Probing of the membrane topology of sarcoplasmic reticulum Ca2+-ATPase with sequence-specific antibodies. Evidence for plasticity of the c-terminal domain.
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 beta,gamma-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.
Moller
J V
JV
Department of Biophysics, University of Aarhus, Ole Worms Allé 185, DK-8000 Aarhus C, Denmark.
Ning
G
G
Maunsbach
A B
AB
Fujimoto
K
K
Asai
K
K
Juul
B
B
Lee
Y J
YJ
Gomez de Gracia
A
A
Falson
P
P
le Maire
M
M
D000818:Animals; D000906:Antibodies / Q000276:immunology*; D000252:Calcium-Transporting ATPases / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D003902:Detergents; D004797:Enzyme-Linked Immunosorbent Assay; D005614:Freeze Fracturing; D006868:Hydrolysis; D007106:Immune Sera; D008565:Membrane Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D016253:Microscopy, Immunoelectron; D015335:Molecular Probes; D016296:Mutagenesis; D017510:Protein Folding; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000201:enzymology* / Q000648:ultrastructure
D000906:Antibodies; D003902:Detergents; D007106:Immune Sera; D008565:Membrane Proteins; D015335:Molecular Probes; D000252:Calcium-Transporting ATPases