Assembly of ATPase protein in sarcoplasmic reticulum membranes

Abstract

Three specimen preparation techniques for electron microscopy were used to investigate the incorporation of the ATPase polypeptide chains in the membranes of fragmented sarcoplasmic reticulum (SR) obtained from rabbit skeletal muscle. Observations were made of both normal vesicles and vesicles exposed to trypsin, which is known to cleave the ATPase protein and to alter the ultrastructure of the vesicles in predictable ways. Freeze-fracture replicas reveal the typical 90-A particles on the concave (PF) faces with a density of 5,730 +/- 520/mum2. On the other hand both negatively stained and deeply etched preparations display outer projections, which are absent on trypsin-incubated vesicles. The etched specimens afford for the first time top views of the vesicles in the absence of any stain. These views reveal outer projections on the PS surface with a density of 21,000 +/- 3,900/mum2, a value nearly approximating the density of the ATPase polypeptide chains (106,000 mol wt) calculated on the basis of protein and membrane area determinations. On the other hand, this value is three to four times higher than that found for the density of the 90-A particles on the concave fracture faces. Since both outer projections and 90-A particles are identified with the ATPase protein, it is suggested that the ATPase polypeptide chains are amphiphilic molecules, with polar ends protruding individually as outer projections on the surface of the vesicles, and hydrophobic ends appearing as 90-A particles on the concave fracture faces. The discrepancy between the densities of the outer projections and the 90-A particles may be attributed either to variable penetration of the polypeptide chains into the membrane bilayer, or to formation of oligomers containing three or four hydrophobic ends and appearing as single 90-A particles. Each ATPase chain forms a complex with 20-30 phospholipid molecules. The remaining phospholipids (approximately 70% of the total SR phospholipids) account for less than half the membrane volume. It is proposed that the outer leaflet of the SR membrane is prevalently composed of the ATPase lipoprotein complex, and the inner leaflet is mostly a phospholipid monolayer.