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Volume 272, Number 46, Issue of November 14, 1997 pp. 29015-29032
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.

Probing of the Membrane Topology of Sarcoplasmic Reticulum Ca²⁺-ATPase with Sequence-specific Antibodies
EVIDENCE FOR PLASTICITY OF THE C-TERMINAL DOMAIN

(Received for publication, May 12, 1997, and in revised form, July 28, 1997)

Jesper V. Møller , Gang Ning ^§ , Arvid B. Maunsbach ^§ , Kazushi Fujimoto , Kouichi Asai , Birte Juul , Young-Jae Lee , Adrienne Gomez de Gracia , Pierre Falson and Marc le Maire

From the  Department of Biophysics, University of Aarhus, Ole Worms Allé 185, DK-8000 Aarhus C, Denmark, the ^§ Department of Cell Biology, Institute of Anatomy, Wilh. Meyers Allé 233, University of Aarhus, DK-8000 Aarhus C, Denmark,  Section de Biophysique des Protéines et des Membranes, Département de Biologie Cellulaire et Moléculaire, CEA et CNRS URA 2096, Centre d'Etudes de Saclay, F-91191 Gif-sur-Yvette Cedex, France, and the  Department of Anatomy, Faculty of Medicine, Kyoto University, Yoshida-Konoecho, Sakyo-ku, Kyoto 606-01, Japan

The topology of Ca²⁺-ATPase in sarcoplasmic reticulum (SR) vesicles was investigated with the aid of sequence-specific antibodies, produced against oligopeptides corresponding to sequences close to the membranous 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, including L7-8, the loop region located between transmembrane segments M7 and M8, with a putative intravesicular position, which had immunochemical properties very similar to those of the C terminus with a documented 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 of Ca²⁺-ATPase with ,-CrATP stabilized the C-terminal domain of Ca²⁺-ATPase against proteinase K proteolysis and reaction with most of the antisera, but immunoreactivity was maintained by the L6-7 and L7-8 loops. Immunoelectron microscopic analyses of vesicles following negative staining, thin sectioning, and the SDS-digested freeze-fracture 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 cytosolic side of the membrane. A preponderant intravesicular location of L7-8 in intact vesicles is suggested by the susceptibility of this region to proteolytic cleavage after disruption of the vesicular barrier with C₁₂E₈ and in symmetrically reconstituted Ca²⁺-ATPase proteoliposomes. In conclusion, our data suggest an adaptable membrane insertion of the C-terminal Ca²⁺-ATPase domain, which under some conditions permits sliding of M8 through the membrane with cytosolic exposure of L7-8, of possible functional significance in connection with Ca²⁺ translocation. On the technical side, our data emphasize that extreme caution is needed when using nondenaturing detergents or other treatments like EGTA at alkaline pH to open up vesicles for probing of intravesicular location with antibodies.

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