Manipulation of activity and orientation of membrane-reconstituted di-tripeptide transport protein DtpT of Lactococcus lactis

The di-tripeptide transport system (DtpT) of Lactococcus lactis was purified to apparent homogeneity by pre-extraction of crude membrane vesicles with octaethylene glycol monodecyl ether (C10E8), followed by solubilization with n-dodecyl-beta-D-maltoside (DDM) and chromatography on a Ni-NTA resin. The DtpT protein was reconstituted into detergent-destabilized preformed liposomes prepared from E. coli phospholipid/phosphatidylcholine. A variety of detergents were tested for their ability to mediate the membrane reconstitution of DtpT and their effectiveness to yield proteoliposomes with a high transport activity. The highest activities were obtained with TX100, C12E8 and DM, whereas DDM yielded relatively poor activities, in particular when this detergent was used at concentrations beyond the onset of solubilization of the preformed liposomes. Parallel with the low activity, significant losses of lipid were observed when the reconstitution was performed at high DDM concentrations. This explained at least part of the reduced transport activity as the DtpT protein was highly dependent on the final lipid-to-protein ratios in the proteoliposomes. Consistent with the difference in mechanism of DDM- and TX100-mediated membrane protein reconstitution, the orientation of the DtpT protein in the membrane was random with DDM and inside-in when TX100 was used. The methodology to determine the orientation of membrane-reconstituted proteins from the accessibility of cysteines for thiol-specific reagents is critically evaluated.     

Reconstitution of the sarcoplasmic reticulum Ca(2+)-ATPase: mechanisms of membrane protein insertion into liposomes during reconstitution procedures involving the use of detergents.

The Ca(2+)-ATPase from skeletal muscle sarcoplasmic reticulum was reconstituted into sealed phospholipid vesicles using the method recently developed for bacteriorhodopsin (Rigaud, J.L., Paternostre, M.T. and Bluzat, A. (1988) Biochemistry 27, 2677-2688). Liposomes prepared by reverse-phase evaporation were treated with various amounts of Triton X-100, octyl glucoside, sodium cholate or dodecyl octa(oxyethylene) glycol ether (C12E8) and protein incorporation was studied at each step of the liposome solubilization process by each of these detergents. After detergent removal by SM-2 Bio-Beads the resulting vesicles were analyzed with respect to protein incorporation by freeze-fracture electron microscopy, sucrose density gradients and Ca2+ pumping measurements. The nature of the detergent used for reconstitution proved to be important for determining the mechanism of protein insertion. With octyl glucoside, direct incorporation of Ca(2+)-ATPase into preformed liposomes destabilized by saturating levels of this detergent was observed and gave proteoliposomes homogeneous in regard to protein distribution. With the other detergents, optimal Ca(2+)-ATPase pumping activities were obtained when starting from Ca(2+)-ATPase/detergent/phospholipid micellar solutions. However, the homogeneity of the resulting recombinants was shown to be dependent upon the detergent used and in the presence of Triton X-100 or C12E8 different populations were clearly evidenced. It was further demonstrated that the rate of detergent removal drastically influenced the composition of resulting proteoliposomes: upon slow detergent removal from samples solubilized with Triton X-100 or C12E8, Ca(2+)-ATPase was found seggregated and/or aggregated in very few liposomes while upon rapid detergent removal compositionally homogeneous proteoliposomes were obtained with high Ca2+ pumping activities. The reconstitution process was further analyzed by centrifugation experiments and the results demonstrated that the different mechanisms of reconstitution were driven predominantly by the tendency for self-aggregation of the Ca(2+)-ATPase. A model for Ca(2+)-ATPase reconstitution was proposed which accounted for all our results. In summary, the advantage of the systematic studies reported in this paper was to allow a rapid and easy determination of the experimental conditions for optimal detergent-mediated reconstitution of Ca(2+)-ATPase. Proteoliposomes prepared by the present simple method exhibited the highest Ca2+ pumping activities reported to date in Ca(2+)-ATPase reconstitution experiments performed in the absence of Ca2+ precipitating agents.     

Effective reconstitution of sarcoplasmic reticulum Ca2+-ATPase using lubrol PX]

Ca2(+)-ATPase of sarcoplasmic reticulum was reconstituted in the proteoliposomes by the salting out procedure. Triton X-100, C12E8 and Lubrol PX were used for the solubilization of the Ca2(+)-ATPase. Using fluorescent probes (diS-C3-(5), chlortetracycline) as well pH-measuring method, the functional of the reconstituted Ca2(+)-ATPase was comparatively studied in three types of proteoliposomes. The efficiency of Ca2(+)-ATPase grew in the following detergent order: Triton X-100, C12E8, Lubrol PX.     

Membrane crystals of Ca2+-ATPase in sarcoplasmic reticulum of developing muscle

The vanadate-induced crystallization of Ca2+-ATPase was analyzed on sarcoplasmic reticulum vesicles isolated between 10 and 28 days of development from pectoralis muscles of chicken. After exposure to Na3VO4 in a Ca2+-free medium, Ca2+-ATPase crystals begin to appear on portions of the surface of a few vesicles, isolated at 18 days of development. Thereafter, the number of vesicles containing Ca2+-ATPase crystals rapidly increases and after 1 week of postnatal development (28 days), it reaches the adult level of about 30% of the vesicle population. These observations are discussed with reference to the mechanism of Ca2+-ATPase crystallization and the regulation of sarcoplasmic reticulum biosynthesis.     

A new method for the reconstitution of membrane proteins into giant unilamellar vesicles

In this work, we have investigated a new and general method for the reconstitution of membrane proteins into giant unilamellar vesicles (GUVs). We have analyzed systematically the reconstitution of two radically different membrane proteins, the sarcoplasmic reticulum Ca(2+)-ATPase and the H(+) pump bacteriorhodopsin. In a first step, our method involved a detergent-mediated reconstitution of solubilized membrane proteins into proteoliposomes of 0.1-0.2 microm in size. In a second step, these preformed proteoliposomes were partially dried under controlled humidity followed, in a third step, by electroswelling of the partially dried film to give GUVs. The physical characteristics of GUVs were analyzed in terms of morphology, size, and lamellarity using phase-contrast and differential interference contrast microscopy. The reconstitution process was further characterized by analyzing protein incorporation and biological activity. Both membrane proteins could be homogeneously incorporated into GUVs at lipid/protein ratios ranging from 5 to 40 (w/w). After reconstitution, both proteins retained their biological activity as demonstrated by H(+) or Ca(2+) pumping driven by bacteriorhodopsin or Ca(2+)-ATPase, respectively. This constitutes an efficient new method of reconstitution, leading to the production of large unilamellar membrane protein-containing vesicles of more than 20 microm in diameter, which should prove useful for functional and structural studies through the use of optical microscopy, optical tweezers, microelectrodes, or atomic force microscopy.     

The binding of monoclonal and polyclonal antibodies to the Ca2(+)-ATPase of sarcoplasmic reticulum: effects on interactions between ATPase molecules.

We analyzed the interaction of 14 monoclonal and 5 polyclonal anti-ATPase antibodies with the Ca2(+)-ATPase of rabbit sarcoplasmic reticulum and correlated the location of their epitopes with their effects on ATPase-ATPase interactions and Ca2+ transport activity. All antibodies were found to bind with high affinity to the denatured Ca2(+)-ATPase, but the binding to the native enzyme showed significant differences, depending on the location of antigenic sites within the ATPase molecule. Of the seven monoclonal antibodies directed against epitopes on the B tryptic fragment of the Ca2(+)-ATPase, all except one (VIE8) reacted with the enzyme in native sarcoplasmic reticulum vesicles in both the E1 and E2V conformations. Therefore these regions of the Ca2(+)-ATPase molecule are freely accessible in the native enzyme. The monoclonal antibody VIE8 bound with high affinity to the Ca2(+)-ATPase only in the E1 conformation stabilized by 0.5 mM Ca2+ but not in the E2V conformation stabilized by 0.5 mM EGTA and 5 mM vanadate. Several antibodies that reacted with the B fragment interfered with the crystallization of Ca2(+)-ATPase in the presence of EGTA and vanadate and at least two of them destabilized preformed Ca2(+)-ATPase crystals, suggesting inhibition of interactions between ATPase molecules. Of five monoclonal antibodies with epitopes on the A1 tryptic fragment of the Ca2(+)-ATPase only one gave strong reaction with the native enzyme, and none interfered with ATPase-ATPase interactions as measured by the polarization of fluorescence of FITC-labeled Ca2(+)-ATPase. Therefore the regions of the molecule containing these epitopes are relatively inaccessible in the native structure. Partial tryptic cleavage of the Ca2(+)-ATPase into the A1, A2 and B fragments did not promote the reaction of anti-A1 antibodies with sarcoplasmic reticulum vesicles, but solubilization of the membrane with C12E8 rendered the antigenic site fully accessible to several of them, suggesting that their epitopes are located in areas of contacts between ATPase molecules. Two monoclonal anti-B antibodies that interfered with ATPase-ATPase interactions, produced close to 50% inhibition of the rate of ATP-dependent Ca2+ transport, with significant inhibition of ATPase; this may suggest a role for ATPase oligomers in the regulation of Ca2+ transport. The other antibodies that interact with the native Ca2(+)-ATPase produced no significant inhibition of ATPase activity even at saturating concentrations; therefore their antigenic sites do not undergo major movements during Ca2+ transport.     

Solubilization, purification and reconstitution of Ca(2+)-ATPase from bovine pulmonary artery smooth muscle microsomes by different detergents: preservation of native structure and function of the enzyme by DHPC

The properties of Ca(2+)-ATPase purified and reconstituted from bovine pulmonary artery smooth muscle microsomes {enriched with endoplasmic reticulum (ER)} were studied using the detergents 1,2-diheptanoyl-sn-phosphatidylcholine (DHPC), poly(oxy-ethylene)8-lauryl ether (C(12)E(8)) and Triton X-100 as the solubilizing agents. Solubilization with DHPC consistently gave higher yields of purified Ca(2+)-ATPase with a greater specific activity than solubilization with C(12)E(8) or Triton X-100. DHPC was determined to be superior to C(12)E(8); while that the C(12)E(8) was determined to be better than Triton X-100 in active enzyme yields and specific activity. DHPC solubilized and purified Ca(2+)-ATPase retained the E1Ca-E1*Ca conformational transition as that observed for native microsomes; whereas the C(12)E(8) and Triton X-100 solubilized preparations did not fully retain this transition. The coupling of Ca(2+) transported to ATP hydrolyzed in the DHPC purified enzyme reconstituted in liposomes was similar to that of the native micosomes, whereas that the coupling was much lower for the C(12)E(8) and Triton X-100 purified enzyme reconstituted in liposomes. The specific activity of Ca(2+)-ATPase reconstituted into dioleoyl-phosphatidylcholine (DOPC) vesicles with DHPC was 2.5-fold and 3-fold greater than that achieved with C(12)E(8) and Triton X-100, respectively. Addition of the protonophore, FCCP caused a marked increase in Ca(2+) uptake in the reconstituted proteoliposomes compared with the untreated liposomes. Circular dichroism analysis of the three detergents solubilized and purified enzyme preparations showed that the increased negative ellipticity at 223 nm is well correlated with decreased specific activity. It, therefore, appears that the DHPC purified Ca(2+)-ATPase retained more organized and native secondary conformation compared to C(12)E(8) and Triton X-100 solubilized and purified preparations. The size distribution of the reconstituted liposomes measured by quasi-elastic light scattering indicated that DHPC preparation has nearly similar size to that of the native microsomal vesicles whereas C(12)E(8) and Triton X-100 preparations have to some extent smaller size. These studies suggest that the Ca(2+)-ATPase solubilized, purified and reconstituted with DHPC is superior to that obtained with C(12)E(8) and Triton X-100 in many ways, which is suitable for detailed studies on the mechanism of ion transport and the role of protein-lipid interactions in the function of the membrane-bound enzyme.     

The regulation of ATPase-ATPase interactions in sarcoplasmic reticulum membrane. II. The influence of membrane potential

Na3VO4 promotes the crystallization of Ca2+-ATPase in sarcoplasmic reticulum vesicles. The rate of vanadate-induced crystallization is dramatically increased by inside positive membrane potential generated through ion substitution. Negative potential caused the transient disruption of preformed Ca2+-ATPase crystals, followed by slower reappearance of the lattice after the potential was dissipated. We propose that positive transmembrane potential alters the conformation of the Ca2+-ATPase molecules in a manner that favors ATPase-ATPase interactions, while negative potential would have the opposite effect. Changes in enzyme conformation caused by potential changes during the contraction-relaxation cycle could regulate ATPase interactions in a similar manner in vivo, with effects upon the Ca2+ transport activity and permeability of the sarcoplasmic reticulum.     

Manipulation of activity and orientation of membrane-reconstituted di-tripeptide transport protein DtpT of Lactococcus lactis

The di-tripeptide transport system (DtpT) of Lactococcus lactis was purified to apparent homogeneity by pre-extraction of crude membrane vesicles with octaethylene glycol monodecyl ether (C₁₀E₈), followed by solubilization with n-dodecyl-beta-D-maltoside (DDM) and chromatography on a Ni-NTA resin. The DtpT protein was reconstituted into detergent-destabilized preformed liposomes prepared from E. coli phospholipid/ phosphatidylcholine. A variety of detergents were tested for their ability to mediate the membrane reconstitution of DtpT and their effectiveness to yield proteoliposomes with a high transport activity. The highest activities were obtained with TX₁₀₀, C₁₂E₈ and DM, whereas DDM yielded relatively poor activities, in particular when this detergent was used at concentrations beyond the onset of solubilization of the preformed liposomes. Parallel with the low activity, significant losses of lipid were observed when the reconstitution was performed at high DDM concentrations. This explained at least part of the reduced transport activity as the DtpT protein was highly dependent on the final lipid-to-protein ratios in the proteoliposomes. Consistent with the difference in mechanism of DDM- and TX₁₀₀-mediated membrane protein reconstitution, the orientation of the DtpT protein in the membrane was random with DDM and inside-in when T₁₀₀ was used. The methodology to determine the orientation of membrane-reconstituted proteins from the accessibility of cysteines for thiol-specific reagents is critically evaluated.     

Reconstitution of the skeletal sarcoplasmic reticulum Ca2(+)-pump: influence of negatively charged phospholipids.

The Ca2(+)-ATPase of skeletal sarcoplasmic reticulum was purified and reconstituted in the presence of phosphatidyl choline using the freeze-thaw sonication technique. The effect of incorporation of negatively charged phospholipids, phosphatidylserine and phosphatidylinositol phosphate, into the phosphatidylcholine proteoliposomes was investigated. Various ratios of phosphatidylserine or phosphatidylinositol phosphate to phosphatidylcholine were used, while the total amount of phospholipid in the reconstituted vesicles was kept constant. Enrichment of phosphatidylcholine proteoliposomes by phosphatidylserine or phosphatidylinositol phosphate was associated with activation of Ca2(+)-uptake and Ca2(+)-ATPase activities. The highest activation was obtained at a 50:50 molar ratio of phosphatidylserine:phosphatidylcholine and at a 10:90 molar ratio of phosphatidylinositol phosphate:phosphatidylcholine. The initial rates of Ca2(+)-uptake obtained at 1 microM Ca2+ were 2.6 +/- 0.1 mumol/min per mg of phosphatidylserine:phosphatidylcholine proteoliposomes and 1.5 +/- 0.1 mumol/min per mg of phosphatidylinositol phosphate:phosphatidylcholine proteoliposomes, compared to 0.9 +/- 0.05 mumol/min per mg of phosphatidylcholine proteoliposomes. These findings suggest that negatively charged phospholipids may be involved in the activation of the reconstituted skeletal muscle sarcoplasmic reticulum Ca2(+)-pump.     

An automated pipeline to screen membrane protein 2D crystallization

Electron crystallography relies on electron cryomicroscopy of two-dimensional (2D) crystals and is particularly well suited for studying the structure of membrane proteins in their native lipid bilayer environment. To obtain 2D crystals from purified membrane proteins, the detergent in a protein–lipid–detergent ternary mixture must be removed, generally by dialysis, under conditions favoring reconstitution into proteoliposomes and formation of well-ordered lattices. To identify these conditions a wide range of parameters such as pH, lipid composition, lipid-to-protein ratio, ionic strength and ligands must be screened in a procedure involving four steps: crystallization, specimen preparation for electron microscopy, image acquisition, and evaluation. Traditionally, these steps have been carried out manually and, as a result, the scope of 2D crystallization trials has been limited. We have therefore developed an automated pipeline to screen the formation of 2D crystals. We employed a 96-well dialysis block for reconstitution of the target protein over a wide range of conditions designed to promote crystallization. A 96-position magnetic platform and a liquid handling robot were used to prepare negatively stained specimens in parallel. Robotic grid insertion into the electron microscope and computerized image acquisition ensures rapid evaluation of the crystallization screen. To date, 38 2D crystallization screens have been conducted for 15 different membrane proteins, totaling over 3000 individual crystallization experiments. Three of these proteins have yielded diffracting 2D crystals. Our automated pipeline outperforms traditional 2D crystallization methods in terms of throughput and reproducibility.     

H(+)/Ca(2+) exchange driven by the plasma membrane Ca(2+)-ATPase of Arabidopsis thaliana reconstituted in proteoliposomes after calmodulin-affinity purification

The plasma membrane Ca(2+)-ATPase was purified from Arabidopsis thaliana cultured cells by calmodulin (CaM)-affinity chromatography and reconstituted in proteoliposomes by the freeze-thaw sonication procedure. The reconstituted enzyme catalyzed CaM-stimulated 45Ca(2+) accumulation and H(+) ejection, monitored by the increase of fluorescence of the pH probe pyranine entrapped in the liposomal lumen during reconstitution. Proton ejection was immediately reversed by the protonophore FCCP, indicating that it is not electrically coupled to Ca(2+) uptake, but it is a primary event linked to Ca(2+) uptake in the form of countertransport.     

The effects of membrane potential and lanthanides on the conformation of the Ca2+-transport ATPase in sarcoplasmic reticulum.

The effects of Ca2+, lanthanide ions (Gd3+, La3+ and Pr3+) and membrane potential on the fluorescence of tryptophan and covalently bound fluorescein were analysed in native and fluorescein isothiocyanate (FITC)-labelled sarcoplasmic reticulum vesicles. The binding of Ca2+ and lanthanides to the Ca2+-ATPase increases the fluorescence intensity of tryptophan and decreases the fluorescence intensity of FITC; the dependence of these effects on cation concentration is consistent with the involvement of the high-affinity Ca2+-binding sites of the Ca2+-ATPase in the cation-induced fluorescence changes. The fluorescence of FITC-labelled sarcoplasmic reticulum vesicles is also influenced by membrane potential changes induced by ion substitution. Inside positive potential increases, while inside negative potential decreases, the fluorescence of bound FITC. Smaller potential-dependent changes in tryptophan fluorescence were also observed. The effects of Ca2+, lanthanides and membrane potential on the fluorescence of tryptophan and FITC are discussed in terms of the two major conformations of the Ca2+-ATPase (E1 and E2), that are assumed to alternate during Ca2+ transport. The observations support the suggestion [Dux, Taylor, Ting-Beall & Martonosi (1985) J. Biol. Chem. 260, 11730-11743] that the vanadate-induced crystals of Ca2+-ATPase represent the E2, while the Ca2+ and lanthanide-induced crystals the E1, conformation of the enzyme.     

Ca~(2+)通过膜脂影响肌质网Ca~(2+)-ATP酶的活性与Ca~(2+)转运功能

重建在大豆磷脂脂质体上的兔骨骼肌肌质网Ca~(2+)—ATP酶在ATP驱动下可将溶液中的Ca~(2+)转运到脂酶体内部;外加EGTA则可除去脂酶体外部的Ca~(2+),由此可得到四种含Ca~(2+)状态不同的脂酶体:(1)内、外都无Ca~(2+);(2)仅外部有Ca~(2+);(3)内、外都有Ca~(2+);(4),仅内部有Ca~(2+).用DPH和AS系列萤光探针对这四种含Ca~+状态不同的脂酶体的膜脂流动性进行了测定,结果表明:脂酶体外部加入Ca~(2+),脂双层外表面的流动性降低.当Ca~(2+)进入脂酶体内部后,内表面膜脂的流动性也降低,而且外层膜脂流动性进一步降低.脂酶体内、外的Ca~(2+)含量不同时,Ca~(2+)—ATP酶功能状态也不同.转运到脂酶体内部的ca~(2+)积累到一定浓度后,通过Ca~(2+)泵向内转运的Ca~(2+)及Ca~(2+)—ATP酶活力都受到了抑制.转运进行到第四分钟时的酶活只有第一分钟的9%.但在相同的实验条件下,失去了完整的膜结构的纯化的Ca~(2+)—ATP酶蛋白没有被抑制.这提示完整的膜结构是这种抑制作用所必需的,而且膜两侧Ca~(2+)浓度的梯差可通过影响膜脂来调节Ca~(2+)—ATP酶的功能.     

Influence of phospholipid fatty acyl composition on sarcolemmal and sarcoplasmic reticular cation transporters

Using solubilization/reconstitution techniques, we have investigated the influence of membrane fatty acyl composition on the activities of sarcolemmal and sarcoplasmic reticular transporters. The sarcolemmal Na(+)-Ca2+ exchanger and Na+, K(+)-ATPase and the sarcoplasmic reticular Ca2(+)-ATPase were reconstituted into phosphatidylcholine:phosphatidylserine:cholesterol (30:50:20% by weight) proteoliposomes of defined fatty acyl composition. Transport activities varied considerably with phospholipid fatty acyl composition. Quite strikingly, the dependence on membrane fatty acyl composition for all three transporters was identical.     

Protein kinase-dependent phosphorylation of cardiac sarcolemmal Ca2(+)-ATPase, as studied with a specific monoclonal antibody

Phosphorylation of the Ca2(+)-pump ATPase of cardiac sarcolemmal vesicles by exogenously added protein kinases was examined to elucidate the molecular basis for its regulation. The Ca2(+)-pump ATPase was isolated from protein kinase-treated sarcolemmal vesicles using a monoclonal antibody raised against the erythrocyte Ca2(+)-ATPase. Protein kinase C (C-kinase) was found to phosphorylate the Ca2(+)-ATPase. The stoichiometry of this phosphorylation was about 1 mol per mol of the ATPase molecule. The C-kinase activation resulted in up to twofold acceleration of Ca2+ uptake by sarcolemmal vesicles due to its effect on the affinity of the Ca2+ pump for Ca2+ in both the presence and absence of calmodulin. Both the phosphorylation and stimulation of ATPase activity by C kinase were also observed with a highly-purified Ca2(+)-ATPase preparation isolated from cardiac sarcolemma with calmodulin-Sepharose and a high salt-washing procedure. Thus, C-kinase appears to stimulate the activity of the sarcolemmal Ca2(+)-pump through its direct phosphorylation. In contrast to these results, neither cAMP-dependent protein kinase, cGMP-dependent protein kinase nor Ca2+/calmodulin-dependent protein kinase II phosphorylated the Ca2(+)-ATPase in the sarcolemmal membrane or the purified enzyme preparation, and also they exerted virtually no effect on Ca2+ uptake by sarcolemmal vesicles.     

Protein kinase C-dependent phosphorylation of sarcolemmal Ca2(+)-ATPase isolated from bovine aortic smooth muscle

We examined the effect of protein kinase C (PKC)-dependent phosphorylation on Ca2+ uptake and ATP hydrolysis by microsomal as well as purified sarcolemmal Ca2(+)-ATPase preparations isolated from bovine aortic smooth muscle. The phosphorylation was performed by treating these preparations with PKC and saturating concentrations of ATP (or ATP-gamma S), Ca2+, and 12-O-tetradecanoyl phorbol-13-acetate (TPA) at 37 degrees C for 10 min. In microsomes, treatment with PKC enhanced a portion of the Ca2+ uptake activity inhibitable by 10 microM vanadate, by up to about 30%. On the other hand, Ca2(+)-dependent ATPase activity in the purified Ca2(+)-ATPase preparation was stimulated by up to twofold. Up to twofold stimulation by PKC was also observed for the Ca2+ uptake by proteoliposomes reconstituted from purified sarcolemmal Ca2(+)-ATPase and phospholipids. Since these effects were evident only at Ca2+ concentrations between 0.1 to 1.0 microM, we concluded that it was the affinity of the Ca2(+)-ATPase for Ca2+ that was increased by the PKC treatment. Under conditions in which PKC increased Ca2+ pump activity, the sarcolemmal Ca2(+)-ATPase was phosphorylated to a level of about 1 mol per mol of the enzyme. There was good parallelism between the ATPase phosphorylation and the extent of enzyme activation. These results strongly suggest that the activity of the sarcolemmal Ca2+ pump in vascular smooth muscle is regulated through its direct phosphorylation by PKC.     

Tryptic digestion of sarcoplasmic reticulum inhibits Ca2+ accumulation by action on a membrane component other than the Ca2+-ATPase

We have reexamined the "uncoupling" of Ca2+ transport from ATP hydrolysis, which has been reported to be caused by trypsin cleavage of the Ca2+-ATPase of sarcoplasmic reticulum (SR) vesicles at the second (slower) of two characteristic tryptic sites (Scott, T. L., and Shamoo, A. E. (1982) J. Membr. Biol. 64, 137-144). We find that the loss of Ca2+ accumulation capacity in SR vesicles is poorly correlated with this cleavage under several conditions. The loss is accompanied by increased Ca2+ permeability but not by changes in the properties of the ATPase or ATP-Pi exchange activities of the vesicles. Proteoliposomes containing purified Ca2+-ATPase which has been cleaved in part at the two tryptic sites are as well coupled and impermeable to Ca2+ as proteoliposomes containing intact Ca2+-ATPase. We conclude that the loss of Ca2+ accumulation capacity in SR vesicles on tryptic treatment is due to cleavage of a SR membrane component other than the Ca2+-ATPase, possibly a component of the gated channels which function in Ca2+ release from SR, which leads to a Ca2+ leak. The hydrolytic and coupled transport functions of the Ca2+-ATPase itself may well be unaffected by the two tryptic cleavages.     

Transmembranous organization of (Ca2(+)-Mg2+)-ATPase from sarcoplasmic reticulum. Evidence for lumenal location of residues 877-888

An antipeptide antibody was produced against a peptide corresponding to residues 877-888 of fast twitch rabbit sarcoplasmic reticulum ATPase. This antipeptide antibody bound strongly to the ATPase in sarcoplasmic reticulum vesicles only after the vesicles had been solubilized with the detergent C12E8 indicating that its epitope was located in the lumen of the sarcoplasmic reticulum. Digestion of sarcoplasmic reticulum or purified (Ca2(+)-MG2+)-ATPase by proteinase K for up to 1 h resulted in a stable ATPase fragment of 30 kDa containing the epitope for the above antibody and the epitope for an antibody directed against the C terminus. Further proteolysis revealed smaller fragments (Mr 19,000 and 13,000) containing both epitopes. By contrast, small fragments of the ATPase (less than 29 kDa) containing the N-terminal epitope were not observed even after short exposures to proteinase K. These data support the view that the (Ca2(+)-MG2+)-ATPase has 10 transmembranous helices.     

The epitope for monoclonal antibody A20 (amino acids 870-890) is located on the luminal surface of the Ca2(+)-ATPase of sarcoplasmic reticulum.

The epitope for monoclonal antibody A20 was mapped to amino acids 870-890 of the Ca2(+)-ATPase of rabbit fast-twitch skeletal muscle sarcoplasmic reticulum. The antibody did not react with the epitope in intact sarcoplasmic reticulum vesicles but reacted with the epitope when the vesicles were solubilized with the detergent C12E8 or made permeable by incubation in a hypotonic medium. By contrast, antibody A52, which binds to a cytoplasmic epitope consisting of amino acids 657-672, reacted with the Ca2(+)-ATPase in vesicular, permeabilized vesicular, and C12E8-solubilized states. These results clearly demonstrate that antibody A20 binds to a luminal epitope and provide the first demonstration that a specific segment of the Ca2(+)-ATPase is located on the luminal surface of the sarcoplasmic reticulum. These results are consistent with, and support, our model for folding of the Ca2(+)-ATPase (Brandl, C. J., Korczak, B., Green, N. M., and MacLennan, D. H. (1986) Cell 44, 597-607) in which residues 657-672 were proposed to form part of the cytoplasmic nucleotide binding domain, while residues 870-890 were proposed to form a luminal loop between proposed transmembrane sequences M7 and M8.