©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Do Transmembrane Segments in Proteolyzed Sarcoplasmic Reticulum Ca-ATPase Retain Their Functional Ca Binding Properties after Removal of Cytoplasmic Fragments by Proteinase K? (*)

(Received for publication, April 18, 1995; and in revised form, June 9, 1995)

Birte Juul (1) Hubert Turc (3) Marie Laure Durand (3) Adrienne Gomez de Gracia (3) Luc Denoroy (2) Jesper Vuust M (1) Philippe Champeil (3)(§) Marc le Maire (3)(§)

From the  (1)Department of Biophysics, University of Aarhus, Ole Worms Allé 185, 8000 Aarhus C, Denmark, the (2)Service Central d'Analyse, CNRS, BP 22, 69390 Vernaison, France, and (3)URA 1290, CNRS, Section de Biophysique des Protéines et des Membranes, Département de Biologie Cellulaire et Moléculaire, Commissariat à l'Energie Atomique, Centre d'Etudes de Saclay, 91191 Gif-sur-Yvette Cedex, France

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The present study was undertaken to investigate the Ca binding properties of sarcoplasmic reticulum Ca-ATPase after removal of the cytoplasmic regions by treatment with proteinase K. One of the proteolysis cleavage sites (at the end of M6) was found unexpectedly close to the predicted membrane-water interphase, but otherwise the cleavage pattern was consistent with the presence of 10 transmembrane ATPase segments. C-terminal membranous peptides containing the putative transmembrane segments M7 to M10 accumulated after prolonged proteolysis, as well as large water-soluble fragments containing most of the phosphorylation and ATP-binding domain. Ca binding was intact after cleavage of the polypeptide chain in the N-terminal region, but cuts at other locations disrupted the high affinity binding and sequential dissociation properties characteristic of native sarcoplasmic reticulum, leaving the translocation sites with only weak affinity for Ca. High affinity Ca binding could only be maintained when proteolysis and subsequent manipulations took place in the presence of a Ca concentration high enough to ensure permanent occupation of the binding sites with Ca. We conclude that in the absence of Ca, the complex of membrane-spanning segments in proteolyzed Ca-ATPase is labile, probably because of relatively free movement or rearrangement of individual segments. Our study, which is discussed in relation to results obtained on Na,K-ATPase and H,K-ATPase, emphasizes the importance of the cytosolic segments of the main polypeptide chain in exerting constraints on the intramembranous domain of a P-type ATPase.


INTRODUCTION

The way in which transmembrane segments of a membrane protein interact to form organized intramembranous domains with functional properties is still a matter of speculation. There is evidence that during the initial stages of protein biosynthesis, insertion into the membrane is governed by local factors in the polypeptide chain such as the presence of hydrophobic segments and neighboring charged amino acid residues, which can function as transfer and stop signals for insertion of the nascent polypeptide chain into the lipid bilayer (Friedlander and Blobel, 1985; Audiger et al., 1987; Lipp et al., 1989; Boyd and Beckwith, 1990; Dalbey, 1990; Sipos and von Heijne, 1993; Anderson and von Heijne, 1994). According to the two-stage hypothesis, as initially proposed by Popot and Engelman (1990), preformed transmembranes helices with independent stability would then pack into the final stable and compact tertiary fold with functional properties. The hypothesis that transmembrane helices behave as autonomous folding domains and acquire most of their native structure independently of the rest of the molecule has prompted a large number of studies. Many laboratories have attempted to reconstitute functional membrane proteins like receptors, bacteriorhodopsin, sugar transporters, or P-glycoprotein, from complementary fragments generally obtained by proteolysis or by co-expression in a host membrane (e.g. Kobilka et al.(1988), Kahn and Engelman(1992), Bibi and Kaback(1992), Popot(1993), Lemmon and Engelman(1994), Cope et al. (1994), and Loo and Clarke(1994)). Judging from published reports, these efforts have up to now been reasonably successful, at least when starting from a small number of large complementary fragments, generally including the extramembranous domains.

In relation to the above mentioned data, it is of interest to know whether autonomous folding applies to all kinds of integral membrane proteins or whether also interaction with cytoplasmic domains is decisive for stability. In this connection it should be noted that several of the proteins previously subjected to study are characterized by a predominance of membrane-embedded segments. In this respect, P-type ion pumps are of particular interest because of the presence of both large cytosolic domains and transmembrane domains that must accommodate the hydrophilic pathways for ion translocation. One example is provided by Na,K-ATPase, involved in the Na/K exchange across the plasma membrane in animal cells. Maturation of newly synthesized Na,K-ATPase, with acquisition of cation transporting properties, occurs after formation of a heterodimeric complex with the glycosylated beta-subunit and export to the plasma membrane (Geering 1991; Fambrough et al., 1994; Schmalzing and Gloor 1994). It has been demonstrated that with mature Na,K-ATPase as well as with the related H,K-ATPase, it is possible after proteolytic removal of the cytosolic domains to retain an intramembranous complex with an intact ability to occlude K (or Rb) and Na; such preparations are often referred to as ``19-kDa'' membranes because they are formed by noncovalent interaction between the remaining separated membrane polypeptide fragments, including a large C-terminal proteolytic fragment of approximately this size (Karlish et al., 1990; Esmann and Sottrup-Jensen, 1992; Rabon et al., 1993). However, reports with Na,K-ATPase 19-kDa membranes suggest that these membranous fragments have an increased susceptibility to thermal inactivation (Or et al., 1993: Shainskaya and Karlish, 1994).

In the closely related SERCA ATPases, Ca transport does not require the presence of a beta-subunit, and the ATPase chain is retained in the SR(^1)/ER membrane after biosynthesis, presumably without undergoing any drastic posttranslational modification other than formation of a few disulfide bonds. In a previous study on the transmembrane organization of the SERCA1a Ca-ATPase polypeptide chain, an ATPase fragment of approximately 30 kDa, relatively resistant to proteolysis, was reported to be formed by incubation with proteinase K (Matthews et al., 1990). Based on the immunoreactivity of this fragment, it was thought to correspond to the whole C-terminal membrane-associated domain of the ATPase. In addition, Matthews et al.(1990) indicated the presence of a smaller fragment (about 19 kDa) with the same immunoreactivity as the 30-kDa peptide, presumably analogous to the C-terminal fragment produced by proteolytic treatment of Na,K-ATPase.

In Ca-ATPase as in Na,K-ATPase, residues critical for cation binding or occlusion are believed to be restricted to the protein transmembrane domain (Clarke et al., 1989; le Maire et al., 1990; Sumbilla et al., 1991; Vilsen and Andersen, 1992; Jewell-Motz and Lingrel, 1993; Vilsen, 1993; Skerjanc et al., 1993; Toyoshima et al., 1993; Andersen, 1994; Andersen and Vilsen, 1994, 1995). Thus, the way seemed open to test if Ca-ATPase, with a simpler structure than Na,K-ATPase, would also be able to retain cation binding after removal of the cytoplasmic domains by proteolytic cleavage. To do so, we first analyzed in detail the cleavage pattern of SR Ca-ATPase with proteinase K. We then proceeded to test how the Ca-binding properties correlated with the extent of proteolytic cleavage at various stages of proteolysis. Our results clearly show that even relatively large transmembrane fragments of Ca-ATPase rapidly experience loss of the ability to control the permeation pathway and to retain high affinity Ca binding, unless a very high Ca concentration keeps the Ca binding cleft permanently occupied during proteolysis. These results provide support for the concept that for P-type ATPases, interaction with stabilizing extramembranous segments, as well as cation binding to the intramembranous sites, is crucial for maintenance of a native-like structure of the transmembrane portion of the protein.


EXPERIMENTAL PROCEDURES

Ca-ATPase Preparations

Tight SR vesicles were prepared from rabbit skeletal muscle as described by de Meis and Hasselbach(1971) or Champeil et al.(1985). When desired, purified, ATPase preparations, unable to accumulate Ca, were obtained by extraction with a low concentration of deoxycholate (Meissner et al., 1973).

Proteolysis

Treatment of SR vesicles or purified Ca-ATPase (1 mg protein/ml) with proteinase K (0.03 mg/ml) generally took place at 20 °C at pH 6.5, with various additions. Our standard calcium medium contained 100 mM bis-Tris buffer and 0.1 mM Ca. Some experiments were performed in the absence of Ca, in an EGTA/Mg medium, which contained 50 mM bis-Tris buffer (pH 6.5), 50 mM NaCl, 10 mM MgCl(2), and 1 mM EGTA (e.g. see Fig. 5A and lane11 in Fig. 1); in some cases (e.g. see Fig. 5, B and C), NaCl and MgCl(2) were omitted. Some experiments were also performed at higher pH, as in the original experiments of Matthews et al.(1990), or under our usual pH 6.5 conditions but with much higher concentrations of Ca (e.g. see Fig. 7). Proteolysis was stopped after appropriate periods by adding concentrated PMSF to a final concentration of 0.5 mM; the samples were maintained ice-cold before further processing to keep residual proteolysis undetectable. In some cases (see Fig. 2A), samples were then centrifuged once or twice at 220,000 g for 1 h at 4 °C (Beckman TL-100, rotor TLA-100, 75,000 rpm) to separate soluble peptides from those still attached to the membranes.


Figure 5: Effect of proteinase K treatment on Ca binding and Ca-dependent phosphorylation from [-P]ATP. A, after incubation of SR vesicles with proteinase K for various periods in EGTA/Mg medium (see ``Experimental Procedures''), we measured their ability to bind Ca (circles) under our standard conditions (buffer A, at pH 7.5, see ``Experimental Procedures''; the protocol was similar to that of the experiment shown in Fig. 4A), as well as the amount of phosphoenzyme (triangles) formed from 10 µM [-P]ATP in buffer A plus 0.2 mM Ca, at 4 °C (see ``Experimental Procedures''). PanelsB and C, SR vesicles were incubated with proteinase K for 15 min in either 100 µM Ca medium (lanes1 and 2) or EGTA medium (without Mg, lanes3 and 4) or left intact (lanes5 and 6) and were then incubated with [-P]ATP either in the presence of Ca (lanes with oddnumbers) or in the presence of excess EGTA (laneswith evennumbers) before being quenched with acid and centrifuged. Resuspended samples were run on a Laemmli gel containing 6.5% acrylamide to enhance resolution between the large fragments, and the gel was both autoradiographed with a PhosphorImager (C) and stained with Coomassie Blue (B).




Figure 1: Time dependence of proteolytic attack by proteinase K in the presence of micromolar Ca and the effect of Ca removal. Left, for SDS-PAGE on a Laemmli gel, lanes were all loaded with 20 µg of total protein: either native SR vesicles (lane9) or SR vesicles incubated with proteinase K for various periods (as indicated) in the presence of 100 µM Ca (see ``Experimental Procedures''). Time zero (lane2) corresponds to a sample for which proteinase K had been inhibited with PMSF before SR was added. Lane1 contains molecular mass markers (LMW Pharmacia kit), one of which (alpha-lactalbumin) is a 14.4-kDa protein with abnormal migration behavior; it is running faster than lysozyme, although both proteins have identical molecular mass (see Fig. 1of le Maire et al., 1993), and also faster than a 14.4-kDa CNBr fragment of myoglobin (data not shown). As a result, alpha-lactalbumin migrates with an apparent molecular mass close to 12 kDa. Right, a different but similar experiment, in which SR proteolysis took place during 15 min either in the Ca-containing medium (lane10) or in an EGTA- and Mg-containing medium (lane11).




Figure 7: Effect of a high Mg or Ca concentration during proteolysis on the Ca sensitivity of intrinsic fluorescence after proteolysis (A) and the proteolysis pattern (B). The 100 mM bis-Tris proteolysis medium (pH 6.5) contained either a, 0.3 mM Ca (left); b, 0.3 mM Ca and 10 mM Mg (center); or c, 10 mM Ca alone (right). A, after the indicated periods, as for the experiment illustrated in Fig. 6A, proteolyzed samples were diluted 20-fold into buffer A and intrinsic fluorescence was recorded. EGTA was first added (closedarrowheads; the EGTA concentration was a, 100 µM; b, 100 µM; or c, 2 mM, since the initial Ca concentration was 0.5 mM in this case), followed by Ca (doubleopenarrowheads; the total concentration of Ca was a, 1, mM; b, 1 mM; or c, 3 mM). B, proteolysis, performed under the same conditions as above, was arrested with PMSF after 5, 15, 30, or 80 min, and the samples were run on a Tricine gel. The lastlane shows molecular mass standards. Note that p19 migrates at approximately the same rate as myoglobin (17 kDa marker) in this gel system.




Figure 2: Characterization of Ca-ATPase fragments after proteolytic attack by proteinase K from their sedimentability (A) and immunoreactivity (B and C). For panelA experiments, SR vesicles were first incubated with proteinase K for 15 min in 100 µM Ca medium. Then, part of the sample was centrifuged at high speed, and the pelletable (Mb.) and soluble (Cyt.) fractions were separated by SDS-PAGE on a Laemmli gel together with an aliquot of the total sample (Tot.) and molecular mass standards (LMW). The gel was stained with Coomassie Blue. For panelB experiment, Ca-ATPase fragments (total sample) obtained after 5 min of proteinase K treatment were separated by SDS-PAGE, again on a Laemmli gel, and blotted onto a PVDF membrane. In one case (lane1), the blot was incubated with Ab 877-888 (an antibody raised against residues 877-888 in the C-terminal part of the ATPase); the asterisk indicates the band referred to as p20. In the other case (lane2), the blot was incubated with Ab 1-12, raised against the very first N-terminal residues of the ATPase. In both cases, bound antibody was revealed first (lanes1a and 2a), and the blots were subsequently stained with Coomassie Blue (lanes1b and 2b, respectively). PanelC experiment was similar to the one illustrated in B, except that 16% gels, prepared according to Schägger and von Jagow(1987), were used. After 10 (lanes1 and 4) or 15 min (lanes2 and 3) proteolysis in the presence of 100 µM Ca, Ca-ATPase fragments, were separated by electrophoresis, blotted onto PVDF, and first revealed by an antibody, either Ab 78(7) (lane2a), which reacts with the N-terminal region ATPase (see ``Experimental Procedures''), or Ab 577-588 (lane3a), which reacts with the central region, or Ab 796-806 (lane4a), which reacts with the C-terminal region. The Western blots were subsequently stained with Coomassie Blue (lanes2b, 3b, and 4b, respectively). Lane1 on the left shows Coomassie Blue staining of the fragments on the gel itself, corrected for gel shrinkage. Note that in Tricine gels, proteinase K migrates at the same speed as p30, so that it cannot be seen in this figure (but see lane1 in Fig. 7B). In addition, in these gels, p54 migrates at a speed intermediate between those of M55 and calsequestrin, the latter protein being the fastest; note also that calsequestrin transfer onto PVDF is not efficient, compared with transfer of other proteins (compare lane1 and lanes2b, 3b, or 4b). The approximate location of molecular mass markers is indicated on the right (LMW).




Figure 4: Loss of Ca binding and sequential dissociation properties of SR vesicles after proteolysis attack in the presence of 100 µM Ca, and parallel quantification of the concomitant appearance of the main Coomassie Blue-stained fragments. For panelsA and B, SR vesicles were incubated with proteinase K in 100 µM Ca medium for various periods before proteolysis arrest by PMSF. A, aliquots (100 µg of protein) were then diluted in a pH 7.5 buffer (buffer A, see ``Experimental Procedures''), adsorbed on a nitrocellulose membrane, and perfused with the same buffer supplemented with Ca (and [^3H]glucose) at pCa 5 so that equilibrium Ca binding to the adsorbed membranes was measured (circles). The results shown here are representative of several experiments (performed in triplicate) leading to the same results. Identical Ca-equilibrated samples were also prepared for subsequent perfusion with 2 ml of the same medium with no Ca, but either 1 mM nonradioactive Ca or 2 mM EGTA (this second perfusion lasted 2 s), and residual Ca was measured (squares and triangles, respectively). For intact SR vesicles, the complete time dependence of the result of this second perfusion is illustrated in panelC. Here, this perfusion was performed with a Biologic rapid filtration apparatus for various periods; the perfusion medium again contained either 1 mMCa (squares) or 2 mM EGTA (triangles). Interpretation for this experiment is shown in D, with Ca being represented by closedcircles and Ca by opencircles. B, proteolyzed samples, similar to those used for the Ca measurements, were run on an SDS gel (cf. Fig. 1) and the main Coomassie Blue-stained bands were scanned and quantified. The ordinateaxis represents integration of the optical density of the main bands: intact ATPase (triangles), p81/83 (circles), p29/30 (diamonds), p27/28 (squares), and p19 (asterisks).




Figure 6: Intrinsic fluorescence level and Ca-induced fluorescence changes of SR vesicles after proteolysis. A, SR proteolysis took place in 100 µM calcium medium. At various times before (t = 0) or after proteinase K addition, as indicated, 100-µl aliquots of proteolyzed SR were diluted in 1.9 ml of buffer A, supplemented with 10 µM Ca, and intrinsic fluorescence was recorded. The final concentration of Ca after SR addition was close to 20 µM, taking contaminating Ca into consideration. 100 µM EGTA was subsequently added (filledarrowhead), followed by 90 µM Ca (final pCa close to 5, singleopenarrowhead) and later on by 1 mM Ca (doublearrowhead). Each addition was performed with 2 µl of concentrated solution, corresponding to a very small dilution that was corrected for (0.1%). PanelsB and C, two batches of SR vesicles were prepared, corresponding to either 30 min proteolysis followed by PMSF quench (C) or to no proteolysis (in this case, proteinase K was quenched with PMSF before addition of SR, panelB). 100 µl of membrane suspension was diluted into 1.9 ml of buffer A, supplemented with 20 µM Ca (final pCa was therefore close to 4.5). Then, starting from this fluorescence level (corresponding to the closed symbols at pCa 4.5), we measured the relative changes in intrinsic fluorescence (expressed as percent of the initial fluorescence level) observed when EGTA or MgEDTA (de Foresta et al., 1994) was added to reduce the free Ca concentration or when Ca was added to increase it.



Gel Electrophoresis

Laemmli gels (Laemmli, 1970) were generally used, with the addition of 1 mM Ca to the gels (usually 11.4% acrylamide) to enhance the resolution of some of the bands (le Maire et al., 1990). Samples were prepared for electrophoresis by adding SDS and beta-mercaptoethanol to final concentrations of 1.8 and 9%. After boiling and addition of glycerol, the samples were deposited in the appropriate wells and run for 1-3 h, depending on gel size, at 30 mA. Gels were stained with Coomassie Blue and scanned with a Vernon PHI5 densitometer or digitalized with a Hewlett Packard Scanjet scanner, images (300 dpi) being handled using a U-lead Systems Photostyler for uniform background correction. In some experiments, Tricine buffers and 16% gels were also used, as described by Schägger and von Jagow (1987) with slight modifications, comparable with those described by Karlish et al.(1990).

CaBinding

The ability of undigested or proteolyzed SR vesicles to bind Ca was determined either by Millipore filtration or by gel equilibration chromatography. For the filtration experiments (see Fig. 4A and 5A), 100 µg of undigested or proteolyzed SR vesicles were diluted in 2 ml of buffer A (100 mM KCl, 1 mM MgCl(2), and 20 mM Tes-Tris (pH 7.5)), layered onto a Millipore HA filter, and equilibrated with Ca by manual perfusion twice of 1 ml of buffer A plus 25 µMCa (including contaminating Ca), 15 µM EGTA (pCa 5), and 250 µM [^3H]glucose (to correct for Ca retention in the wet volume of the filter, see Champeil and Guillain(1986) or Yamaguchi and Watanabe (1989)). Filters were double-counted with no rinsing. The ability of undigested or proteolyzed SR vesicles to retain the sequential dissociation properties of native Ca-ATPase was evaluated in Ca/Ca exchange experiments from the residual amount of Ca found on the filter after a 2-s perfusion with buffer A plus 1 mMCa of the filter-adsorbed Ca-equilibrated samples. A control 2-s perfusion of the same samples with buffer A plus 2 mM EGTA (a situation corresponding to simple Ca dissociation) was also performed to check that it resulted in loss of all bound Ca.

In some cases, Ca binding was also examined by gel equilibration chromatography (Inesi et al., 1980) after separation of soluble fragments and proteinase K from the membranes by ultracentrifugation. The pelleted membranes were resuspended in a pH 7.5 buffer containing 100 mM KCl and 10 mM Tes-Tris, to which 55 µMCa (740 kBq/liter) plus 40 µM EGTA were added. This sample was applied to a column (1 30 cm of Sephadex G-50, Pharmacia Biotech Inc.), which had been preequilibrated with the same buffer. Ca binding was calculated from the rise in Ca in the eluted fractions. The results obtained with both techniques for Ca binding measurement were quantitatively similar, both before and after proteinase K treatment.

Retention on Nitrocellulose Filters of the Membranous Portion of Proteolyzed SR Vesicles

In preliminary experiments, we tested whether SR membranes could still adsorb to Millipore nitrocellulose filters despite ongoing proteolysis, a prerequisite for the above-mentioned filtration experiments. Using a very sensitive phospholipid assay based on the lipid-enhanced fluorescence of diphenylhexatriene (see London and Feigenson(1978)), no lipid was found in the filtrate, showing that the membranous portion of the ATPase did adsorb to the filter, even after a 1-h proteolysis. This finding, together with the gel chromatographic Ca binding determinations mentioned above, justifies our use of filtration to measure Ca binding to the membranous domain of proteolyzed Ca-ATPase. Note, in contrast, that when we repeated similar experiments with SR vesicles labeled with fluorescein isothiocyanate at Lys-515, i.e. in the cytoplasmic domain, a significant portion of the total fluorescein isothiocyanate fluorescence was found in the filtrate, implying that a large fraction of the released water-soluble peptides originating from this region had not adsorbed to the nitrocellulose filters.

Phosphorylation from [-P]ATP

To undigested or proteolyzed Ca-ATPase (50 µg/ml in buffer A at 4 °C, plus either Ca (0.2 mM free Ca) or 2.5 mM EGTA), 10 µM [-P]ATP was added; after 10 s, the reaction was quenched with trichloracetic acid. Incorporated P was determined after removal of nonincorporated P either by Millipore filtration and extensive rinsing (e.g. see Fig. 5A) or by centrifugation of the denatured protein, resuspension of the pellet, and gel electrophoresis to separate the phosphorylated peptides (e.g.Fig. 5C). In this case, the gel was either a modification of the phosphate gel system described by Weber and Osborn(1969) but with a lower pH (6 instead of 7) to reduce dephosphorylation (see also Sarkadi et al.(1986)), or a 6.5% Laemmli gel for better separation of p83 and p81 (see Fig. 5, B and C). Phosphorylation of the bands was detected using a PhosphorImager detector (Molecular Dynamics).

Intrinsic Fluorescence Measurements

Tryptophan fluorescence in native or proteolyzed SR was measured with a Perkin-Elmer MPF44A spectrofluorometer, with excitation and emission wavelengths set at 290 and 330 nm, respectively, and bandwidths set at 10 nm (Orlowski and Champeil, 1991). 100 µl of SR protein suspension in the proteolysis medium was diluted into 2 ml of buffer A, with various additions of either Ca, EGTA, or MgEDTA to titrate the sensitivity to free Ca. MgEDTA (an equimolar mixture of Mg and EDTA), and not EGTA, was used to buffer the pH 7.5 buffer A in the pCa 5 to pCa 6.5 range reliably, as its apparent dissociation constant for Ca is about 12.5 µM under these conditions (de Foresta et al., 1994).

Preparation of Sequence-specific Antibodies

Oligopeptides, corresponding to selected sequences located in the N terminus, the large cytosolic domain, and the C-terminal domain of fast twitch rabbit muscle Ca-ATPase (Brandl et al., 1986; see Fig. 3A for the approximate location on the polypeptide chain), were synthesized by Fmoc (N-(9-fluorenyl)methoxycarbonyl) solid phase chemistry by Neosystem A.S., Strasbourg. The peptides (all of which contained a single N-terminal or C-terminal cysteine residue) as part of, or added to the Ca-ATPase sequence, were coupled to keyhole limpet hemocyanin by m-maleimide benzoyl-N-hydroxysuccinimide ester according to Green et al.(1982). Rabbits were boosted with injections of 0.2-0.3 mg of protein conjugate, given subcutaneously with 2-week intervals, together with Freund's incomplete adjuvant. The injections were then extended to 4-week intervals, followed by collection of blood 1 week after each injection. The development in the sera of a titer against Ca-ATPase was screened in enzyme-linked immunosorbent assay experiments, employing purified SDS-denatured Ca-ATPase adsorbed to the enzyme-linked immunosorbent assay wells. A maximal titer was reached after 6-12 immunizations. The peptides used are referred to on the basis of their location in the Ca-ATPase sequence, e.g. peptide 403-417 refers to the pentadecameric peptide Arg-403-Cys-417, and antibody 403-417 refers to the antibody prepared against this peptide. Some of our initial experiments were performed with antisera generously given by Dr. Malcolm East and which had been raised against the N terminus, C terminus, and the 877-888 sequence of the ATPase in a similar way as that described here. The polyclonal antibody 78(7) was a kind gift from A. M. Lompré (Enouf et al., 1988). Although this antibody was raised against entire purified Ca-ATPase, its main epitope is located in the N-terminal one-fifth of the ATPase since it reacts with tryptic fragments A (residues 1-505) and A2 (residues 1-198), but not with A1 fragment (residues 199-505) (data not shown).


Figure 3: Schematic representation of the location of various cleavage sites, relative to the predicted topology of ATPase (A), and linear map of the various fragments identified after proteolytic attack by proteinase K (B). A, schematic view of Ca-ATPase predicted folding (numbers for the residues predicted to lie at each membrane/water interface are taken from Clarke et al. (1990); the dashedhorizontalline schematizes the position of the top of the stalk) with the position of the main sites for proteinase K attack. The indicated numbers correspond to the residues immediately following the cleavage sites (120 refers to the Leu-119-Lys-120 peptide bond, 243 refers to the Thr-242-Glu-243 peptide bond). Sites for V8 cleavage (V8(1) at Glu-231-Ile-232 and V8(2) at Glu-715-Ile-716) and tryptic cleavage (T(1) at Arg-505-Ala-506 and T(2) at Arg-198-Ala-199) are also indicated, as well as the phosphorylation site (P) at Asp-351. Openboxes in the membranous domain correspond to critical residues, Glu-309, Glu-771, Asn-796, Thr-799, Asp-800, and Glu-908 (Clarke et al., 1989), the latter being controversial (Vilsen and Andersen, 1992). The approximate locations of the epitopes for our antibodies (1-12; 403-417; 577-588; 796-806; 877-888; 985-994) are indicated by filledboxes. Questionmarks indicate cleavage sites not precisely determined. B, linear map for the main Ca-ATPase fragments identified after proteinase K treatment.



Western Blotting

For immunocharacterization of the peptides separated by electrophoresis, these peptides (on nonstained gels) were first transferred (Bio-Rad) to PVDF membranes, either by semidry blotting (1 h at 200 mA for large gels in a Kem-En-Tec apparatus) or by wet blotting (mini gels in a Mini Protean II apparatus for 1 h at 400 mA or large gels in a Transblot apparatus for 1.5 h at 500 mA, see Garrigos et al.(1991)). In both cases, a 10 mM CAPS buffer at pH 11, containing 10% methanol, was used for the transfer. In a number of experiments, appropriate portions of the PVDF membranes were then blocked for 10-30 min at room temperature with 0.5% Tween 20 in phosphate-buffered saline (140 mM NaCl, 8 mM Na(2)HPO(4), 1 mM KH(2)PO(4) (pH 7.2)), and all of the following procedures were performed in this buffer. Alternatively, the membranes were blocked overnight at 4 °C in phosphate-buffered saline, 5% bovine serum albumin, rinsed in phosphate-buffered saline, 0.1% bovine serum albumin, and incubated in phosphate-buffered saline, 1% bovine serum albumin. The membranes were incubated with primary antibodies for 1-2 h at room temperature. Detection of bound antibody was done either with S-labeled protein A (Amersham Corp.) and autoradiography (Kodak X-Omat AR) or by reaction with a horseradish peroxidase-coupled secondary antibody (goat anti-rabbit or anti-guinea pig) and visualized either by staining with aminoethylcarbazole (Kem-En-Tec Ltd.) or by the ECL kit (Amersham Corp.). In some cases, the same portion of the PVDF membranes was subsequently stained with Coomassie Blue (LeGendre and Matsudaira, 1989) so that the autoradiography or ECL result could be directly compared with the Coomassie Blue-stained bands, permitting unambiguous identification of the peptide bands recognized by the primary antibody (e.g. see Fig. 2, B and C).

N-terminal Sequencing

After electrophoresis, transfer to PVDF membranes, and Coomassie Blue-staining, the bands corresponding to the various peptides were cut from the stained membranes and analyzed by Edman degradation. In some cases, two or three amino acids were revealed at each step, indicative of the presence of a mixture of peptides in the Coomassie Blue-stained band (minor sequences are indicated in the legend to Table 1). For all bands sequenced, the average amino acid recovery, estimated as described by Shin et al.(1994), was in the range of 1-40 pmol/100 µg of SR protein.



Electrospray Ionization Mass Spectrometry

Mass spectrometry was used to evaluate the exact length of peptides in two cases: p29/30 and p19. In the first case, the p29/30 cytosolic fragments were separated by reverse phase HPLC; after proteolysis and centrifugation of membranous fragments, as described above, the supernatant, containing a mixture of p30, p29, proteinase K, and smaller peptides, was chromatographed on a C-18 Vydac column (4.6 300 mm) and eluted in a water/acetonitrile gradient in the presence of 0.1% trifluoroacetic acid. The collected fractions were lyophilized (Speedvac) and dissolved in 20 µl of acetonitrile/water/formic acid, 50:49:1 (v/v/v). The samples were then injected in an electrospray mass spectrometer as described previously (le Maire et al., 1993). In the second case, the membranous p19 peptide was electroeluted from Laemmli gels and prepared for ESI-MS as described in le Maire et al.(1993) for bacteriorhodopsin, with the following modifications. 1) To reduce cysteine reaction with acrylamide monomers, gels were aged overnight before use and preelectrophoresed for 15 min, and 1 mM thioglycollic acid was added to the upper reservoir. 2) To facilitate peptide precipitation after desalting, 7 mM acetic acid was added before acetone precipitation.


RESULTS

Proteolysis of SR Vesicles with Proteinase K

The gel electrophoresis results presented in Fig. 1give an overview of the time course of formation and degradation of characteristic proteolytic fragments, arising from digestion of intact SR vesicles with proteinase K. For the experiment illustrated on the left, the proteolytic treatment took place at 20 °C in the presence of 100 µM Ca in a pH 6.5 bis-Tris buffer that, on the basis of our previous experience with V8 protease, was found to be favorable for protecting vesicle integrity (le Maire et al., 1990). It is seen from Fig. 1that immediately after the addition of proteinase K, Ca-ATPase is rapidly degraded, giving rise to a number of major bands corresponding to polypeptide fragments of both high (apparent molecular masses of about 81/83 and 54 kDa), low (19 kDa, 14 kDa), and intermediate molecular masses (29/30 kDa, 27/28 kDa). Slightly above the latter bands, proteinase K itself is visible. The bands appearing at 81/83 and 27/28 kDa presumably represent primary split products of intact Ca-ATPase, since they are characterized by a rapid initial increase in concentration followed by a decline after 10 min, when the major part of the intact Ca-ATPase has been degraded (see scan of this gel in Fig. 4B, below). By contrast, in the case of p29/30 a relatively slow rise is observed initially, and the production of p19 only becomes apparent after a 5-10 min delay; then, p19 and p29/30 steadily accumulate and reach a maximum during the first hour of incubation. Subsequently, there is a slight decrease of p19, and, for p30, a complete conversion to p29.

Bands of lower molecular mass (less than 14 kDa) also appear in significant amount as a result of proteolysis (data not shown). Bands representing components with apparent molecular masses about 43 and 37/38 kDa are also visible on Fig. 1after proteolysis. Finally, the bands in the 55-60 kDa region in Fig. 1, visible both in native SR and after proteolysis, correspond to calsequestrin and M55 glycoprotein, as documented previously (le Maire et al., 1990).

When similar proteolysis experiments were repeated in the presence of EGTA together with Mg and Na (to minimize the EGTA-induced instability of Ca-ATPase), the rate of proteolytic degradation was slowed down; otherwise, we observed essentially the same pattern of proteolytic degradation as in the presence of Ca, except that a new protein component (p95) became prominent (see lane11 in Fig. 1). Binding of Ca to sarcoplasmic reticulum Ca-ATPase was previously found to greatly affect its sensitivity to V8 protease also, especially at Glu-231-Ile-232 (le Maire et al., 1990), as well as the sensitivity to trypsin of the peptidic bond located after Arg-198 (Andersen et al., 1986). Thus, it appears that the rate of proteolysis attack at various sites is modified by Ca, but, as in the case of V8, the same peptides are probably formed, as shown by the similar mobility of the bands formed in the presence or absence of Ca (see lanes10 and 11 in Fig. 1).

We then focused on the region corresponding to molecular masses around 20-30 kDa, which were reported by Matthews et al.(1990) to contain the C-terminal membranous fragments. In the experiment illustrated in Fig. 2A, SR vesicles were first incubated with proteinase K for 15 min. An aliquot of the total sample was kept for electrophoretic analysis (Tot.), while another aliquot was submitted to high speed centrifugation to separate membrane-bound (Mb.) and cytosolic soluble (Cyt.) fragments. To our surprise, a vast majority of the p29/30 component accumulating after long proteolysis periods (Fig. 1) was found in the soluble fraction, excluding that it could represent the fragment corresponding to the C-terminal transmembrane segments of the ATPase. The supernatant was extraordinarily enriched in this p29/30 peptide, whereas, besides proteinase K, the only other peptide of significant length present was p14. All the other proteolytic peptides, including p27/28, p19, and part of p14, were recovered in the pelletable fraction (Fig. 2A, lane3). This was also the case for the intravesicular proteins calsequestrin and M55, suggesting that the vesicles remained sealed after treatment with proteinase K.

The bands were further characterized after transfer to PVDF membranes and immunodetection with various antibodies. PanelB shows two blots, incubated with an antibody directed against peptide 877-888 (lane1a) and with an antibody directed against peptide 1-12 (lane2a), respectively. The same blots were subsequently stained with Coomassie Blue (lanes1b and 2b, respectively). The C-terminal antibody (lane1a) reacted with p28, p27, and p19 as well as with a fainter band referred to as p20 (see asterisk), slightly larger than p19 and hardly visible in the Coomassie Blue-stained gel under these conditions. Similar results were obtained using an antibody raised against the very last residues of the ATPase, namely against peptide 985-994 (data not shown). The N-terminal antibody (lane2a) reacted strongly with the p28 region, as well as with a 14-kDa component. In addition, both antibodies reacted with undegraded ATPase and p81/83, but not with p29/30 (see below). Results similar to those observed with the antibody against peptide 1-12 were obtained using the polyclonal antibody 78(7), whose main epitope is located in the N-terminal one-fifth of the ATPase (see ``Experimental Procedures''). In this case, Ab 78(7) decorated the lower edge of the 81/83 band, indicating the presence of two components in this region; these two bands could be clearly differentiated when a Laemmli gel with a lower acrylamide content was used (e.g. see Fig. 5B, below). To be able to resolve the 27/28-kDa components, it turned out to be important to use, instead of the Laemmli system, the Tricine gel system devised by Schägger and von Jagow(1987). The latter gel system resulted in a higher relative mobility for the C-terminal fragments and thus allowed us to differentiate the N- and C-terminal components of p28 (Fig. 2C, lanes2a and 4a). Coomassie Blue staining intensities, in turn (Fig. 2C, lane1), led to the conclusion that the major part of p28 is represented by the N-terminal fragment. In the same series of experiments, the p29/30 fragments were clearly stained by an antibody against peptide 577-588 in the central cytosolic domain of Ca-ATPase (Fig. 2C, lane3a), as expected from the fact that these fragments were recovered in the supernatant (panelA). Thus, after proteolysis in our standard 100 µM Ca medium, the major peptides products present in the 27-30 kDa region originate from the N-terminal and central regions, whereas peptides originating from the C-terminal region are mainly present as smaller 19-kDa fragments, as was previously found for Na,K-ATPase and H,K-ATPase.

Identification of Proteolysis Sites and Resulting Fragments

Precise identification of the various bands described above was then attempted. In all cases, the N-terminal sequence(s) determined by Edman degradation of the Coomassie Blue-stained blots were unambiguously recognized in the published ATPase sequence, even when the cut band turned out to contain two or three peptides, e.g. for p27/28. For peptides starting at the (blocked) N terminus of the ATPase, identification was based on Western blots, using antibodies raised against the N-terminal 1-12 residues of ATPase. The C-terminal residue of each peptide was deduced from the existence of a proteolysis site located at an appropriate distance from the N-terminal residue, compatible with the peptide migration rate, and from the immunoreactivity of the peptide with a series of antibodies. As the antibody series included Ab 985-994, raised against the C-terminal residues of ATPase, we could conclude that none of the C-terminal peptides had been cut close to the ATPase C terminus. This was confirmed by mass spectrometry, which was used to estimate the final amino acid of peptide p19 after electroelution from the gels as described previously for other Ca-ATPase membrane fragments (le Maire et al., 1993). The size of the p19 peptide, starting at Asp-818, was found to be 20,116 ± 13, close to the expected 20,112. The sizes of the water-soluble p29/30 peptides, starting at Ser-350 and Thr-357, respectively, were also determined by ESI-MS after HPLC purification (to ±90 kDa) and were best compatible with Ser-610 being a common C-terminal amino acid for these peptides. The data obtained are assembled in Table 1, which also shows the proposed sequences for the various peptides that we have characterized.

Fig. 3illustrates the location of all of these proteolysis sites in the Ca-ATPase sequence. The formation of primary and secondary proteolytic degradation products can be accounted for by the presence of seven characteristic main proteolytic regions, five of which are unambiguously localized: 1) the Leu-119-Lys-120 peptide bond located after M2 in the S2 region, 2) the Thr-242-Glu-243 peptide bond at the beginning of the S3 region, 3) the Cys-349-Ser-350 and Leu-356-Thr-357 peptide bonds immediately before and after the phosphorylation site (Asp-351) (cleavage at Thr-345-Ser-346 and Asn-359-Gln-360 was also detected, in low amounts), 4) various peptide bonds in the 733-747 region, preceding S5/M5, and 5) the Met-817-Asp-818 peptide bond between M6 and M7, as well as the nearby Leu-807-Gly-808 bond. Furthermore, based on ESI-MS, p29/30 presumably ends at Ser-610-Ile-611 (see above). Since by N-terminal sequencing we did not detect any peptide starting at this position and ending at 733-747 (the molecular mass of such peptide would be close to 14 kDa), additional cleavage sites must be present in the intervening sequence (comprising the ``hinge'' region), resulting in the formation of small peptides not detected by gel electrophoresis and probably released into the supernatant. The observed sedimentability of the peptides (Table 1, ninth column) generally agreed with what is expected from the topology predicted for Ca-ATPase. Note, however, that p14b and p43 (and possibly also a small fraction of p29/30) were found both in the soluble fraction and in the pellet, suggesting noncovalent interaction of these peptides with the rest of the molecule.

Effect of ATPase Proteolysis under Standard Conditions on CaBinding Properties

On the basis of the preceding analysis, it is evident that our preparation after long-term treatment with proteinase K is degraded down to fragments similar to those found in the 19-kDa membranes that can be produced by proteolytic treament of Na,K-ATPase with retention of cation occlusion as well as Rb-Rb exchange properties (Karlish et al., 1990; Capasso et al., 1992). It was therefore of particular interest to examine what happened to the Ca-binding properties of Ca-ATPase during treatment with proteinase K. As shown in Fig. 4, the ability of SR vesicles to bind Ca at pH 7.5 dropped precipitously after addition of proteinase K (circles in panelA), approximately concomitant with the disappearance of intact ATPase, as recorded by gel scanning measurements (triangles in PanelB). Moreover, in parallel experiments, we found that SR vesicles, passively loaded with Ca by overnight incubation, rapidly lost their normal permeability barrier to Ca during proteinase K treatment (data not shown), suggesting that control of the Ca channel involved in the translocation pathway had become lost already after mild proteolytic treatment. We also examined the effect of proteinase K treatment of SR vesicles on the kinetics of Ca release from the binding sites. It is well known that intact Ca-ATPase binds two Ca ions/polypeptide chain and that in a Ca/Ca exchange experiment, Ca bound to the superficial subsite of the binding pocket prevents the deeply bound Ca ion from dissociating; this is the ``sequential dissociation'' mechanism (Dupont, 1984; Forbush, 1987; Inesi, 1987; Petithory and Jencks, 1988; Orlowski and Champeil, 1991), analogous to phosphorylation-induced ``deocclusion'' of K or Rb in Na,K-ATPase. We first repeated this basic Ca/Ca exchange experiment with intact Ca-ATPase (Fig. 4C, see panelD). Then, with proteinase K-treated ATPase, we found that the ability to retain one Ca ion in a slowly dissociating state was already very strongly reduced 5 or 10 min after the start of the proteinase K treatment (squares in Fig. 4A), even before accumulation of appreciable amounts of p19 had occurred (compare with asterisks in Fig. 4B). The ability of Ca-ATPase to bind two Ca ions/polypeptide chain with high affinity at equilibrium and to retain one of them on a 2-s time scale during Ca/Ca exchange was thus strongly reduced after the formation of some of the early split products, and there is no doubt that it was completely absent well before maximal accumulation of the p19 fragment had occurred (30-60 min). Note that on a molar basis, the p19 fragment accumulated to a significant extent in our experiments; assuming as a first approximation that Coomassie Blue staining of ATPase fragments was proportional to their mass, 8% dye in this 19-kDa peptide (as estimated after 30 min of proteolysis, asterisks in panelB) would imply that about 40%, on a molar basis, of the intact ATPase (110 kDa) was recovered as this peptide.

Effect of ATPase Proteolysis on Ca-dependent Phosphorylation from [-P]ATP

Careful examination of our data suggested that during the initial stage of proteolysis, Ca binding (circles in Fig. 4A) was in fact reduced to a slightly smaller extent than that corresponding to the amount of intact ATPase remaining in the preparation (triangles in Fig. 4B); thus, not all cuts were critical for Ca binding. But since this difference was small, it was not straightforward to establish from the time course of appearance and disappearance of the main ATPase fragments during proteinase K treatment (see the various symbols in Fig. 4B) which cuts were the critical ones and which fragments had lost or retained their Ca binding properties. To study in more detail the Ca binding properties of the intermediate fragments resulting from proteinase K treatment, before production of the 19-kDa membranes, proteolytic treatment was also performed in the EGTA/Mg medium, in order to slow down ATPase proteolysis. Under these conditions, we found that the reduction in the ability to bind Ca (opencircles in Fig. 5A) occurred at a rate that was slower, as expected, than when proteolysis was performed in the presence of Ca and the absence of Mg. Probably for this reason, a slight delay was observed before reduction of Ca binding could be detected, confirming that certain proteolysis products have intact binding properties. Another interesting observation in this experiment was that Ca-dependent phosphorylation from [-P]ATP (triangles), like Ca binding, was relatively resistant to proteolysis and was subsequently reduced roughly concomitant with the reduction in Ca binding. This provided an opportunity to recognize cleavage sites not affecting Ca binding by establishing, by SDS-PAGE followed by autoradiography, which ATPase fragments were still phosphorylated from [-P]ATP in a Ca-dependent fashion. To this end, we first treated SR vesicles with proteinase K in the presence of either Ca or EGTA, subsequently incubated the proteolyzed samples with [-P]ATP in the presence or absence of Ca, quenched the reaction with acid, and then separated the various peptides on an SDS gel that was finally submitted to P autoradiography. No radioactivity migrated with the smallest fragments; in particular, p30, the soluble fragment starting close to the phosphorylation site, and p54, a larger central fragment of the ATPase, were not phosphorylated (not shown). Fig. 5, B and C, illustrates a similar experiment in which particular attention was given to the largest fragments. The Coomassie Blue-stained gel is shown in panelB, and the result of autoradiography is shown in panelC. Three phosphorylated bands were observed, as shown in panelC; intact ATPase, p95 (produced after proteolysis in the presence of EGTA only, i.e. in lanes3 and 4 but not in lanes1 and 2, see panelB), and p83(C), which, as the previous fragment, resulted from a cut in the N-terminal putative beta-strand region. By contrast, p81(N), present in low but distinct amounts on the Coomassie Blue-stained gel after proteolysis in the presence of Ca (Fig. 5B, lanes1 and 2), did not appear to be phosphorylated from [-P]ATP (Fig. 5C). These results establish that the p83(C)bulletp28(N) complex does bind Ca, at least in the presence of ATP, but at the same time they suggest that the cut in the 734-747 region, leading to the formation of p81(N) and p28(C), may have been deleterious for Ca binding.

Effect of ATPase Proteolysis on Intrinsic Fluorescence Properties of CaATPase and Its Calcium Sensitivity

In connection with the data shown in Fig. 4, the question remains, whether the residual small binding of Ca observed after long term proteolysis (see circles in Fig. 4A, above) is due to a small extent of nonspecific binding to the membranes, as has been observed to take place at alkaline pH and low Mg (e.g. Forge et al.(1993)) or whether it reflects low affinity binding to the Ca translocation sites still remaining on the ATPase fragments. Since it is known that the intrinsic fluorescence level of Ca-ATPase in native SR is sensitive to high affinity Ca binding to the translocation sites but not to Ca binding to nonspecific sites (e.g. Dupont (1976) and Orlowski and Champeil(1991)), we investigated the Ca sensitivity of Ca-ATPase intrinsic fluorescence in proteolyzed SR. A favorable circumstance was that most of the tryptophan residues in Ca-ATPase, including those responsive to Ca, are located in the protein transmembrane domain (Brandl et al., 1986; de Foresta et al., 1990). After various proteolysis periods, SR vesicles were diluted in the pH 7.5 buffer, supplemented with Ca (final free Ca was 20 µM), and both the intrinsic fluorescence level and the changes induced by subsequent addition of EGTA were recorded. The total intrinsic fluorescence level was reduced by 25-30% after proteolysis, consistent with exposure of the membranous tryptophan residues to a less hydrophobic or less ordered environment after proteinase K treatment (see traces in Fig. 6A). In addition, the fluorescence drop induced by EGTA (filledarrowheads in Fig. 6A) was reduced to a low value after proteolysis, in agreement with the reduction of high affinity Ca binding illustrated in Fig. 4A. A key part of the experiment consisted of subsequently raising the Ca concentration by adding Ca back to the Ca-deprived proteolytic fragments, which was done in two steps; with native SR, as expected, we observed that addition of Ca up to pCa 5 (toptrace in Fig. 6A, singleopenarrowhead) was sufficient to recover almost maximal fluorescence level. In contrast, after proteolysis, this first addition only increased intrinsic fluorescence to a small extent, whereas increasing the Ca concentration up to pCa 3 (doubleopenarrowhead) raised the fluorescence level much further. This was especially the case after intermediate proteolysis periods. After half an hour of treatment, the Ca-dependent fluorescence signals were small, but their dependence on free Ca could be quantitated reliably. Fig. 6, B and C show that the affinity of the ATPase sites for Ca changed from a submicromolar to a millimolar value as a result of proteinase K treatment. Although it cannot be completely excluded that tryptophan residues in proteolyzed SR have acquired an unexpected sensitivity to nonspecific Ca binding to the membranes after proteolysis, the outcome of the experiments illustrated in Fig. 6is best explained by transformation of the original high affinity Ca translocation sites into Ca binding sites of intermediate or low affinity, early after the start of proteolysis.^2

Effect of Having a High CaConcentration during Proteolysis on Preservation of CaBinding Properties

In view of the possibility that ion binding per se could stabilize the Ca binding domain (see ``Discussion'') and because of the apparently very low affinity for Ca of proteolyzed SR (Fig. 6C), we finally also performed proteolysis experiments under other conditions, including in the presence very high concentrations of divalent cations, and again measured Ca-ATPase intrinsic fluorescence after various proteolysis periods (Fig. 7A, columnsa-c). When proteolysis was carried out in the presence of 10 mM Ca, we found that a large fraction of the original EGTA-induced decrease in intrinsic fluorescence could be retained, even after 80-min proteolysis (Fig. 7A, see filledarrowheads in tracesc), despite significant reduction of the total fluorescence level. Proteolysis in the presence of 10 mM Mg (tracesb) initially slightly retarded the reduction of the response to EGTA, as compared with a medium with a low concentration of Ca only (0.3 mM, tracesa), but at the end of the 80 min incubation period, in contrast to what was observed with 10 mM Ca, the response to EGTA had vanished. As to the effect of 10 mM Ca on proteolyzed ATPase, it should be noted that events subsequent to the addition of EGTA showed particular features. First, the drift in intrinsic fluorescence of proteolyzed ATPase was faster than before EGTA addition. This finding can probably be interpreted as resulting from conformation-sensitive photolysis and/or inactivation of ATPase, as for intact SR (e.g. Andersen et al. (1986)); it therefore confirms that Ca was bound to the proteolyzed membranes before EGTA addition. Second, and more importantly, after readdition of Ca, up to a final concentration even higher than the initial value of 0.5 mM (doubleopenarrowhead; Ca was now 1.5 mM), the fluorescence only rose slowly and did not reach the level recorded before the addition of EGTA. This implies that after being left vacant for a short period (10 s was sufficient) the Ca binding sites of proteolyzed ATPase had experienced an evolution from which many of them did not recover, even by readdition of Ca in large excess.

The ATPase fragments formed after proteolysis under the conditions of the Fig. 7A experiment were separated by gel electrophoresis. Both Laemmli gels (not shown) and Tricine gels (Fig. 7B) indicated a general stabilization of the long ATPase fragments (p81/83, p54, and the intact ATPase itself) in the presence of 10 mM of either Ca or Mg. But Tricine gels showed that 10 mM Ca, and not Mg, exerted a specific stabilization of p28N and p27C fragments (see arrows) over long proteolysis periods (compare lane13 and lanes9 or 5 in Fig. 7B, see asterisks), with a concomitant reduction in the amount of p19 formed.


DISCUSSION

Sites for Cleavage by Proteinase K

Fig. 3summarizes the proposed origin of the main peptidic fragments that we have identified after treatment of sarcoplasmic reticulum ATPase with proteinase K. In the presence of 100 µM Ca, primary split products appeared after cuts at Asp-243 (resulting in the formation of p28N and p83C) and in the Val-734-Val-747 region (resulting in the formation of p27/28C and p81N). In the presence of EGTA/Mg, proteolysis gave rise to a prominent cut at Lys-120, leading to accumulation of p95 and its complementary peptide, p14a; otherwise the degradation pattern resembled that observed in the presence of Ca, apart from a general reduction in the proteolysis rate caused by the presence of 10 mM Mg and Na in the EGTA/Mg medium. In the region of molecular masses slightly smaller than 30 kDa, we found, contrary to our initial expectation, only small amounts of the C-terminal fragments containing the last 6 putative transmembrane helices as mentioned in Matthews et al.(1990). Instead, the major bands accumulating in the 29-30 kDa region corresponded to soluble fragments (Fig. 2A), starting close to the catalytic residue Asp-351 and probably ending around Ser-610. The paucity of p27/28C presumably was caused by splits at Asp-818 and slightly before, at Gly-808, resulting in the formation of a C-terminal 19-kDa membranous fragment plus a slightly larger 20-kDa fragment. The fact that several of the main bands, including p81/83, p27/28, and p14, were heterogenous, while migrating at similar speeds on Laemmli gels, reflects the ability of proteinase K to cut the ATPase at almost symmetrically located or nearly equidistant sites. Fortunately, we found that p28(N) and p28(C) migrated at distinct (although very close) positions when a Tricine gel system was used (see Fig. 2C, lanes2a and 4a, and Fig. 7B). It is also worth pointing out that the immunological data indicate that all C-terminal fragments described above have an intact C terminus, as revealed by strong reactivity with the 985-994 epitope (Table 1), a finding that for the p19 peptide was confirmed by mass spectrometry. The fact that p95 was visible only when proteolysis was performed in the absence of Ca can probably be explained in part by an increased susceptibility of the Thr-242-Glu-243 peptide bond in the presence of Ca, which prevented p95 from accumulating to any significant extent under the latter conditions. Since V8 cleavage of the nearby Glu-231-Ile-232 peptide bond was previously also shown to take place only in the presence of Ca (le Maire et al., 1990), this region of the ATPase stalk and just above it is obviously sensitive to Ca binding, becoming more exposed to the outer medium after Ca binding.

Most peptides of low molecular mass produced as a result of long term proteinase K treatment (running at the front of Laemmli gels, as shown in Fig. 1, or separated by SDS-PAGE in Tricine gels, data not shown) reflect further degradation of the membranous peptides mentioned above, particularly in the N-terminal region, which nevertheless, as in the case of SR treatment with V8 protease (le Maire et al., 1993), left the membrane-spanning segments M1-2, M3-4, and M5-6, together with the attached stalk segments, intact. (^3)By contrast, p19 was only slowly degraded, and, on a molar basis, accumulated to a significant extent after 0.5-2 h of proteinase K treatment. Thus, our proteinase K-treated SR membranes, in the latter stages of proteolysis, are analogous to the 19-kDa membranes, which can be obtained after proteolytic treatment of Na,K-ATPase and H,K-ATPase (Capasso et al., 1992; Rabon et al., 1993). It is of note that accumulation of ATPase fragments took place without proteolytic degradation of protein components, known to be localized inside the SR vesicles (calsequestrin and M55 glycoprotein; see Fig. 1and Fig. 7B). These proteins were fully recovered in the membranous pellet obtained after ultracentrifugation (see Fig. 2A), whereas they were completely degraded as soon as the vesicular membrane was disrupted, either by detergent solubilization or by alkaline EDTA treatment (data not shown). Electron microscopy ultrastructural studies (not shown) also indicated that the SR vesicular structure remained intact after prolonged treatment with proteinase K. As a result, it can be concluded that the cut between Met-817 and Asp-818 in SR vesicles, leading to the p19 fragment, occurred on the exterior of the vesicles, which corresponds to the cytosolic side. A similar result was obtained with Na,K-ATPase (Karlish et al., 1993), and both results, for P-type ATPases, are in agreement with a cytosolic location of the M6-M7 loop (see also Mata et al., 1992; Met al., 1993; Shin et al., 1994), for which the exact location has been controversial in Na,K-ATPase (Ovchinnikov, 1987; Mohraz et al., 1994). Note, however, that the cleavage observed between Leu-807 and Gly-808, leading to formation of p20, is located unexpectedly close to the C-terminal border of the predicted M6 segment, in fact just before it (e.g. Clarke et al., 1990; Toyoshima et al., 1993).

CaBinding to Proteolyzed Ca-ATPase

As mentioned above, the membranous fraction that we obtained as a result of prolonged treatment of SR vesicles with proteinase K can be compared with the 19-kDa membranes that under appropriate conditions are obtained by tryptic treatment of Na,K-ATPase or H,K-ATPase and that have an intact ability to occlude K or Rb and an almost unaltered ability to occlude Na in the presence of oligomycin (Karlish et al., 1990; Capasso et al., 1992; Esmann and Sottrup-Jensen, 1992; Rabon et al., 1993; Or et al., 1993; Shainskaya and Karlish, 1994). The N terminus of the Na,K-ATPase 19-kDa C-terminal fragment, Asn-830, is homologous to Ser-823 on Ca-ATPase, so that our p19 fragment, which starts at Asp-818, is even slightly longer than its Na,K-ATPase congener. Previous freeze-fracture studies have suggested that intramembranous particles in P-type ATPases are relatively well preserved even after prolonged proteolytic treatment (Yamanaka and Deamer, 1976; Ning et al., 1993). Because of the intact occlusion properties of the Na,K-ATPase 19-kDa membranes, and in line with the suggestion that transmembrane segments may behave as autonomous folding domains (Popot, 1993), we thus initially wondered whether the membranous fraction of proteinase K-treated SR vesicles would also retain most of the Ca binding properties characteristic of intact Ca-ATPase. However, this was not the case after proteolysis under our standard conditions in the presence of 100 µM Ca; after a sufficiently long incubation time with proteinase K, when the p19 fragment had accumulated to a significant extent on a molar basis (Fig. 1), we observed that the SR membranes had completely lost their characteristic ``sequential Ca dissociation'' properties (at least on the 2-s time scale), together with most of their equilibrium Ca binding ability (squares and circles, respectively, in Fig. 4A). Even limited proteolysis of the C-terminal part of Ca-ATPase ruined the ability of Ca to prevent Ca dissociation from the deeply buried site of Ca-ATPase. Simultaneously, proteolysis reduced to almost zero the drop in intrinsic fluorescence observed upon the addition of EGTA to SR fragments, diluted in a medium containing 20 µM Ca (Fig. 6A). We considered the possibility that this apparent discrepancy with the ability of proteolyzed Na,K-ATPase or H,K-ATPase to occlude Rb could be due to the fact that interaction of Ca with Ca-ATPase is not homologous to interaction of K or Rb with these other ATPases. However, in our case, performing proteolysis of Ca-ATPase in the absence of Ca, and also in some cases at more acidic pHs (as H in Ca-ATPase is the likely homologue of K in Na,K- or H,K-ATPase), did not favor accumulation of the p19 fragment (data not shown). In addition, it has also been reported that Na is as efficient as K or Rb in protecting Na,K-ATPase 19-kDa fragments from further proteolysis (Capasso et al., 1992).

Inactivation of CaBinding

To further elucidate the relationship between Ca-ATPase proteolysis and Ca binding ability, we studied the Ca-dependence of the intrinsic fluorescence of Ca-ATPase fragments. The residual small binding of Ca that we observed after prolonged SR proteolysis (see Fig. 4A above) could have been due either to low affinity binding to the ATPase fragments or to nonspecific binding to the membranes. Based on the known fact that Ca-ATPase intrinsic fluorescence is not influenced by nonspecific Ca binding to SR membranes, the fluorescence experiments permitted discrimination between these two possibilities and clearly suggested that the Ca translocation sites on Ca-ATPase have not disappeared completely after proteinase K treatment but have now acquired a very low affinity for Ca (Fig. 6, B and C). This large reduction in affinity, in turn, is consistent with a loss of the gating properties normally responsible for sequential Ca dissociation from native Ca-ATPase.

In addition, the intrinsic fluorescence experiments shown in Fig. 7suggest that the inability of Ca-ATPase, proteolyzed under standard conditions, to retain Ca binding properties reflects a lability of the bundle of transmembrane fragments. This we conclude from the fact that when proteolysis was performed in the presence of a high (10 mM) Ca concentration, Ca binding to ATPase fragments after 20-fold dilution of the proteolysis medium was first retained, as shown by EGTA-induced fluorescence changes, but chelation of Ca irreversibly reduced the ability of these fragments to rebind Ca in a subsequent step (compare single and doublearrowheads in columnc of Fig. 7A). This occurred within a few seconds, implying that proteolysis after dilution had not proceeded much further and thus was not responsible for the observed loss in Ca sensitivity. Since the presence of 0.3 mM Ca during 80-min proteolysis, either in the absence or the presence of Mg (columnsa and b of Fig. 7A), was not sufficient to retain the EGTA-induced fluorescence change, the requirement for a high Ca concentration presumably derives from a need for keeping the Ca binding cleft permanently occupied by Ca to avoid irreversible inactivation after proteolysis. Thus, it appears that, in this respect, proteolyzed SR membranes behave like Na,K-ATPase 19-kDa membranes, for which it has been clearly demonstrated that a major effect of proteolysis is to increase the sensitivity to thermal inactivation, an inactivation that is antagonized by cation occlusion within the membrane-spanning segments, Na being as efficient as K or Rb in this respect (Or et al., 1993; Shainskaya and Karlish, 1994). In fact, the above-quoted work on trypsinized Na,K-ATPase was a strong impetus for us to perform the experiments illustrated in Fig. 6and Fig. 7. The rather similar relative effects of Ca and Mg in protecting Ca-ATPase during proteolysis at early times, between 0 and 12 min (Fig. 7), suggest that, at early stages of proteolysis, Ca and Mg at high concentrations are probably both recognized by a common site, e.g. the Mg binding site in the catalytic center on the cytoplasmic portion of the ATPase, whereas the significantly different effects of these ions at later stages of proteolysis, between 30 and 80 min, suggest that only Ca stabilizes the ATPase polypeptide chain by binding to the Ca binding intramembranous cleft (see a related discussion with Na,K-ATPase in Or et al.(1993)). Note that Fig. 7B shows that in the presence of 10 mM Ca, 19-kDa peptides were virtually absent throughout the 80-min proteolysis period, while the membranous p28N and p27C peptides were stabilized, suggesting that stabilization of the Ca-ATPase polypeptide chain by cations bound to the translocation sites seems to favor the formation of Ca-ATPase fragments slightly longer than those resulting from ligand stabilization of the Na,K-ATPase 19-kDa membranes.

General Conclusions on Membrane Stability of P-type ATPases

From the point of view of the putative spontaneous assembly of individual transmembrane segments in a membrane protein (Popot, 1993; Lemmon and Engelman, 1994), our data are somewhat unexpected since a recent review emphasizes the thermal stability of the membrane-embedded segments of membrane proteins, as compared with that of the cytoplasmic regions (Haltia and Freire, 1995). In this connection, it should be noted that loss of high affinity Ca binding does not necessarily denote major structural changes; slight but irreversible changes are probably sufficient to perturb the Ca binding sites and the gating mechanism. Nevertheless, our results suggest that when the cytoplasmic portion of a membrane protein like a P-type ATPase is ``shaved off,'' transmembrane segments may not retain the same relative positions as in native ATPase, where the extramembranous portions of the protein presumably exert large constraints and prevent significant reorganization. The fact that the stability of the Ca-ATPase transmembrane domain depends on its interaction with the extramembranous regions corroborates recent thermal unfolding experiments with intact ATPase (Merino et al., 1994), which do not support the hypothesis that two distinct domains of the ATPase unfold separately.

In proteolyzed membranes, thermal agitation probably allows larger movements of the transmembrane segments with respect to each other than in the intact protein. Under conditions where these transmembrane segments are not held together properly oriented by cations bound to the translocation sites, they may well change their relative orientations and fall into energy ``traps'' from which a return to the original cation binding topography is either irreversibly lost or only occurs slowly ( Fig. 6and Fig. 7). Proteolyzed Ca-ATPase fragments seem to be especially sensitive to such thermal inactivation, while 19-kDa Na,K-ATPase membranes with occluded K or Rb evidently are more stable. It is not easy to evaluate to what extent this is a qualitative or a quantitative difference. Note in this connection that Ca dissociates from intact Ca-ATPase much more rapidly (less than 100 ms half-time, see triangles in Fig. 4C) than K or Rb does from Na,K-ATPase. As the beta chain in Na,K- or H,K-ATPase is known to modulate K-dependent events, its presence may be important for stabilization of the occluded form of these ATPases in both native and proteolyzed membranes (see Jaisser et al., 1992; Schmalzing et al., 1992; Eakle et al., 1992; Capasso et al., 1992; Lutsenko and Kaplan, 1993; Shainskaya and Karlish, 1994).

Irrespective of the detailed explanation for the difference between Na,K-ATPase and Ca-ATPase, it is clear from our results that cuts outside the transmembrane region are deleterious for the subsequent stability of the cation binding sites, implying that these sites are very dependent on the conformation or rigidity of segments of the protein outside the membrane. In fact, this is a necessary corollary of the functional ion transport properties of intact ATPases, in which at one point during turnover (corresponding to the release of occluded ions to the other side of the membrane), the affinity of the binding sites for cation is reduced and their topological orientation is altered after phosphorylation of an aspartyl residue located far away in the catalytic domain.


FOOTNOTES

*
This work was supported by grants from European Economic Community (Science contract ERB-SC1-CT92-0783), from the Association Française Contre les Myopathies, and from the Danish Medical Research Council and Biotechnology Program. 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 may be addressed. Tel.: 33-1-6908-3731; Fax: 33-1-6908-8139.

(^1)
The abbreviations used are: SR, sarcoplasmic reticulum; ER, endoplasmic reticulum; bis-Tris, bis(2-hydroxyethyl)iminotris(hydroxymethyl)methane; PMSF, phenylmethylsulfonyl fluoride; PVDF, polyvinylidine difluoride; Tes, 2-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino}ethanesulfonic acid; CAPS, 3-(cyclohexylamino)propanesulfonic acid; HPLC, high performance liquid chromatography; Tricine, N-[2-hydroxy-1,1,bis(hydroxymethyl)ethyl]glycine; ESI-MS, electrospray ionization mass spectrometry; PAGE, polyacrylamide gel electrophoresis.

(^2)
Note that in contrast to intrinsic fluorescence measurements, Ca binding experiments at high Ca concentration after proteolysis would have been both 1) unrealistic because of too high background noise level and 2) useless for discrimination between nonspecific binding to the membrane and low affinity binding to residues initially involved in the Ca binding sites.

(^3)
B. Juul, L. Denoroy, J. V. M, and M. le Maire, manuscript in preparation.


ACKNOWLEDGEMENTS

We thank Birte Nielsen for precious help; M. East and A. M. Lompré for initial gifts of antibodies; F. Penin and S. Deschamps for purifying p29/30 before ESI-MS; J. P. Le Caer (Institut Alfred Fessart, CNRS) for help with ESI-MS; and M. East, A. G. Lee, B. de Foresta, and S. Orlowski for critical reading of a preliminary version of our manuscript. We also thank A. Shainskaya and S. Karlish for illuminating discussions about their work with Na,K-ATPase.


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