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.
Lévy
D
D
Service de Biophysique, Département de Biologie et URA-CNRS (D 1290), CEN Saclay, Gif-sur-Yvette, France.
Gulik
A
A
Bluzat
A
A
Rigaud
J L
JL
D002118:Calcium / Q000502:physiology; D000252:Calcium-Transporting ATPases / Q000502:physiology*; D003902:Detergents / Q000494:pharmacology*; D005614:Freeze Fracturing; D007700:Kinetics; D008081:Liposomes; D008565:Membrane Proteins / Q000502:physiology*; D008854:Microscopy, Electron; D010743:Phospholipids / Q000502:physiology; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*
D003902:Detergents; D008081:Liposomes; D008565:Membrane Proteins; D010743:Phospholipids; D000252:Calcium-Transporting ATPases; D002118:Calcium