PROBING OF THE MEMBRANE TOPOLOGY OF SARCOPLASMIC-RETICULUM CA2-ATPASEWITH SEQUENCE-SPECIFIC ANTIBODIES - EVIDENCE FOR PLASTICITY OF THE C-TERMINAL DOMAIN()

The topology of Ca2+-ATPase in sarcoplasmic reticulum (SR) vesicles wa s investigated with the aid of sequence-specific antibodies, produced against oligopeptides corresponding to sequences close to the membrano us portions of the protein, The antisera in competitive enzyme-linked immunosorbent assays only reacted with intact SR vesicles to a limited extent, but most epitopic regions were exposed by low concentrations of nondenaturing detergent, octaethylene glycol dodecyl ether (C12E8) or after removal of cytosolic regions by proteinase K, In particular, these treatments exposed the loop regions in the C-terminal domain, in cluding L7-8, the loop region located between transmembrane segments M 7 and M8, with a putative intravesicular position, which had immunoche mical properties very similar to those of the C terminus with a docume nted cytosolic exposure, In contrast to this, the reactivity of the N- terminal intravesicular loop regions L1-2 and L3-4 was only increased by C12E8 treatment but not by proteinase K proteolysis, Complexation o f Ca2+-ATPase with beta,gamma-CrATP stabilized the C-terminal domain o f Ca2+-ATPase against proteinase K proteolysis and reaction with most of the antisera, but immunoreactivity was maintained by the L6-7 and L 7-8 loops, Immunoelectron microscopic analyses of vesicles following n egative staining, thin sectioning, and the SDS-digested freeze-fractur e labeling method suggested that the L7-8 epitope, in contrast to L6-7 and the C terminus, can be exposed on either the intravesicular or cy tosolic side of the membrane. A preponderant intravesicular location o f L7-8 in intact vesicles is suggested by the susceptibility of this r egion to proteolytic cleavage after disruption of the vesicular barrie r with C12E8 and in symmetrically reconstituted Ca2+-ATPase proteolipo somes. In conclusion, our data suggest an adaptable membrane insertion of the C-terminal Ca2+-ATPase domain, which under some conditions per mits sliding of M8 through the membrane with cytosolic exposure of L7- 8, of possible functional significance in connection with Ca2+ translo cation, On the technical side, our data emphasize that extreme caution is needed when using nondenaturing detergents or other treatments lik e EGTA at alkaline pH to open up vesicles for probing of intravesicula r location with antibodies.