Correlation of ultrastructure of reconstituted sarcoplasmic reticulum membrane vesicles with variation in phospholipid to protein ratio

Abstract

We have previously described the reconstitution of functional membrane vesicles with lipid content similar to that of the normal sarcoplasmic reticulum membrane (approximately 1.0 mumol of phospholipid/mg of protein). The present study describes methodology to prepare reconstituted membrane vesicles with defined phospholipid to protein ratio, both lower and higher than that of the original membrane. The Ca2+ loading rate and efficiency are greatest in the membranes of highest protein content (0.38 mumol of phospholipid/mg of protein), decline slowly as the lipid content is quadrupled, and decrease markedly as the lipid content is quadrupled again. Such membranes of defined composition can be used to study lipid-protein interaction and to correlate membrane structure with composition. The number of particles observed by freeze-fracture electron microscopy can be correlated with protein content, whereas the percentage of smooth domain is proportional to the lipid content of the reconstituted membrane. Since 90% or more of the protein of the reconstituted membrane is the calcium pump protein, the number of particles observed by freeze-fracture is directly proportional to the amount of calcium pump protein in the membrane. The number of pump molecules calculated to be in the membrane is greater by a factor of two than the number of particles which we observed. This multiplicity ratio could be greater depending upon the assumptions made regarding the width of the membrane (see "Appendix"). Thus, it would appear that the particles consist of two or more molecules of pump protein. The change in protein concentration of the membrane is reflected also in thin sections and by negative staining. In thin sections, the broad inner and outer 70 A bands become discontinuous and patchy and, in the limit, approach a symmetrical 20,20,20 A trilayer as the protein content of the membrane becomes small. In an analogous fashion, the concentration of particles at the surface of the membrane, observed by negative staining, decreases with increasing lipid concentration in the membrane. Thus, the correlation of composition with structure can be observed by each of the three methods of sample preparation for electron microscopic analysis.