragment is not a cross-linked species and does not contain the PKA site at Ser936 (compare L7-8 with 32Pi).


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Fig. 5.   Phosphorylation and chymotryptic digestion of the CuP-catalyzed cross-linked product. The CuP cross-linked product (10 µg, 0.25 mg/ml) was labeled with 15 µCi of [gamma -32P]ATP using PKA (see "Experimental Procedures"). The protein was precipitated with methanol and then digested with chymotrypsin at 0.2 mg/ml for 1.5 h. The digest was divided and either reduced (Red) or not (Non-red). The 19-kDa fragment (2 µg, 0.1 mg/ml) was also labeled with [gamma -32P]ATP, digested with chymotrypsin, and then reduced. Samples were resolved on a short 16.5% T, 6% C gel, and blotted onto PVDF. The sheet was autoradiographed using a Fuji BAS 1000 PhosphorImager (32Pi) and then immunostained with an antibody recognizing residues Asn889-Gln903 (L7-L8).

                              
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Table III
Sequence of 32P label chymotryptic fragment derived from the major cross-linked product

In principle, the 6.5-kDa 32Pi-labeled cross-linked fragment might extend forward from Ser936 and include M9 (containing Cys964) or backward from Ser936 and include M8 (containing Cys911 and Cys930). In order to distinguish between these possibilities we have sequenced the fragment, repeating the experiment of Fig. 5 with 50-fold more protein (Fig. 6). The CuP cross-linked product (0.3 mg) was labeled with 32Pi using PKA, digested with chymotrypsin, and protein was separated on a 16.5/6% gel. Most of the digest was separated under reducing conditions (Red), but for comparison a portion was separated in nonreducing conditions (Non-red). Because a large amount of protein was loaded onto each lane, the labeled fragment could not be expected to run as a sharp band even in the reducing conditions, as in Fig. 5, and indeed the autoradiograph in Fig. 6 (Red) showed a relatively broad band of labeled material in the region of 6-7 kDa. The broad labeled band was transferred to PVDF, and sequenced (Table III). A mixture of two sequences was found, N terminus Glu902 corresponding to a fragment containing M8 and N terminus Arg972 corresponding to a fragment containing M10, respectively. Only the former fragment contains Ser936, and hence the radioactive label. Thus, Figs. 5, 6, and Table III demonstrate that Cys44 of the beta  subunit is cross-linked to either Cys911 or Cys930 in M8 (see "Discussion").


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Fig. 6.   Preparation of the 32Pi-labeled chymotryptic fragment of the cross-linked product used for N-terminal sequencing. The CuP cross-linked product (300 µg, 0.2 mg/ml) was labeled with 51 µCi of [gamma -32P]ATP by PKA, precipitated with methanol, and then digested at 1 mg/ml with chymotrypsin for 70 min. Samples (50 µg) were resolved on a long 16.5% T, 6% C gel under reducing (Red) or nonreducing conditions (Non-red). The gel was autoradiographed using a Fuji BAS 1000 PhosphorImager. The 6-7-kDa 32Pi-labeled band was transferred to PVDF and sequenced.


    DISCUSSION

Cross-linked Fragments of 19-kDa Membranes

CuP treatment of 19-kDa membranes solubilized with C12E10 in the presence of Rb ions and ouabain led to appearance of one major cross-linked band, which consists of a mixture of beta :19-kDa dimers (approx 65%) and beta :19 kDa:11.7-kDa trimers (approx 35%) (Figs. 1 and 3, Table I). These cross-links reflect proximities of fragments within the intact detergent-solubilized complex. The fact that cross-links were formed in detergent-solubilized but not in native 19-kDa membranes could imply either that the detergent induced a degree of rearrangement of trans-membrane segments which permit cross-links between the relevant cysteines or that cysteines embedded in lipid, such as Cys44 of the beta  subunit, are not reactive in native membranes due to insufficient exposure to oxygen and CuP, and only become exposed to oxygen or CuP after solubilization. However, the crucial point is that Rb occlusion is fully preserved in these C12E10-solubilized 19-kDa membranes (1). Therefore the organization of trans-membrane segments in the soluble complex of fragments, and proximities revealed by cross-linking (M8/Mbeta , M9/M10), must be essentially similar to those in native 19-kDa membranes and Na,K-ATPase.

CuP treatment of digitonin-solubilized proteolyzed dog kidney Na,K-ATPase produces cross-linked fragments corresponding to a trimer of beta :22 kDa:11 kDa (1:1:1) and, in lower amounts, a dimer of 22 kDa:11 kDa3 (23). Our study confirms the finding of a beta :19 kDa:11.7-kDa trimer, although in lower yield than a beta :19-kDa dimer, but a 19 kDa:11.7-kDa dimer was not observed (see Fig. 1). Presumably, the similarities and differences between the two sets of observations reflect the state of the soluble complex. Solubilization of pig kidney 19-kDa membranes with C12E10, in the absence of Rb ions and ouabain, leaves the beta -M7/M10 pair tightly associated but the M5/M6 and M3/M4 fragments dissociate, while the M1/M2 fragment shows an intermediate degree of interaction with the beta :19-kDa pair (1). Thus, solubilization of 19-kDa membranes by digitonin in the absence of Rb ions and ouabain (23) should not have preserved an intact complex. Tighter association of the 11-kDa fragment with the beta :22-kDa pair could explain why a beta :22 kDa:11-kDa trimer was observed even in the absence of Rb ions and ouabain (23).

CuP treatment of unsolubilized 19-kDa membranes revealed a heterogeneous mixture of cross-linked products containing the 19-kDa fragment, in the absence of Rb ions, and suppression of the cross-links in the presence of Rb ions (Fig. 2). These cross-links are probably the products of non-native interactions since the same fragments were observed after thermal inactivation which disorganizes the fragments (30, 31). K+, Na+, or ouabain protect against CuP-catalyzed cross-linking of fragments of unsolubilized 19-kDa membranes (24). These ligands also protect 19-kDa membranes against thermal inactivation of Rb occlusion (30, 31). Thus protection against CuP-catalyzed cross-linking presumably reflects cation- or ouabain-induced stabilizing interactions between fragments which prevent their disorganization.

Identity of Cross-linked Regions

The conclusion that Cys911 or Cys930 in M8, is cross-linked to Cys44 of the beta  subunit rests on a combination of direct evidence and exclusion of alternatives. The inference of the internal cross-link (Fig. 4, Table II, Fig. 1) suggest that M9 and M10 form a hairpin with Cys964 and Cys983 juxtaposed, and excludes both residues as partners for the beta  subunit. Direct evidence has been obtained by utilizing PKA to label Ser936 with 32Pi (32, 33). After chymotryptic digestion of the 32Pi-labeled cross-linked product, reducing conditions released a 6.5-kDa 32Pi-labeled fragment, with N terminus Glu902 (Figs. 5 and 6, Red; Table III). This 6.5-kDa fragment includes Cys911 and Cys930 in M8 and ends before Cys964 in M9. In nonreducing conditions the fragment is cross-linked to a mixture of partially digested glycosylated fragments of the beta  subunit, held together by internal S-S bridges (Figs. 4-6).

Proximity of M8 and Mbeta is compatible with prior evidence that alpha  and beta  subunit interact strongly at the extracellular surface primarily within the short sequence SYGQ outside M8 (34, 35). It is also compatible with our finding that o-phthalaldehyde cross-links alpha  and beta  subunits near M8 (2). Although we cannot say which cysteine in M8 is cross-linked to the beta  subunit, Cys911 is a more likely candidate. A helical wheel representation of Mbeta (Fig. 7) reveals a sector of about 100° with short or non-hydrophobic side chains (Gly43, Cys44, Gly47, Gly51, Gln54, shaded boxes) near the extracellular surface. The nonhydrophobic sector could participate in a protein-protein interface with the remaining hydrophobic surface facing the lipid. Thus Mbeta may contact M8, including Cys911, near the extracellular surface.


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Fig. 7.   Helical wheel representation of the membrane spanning helix of the beta  subunit.

The cross-link between M7/M10 and M1/M2 fragments has not been identified. Based on topological considerations, Sarvazyan et al. (23) proposed that Cys983-Cys104 (M1-M10) or Cys138-Cys930 (M2-M8) are likely pairs. At first sight a Cys983-Cys104 cross-link appears incompatible with the Cys983-Cys964 internal cross-link (Table II). However, the observation of a mixture of beta -M7/M10 dimers and beta -M7/M10-M1/M2 trimers (Figs. 1 and 3 and Table I) leads to the following hypothesis. Assume, that M1, M9, and M10 are in proximity, so that S-S bridges can form either between Cys983 in M10 and Cys964 in M9 or between Cys104 in M1 and either Cys983 or Cys964. Then 19-kDa fragments containing the Cys983-Cys964 internal cross-link could not be cross-linked to the M1/M2 fragment via Cys983-Cys104 or Cys964-Cys104. Thus, the mutually exclusive formation of S-S bridges provides an economical explanation of the mixture of cross-linked product. By contrast, the assumption of a Cys138-Cys930 cross-link does not readily explain this observation. Sarvazyan et al. (23, 24) observed the beta :22 kDa:11 kDa trimer with 1:1:1 stoichiometry and the 22 kDa:11-kDa dimer, but no beta :22-kDa dimer. This could imply that the internal Cys964-Cys983 cross-link was not formed in their conditions.

Spatial Organization of Membrane Spanning Helices of alpha  and beta  Subunits

Fig. 8 presents a tentative proposal for spatial organization of trans-membrane segments of Na,K-ATPase. The arrangement is based on direct and indirect evidence on helix proximity, indications that trans-membrane helices of class II P-type pumps are separated into domains, and the necessity for M1/M2, M3/M4, M5/M6, and M9/M10 to be paired due to their short extracellular loops. Obviously, Fig. 8 depicts only an approximate arrangement, due to lack of detailed information on helix proximity and tilt. The major objective is to illustrate the structural constraints discussed below.


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Fig. 8.   Spatial organization of trans-membrane helices of Na,K-ATPase. Regions cross-linked by o-phthalaldehyde (2) or cysteine residues cross-linked by CuP in this study are joined by gray lines. The gray rectangle in the ectodomain of the beta  subunit represents a region which interacts within the extracellular loop between M7 to M8 (SYGQ). The division between two membrane domains is depicted by the thick dashed line.

The thick dashed line in Fig. 8 demarcates separate domains comprising M1 to M6 and M7 to M10 with Mbeta , respectively, evidence for which is provided by the following observations. (a) Compared with class II eukaryotic P-type pumps with 10 trans-membrane helices, prokaryotic class I P-type heavy metal pumps contain the equivalent of only the first six helices and conserved cytoplasmic regions (with two extra helices at the N-terminal side). It has been proposed that sequences corresponding to M1 to M6 of class II pumps represent a core structural and functional unit for cation transport and energy transduction, while the extra four C-terminal segments of class II pumps evolved to serve additional functions (3, 36). (b) We have shown recently that Fe2+ (37) and Cu2+ (38) ions catalyze selective oxidative cleavages of Na,K-ATPase, providing information on spatial organization around the bound metal ions. Iron-dependent cleavages of the alpha  subunit occur at the cytoplasmic surface in conserved sequences located between M2 and M3 and between M4 and M5 (37). Copper-dependent cleavages occur at the extracellular surface between M7 and M8, to a small extent between M5 and M6, and between M9 and M10, and in the beta  subunit (38). A comparison of iron- and copper-catalyzed cleavages is highly suggestive of division of the alpha  subunit into two domains comprising M1-M6 and M7-M10/Mbeta , respectively (38). (c) High resistance to digestion by selective and unselective proteases of the C-terminal fragment (M7-M10) of Na,K-ATPase (8, 22) and H,K-ATPase (39) implies that it is folded into a compact domain.

Division into domains of M1-M6 and M7-M10 provides a strong constraint on the way helices can or cannot be packed. For example, M7 and M8 are unlikely to be widely separated in the molecule although they are connected by a relatively long extracellular loop (approx 40 residues) which, in principle, could permit such a separation. The positions allocated to M1-M10 of the alpha  subunit and Mbeta are based on the following considerations.

M4, M5, and M6-- Site-directed mutagenesis indicates that these helices form the main cation occlusion sites in Ca-ATPase and Na,K-ATPase, supplying ligating groups to occluded cations (6, 11, 40, 41). Recent cross-linking studies on Ca-ATPase have shown directly that M4 and M6 interact intimately (15). Thus M4, M5, and M6 are drawn in triangular contact (as in Refs. 14 and 15).

M7, M8, and Mbeta -- Proximity of M7 to M5 is strongly implied by our recent finding of a cross-link between cytoplasmic segments just preceding M7 and M5 (2). M6 and M7 are connected by a fairly short cytoplasmic loop, and charged residues within the loop may form the entrance to occlusion sites in M4, M5, and M6 (42). Therefore M7 is also placed near M6. As argued above, M7 and M8 are unlikely to be widely separated and proximity of M8 to M4, M5 and M6, is suggested by mutations of Glu908 in Ca-ATPase (9, 43). Thus M7 and M8 are placed together. Mbeta interacts with M8 as required by the CuP cross-linking (beta Cys44-Cys911 or perhaps beta Cys44-Cys930), and the evidence for interaction between beta  and alpha  subunits near the entrance to M8 (35, 2). As seen in Fig. 7 a likely surface of interaction of Mbeta occupies a sector of only 100° with the remaining hydrophobic sector facing the lipid. This is suggestive of the triangular contact in Fig. 8 with Mbeta placed between M7 and M8.

M9 and M10-- The M9/M10 pair are internally cross-linked through Cys964 and Cys983 (Table II). Helices M8 and M9 are connected by a fairly short cytoplasmic loop. Thus, M9 is placed near M8. M9 is also placed near cation sites, i.e. M4, M5, and M6, in order to explain cation-protected modification of Glu953 by dicyclohexylcarbodiimide (44), conformation-dependent changes in fluorescence of N-(p-(2-benzimidazolyl)phenyl)-maleimide, covalently attached to Cys964 (45), and chemical modification of Cys964 (46). A direct interaction of M10 with M5/M6 is suggested by chemical modification of Cys983 following dissociation of the M5/M6 hairpin from dog kidney 19-kDa membranes (46, 47) and there is evidence implying association of the M5/M6 and M9/M10 hairpins of H,K-ATPase (39).

M1, M2, and M3-- The major constraints on the locations of these helices are the indications for the two domains and the necessity for M3 to be close to M4 and M2 to M1. We have proposed above that M1 is cross-linked either to M10 (Cys104-Cys983) or M9 (Cys104-Cys964), the M1-M10 interaction (Cys104-Cys983) depicted in Fig. 8 is a more likely possibility due to other constraints on the interactions of M9 referred to in the previous paragraph.

Comparison of Fig. 8 with the helix packing model of Ca-ATPase (12) shows a general similarity in positions of M1-M7 as well as a major difference in that M8, M9, and M10 are placed on opposites sides of the molecules. Conceivably the position of M7 relative to M8-M10 is affected by the beta  subunit which interacts strongly with the L7/8. However, it is equally likely that the uncertainty is due to lack of detailed information. Two structural constraints used in Ref. 12, namely proximity of M4-M6 based on S-S bridges (15) and orientation of nonconserved residues of trans-membrane helices toward the lipid, fit either arrangement. A third line of evidence involved placing helices identified as the cytoplasmic stalk, S2-S5, above M2-M5 in the middle of the molecule (12). Without comparable structural data for Na,K-ATPase it is not known whether this constraint applies. Specific predictions in Fig. 8, are that M4 and M6 are largely surrounded by neighboring helices rather than lipid (see Ref. 47), M1 is close to M10, and there is no proximity between M1/M2 and M7/M8. Clearly, it now becomes necessary to devise a means to test specific predictions and assumptions in order to refine the models.

    ACKNOWLEDGEMENTS

We thank Prof. Jack Kyte (University of California at San Diego, La Jolla, CA) for providing anti-KETYY, Prof. Jesper V. Møller (University of Aarhus, Aarhus, Denmark) for providing anti-Asn889-Gln903, and Prof. Shmuel Shaltiel (Department of Biological Regulation, Weizmann Institute of Science, Rehovot, Israel) for a gift of the catalytic subunit of PKA.

    FOOTNOTES

* This work was supported by Grant 96-00022 from the US-Israel Binational Science Foundation.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 Present address: Dept. of Biochemistry, Biozentrum, University of Basel, Klingelbergstr. 70, Basel, Switzerland.

§ To whom correspondence should be addressed. Tel.: 972-8-9342278; Fax: 972-8-9344118; E-mail: bckarlis{at}weizmann.weizmann.ac.il.

The abbreviations used are: CuP, Cu2+-phenanthroline; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; PAGE, polyacrylamide gel electrophoresis; PVDF, polyvinylidene difluoride; C12E10, polyoxyethylene 10-laurylether; PKA, protein kinase A.

2 Chymotrypsin was used rather than trypsin in order to avoid cleavage before the PKA phosphorylation site.

3 The 22- and 11-kDa fragments of proteolyzed dog kidney enzyme are equivalent to the 19- and 11.7-kDa fragments of proteolyzed pig kidney enzyme.

    REFERENCES
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Abstract
Introduction
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