Topography of succinate dehydrogenase in the mitochondrial inner membrane. A study using limited proteolysis and immunoblotting.
The arrangement of the large (70,000-Mr) and small (30,000-Mr) subunits of succinate dehydrogenase in the mitochondrial inner membrane was investigated by immunoblot analysis of bovine heart mitochondria (right-side-out, outer membrane disrupted) or submitochondrial particles (inside-out) that had been subjected to surface-specific proteolysis. Both subunits were resistant to proteinase treatment provided that the integrity of the inner membrane was preserved, suggesting that neither subunit is exposed at the cytoplasmic surface of the membrane. The bulk of the small subunit appears to protrude into the matrix compartment, since the 30,000-Mr polypeptide is degraded extensively during limited proteolysis of submitochondrial particles without the appearance of an immunologically reactive membrane-associated fragment: moreover, a soluble 27,000-Mr peptide derived from this subunit is observed transiently on incubation with trypsin. Similar data obtained from the large subunit suggest that this polypeptide interacts with the matrix side of the inner membrane via two distinct domains; these are detected as stable membrane-associated fragments of 32,000 Mr and 27,000 Mr after treatment of submitochondrial particles with papain or proteinase K, although the 27,000-Mr fragment can be degraded further to low-Mr peptides with trypsin or alpha-chymotrypsin. A stable 32,000-34,000-Mr fragment is generated by a variety of specific and non-specific proteinases, indicating that it may be embedded largely within the lipid bilayer, or is inaccessible to proteolytic attack owing to its proximity to the surface of the intact membrane, possibly interacting with the hydrophobic membrane anchoring polypeptides of the succinate: ubiquinone reductase complex.
Clarkson
G H
GH
Department of Biochemistry, University of Glasgow, Scotland, U.K.
Neagle
J
J
Lindsay
J G
JG
D000818:Animals; D002417:Cattle; D002458:Cell Fractionation; D010450:Endopeptidases / Q000378:metabolism; D015151:Immunoblotting; D007425:Intracellular Membranes / Q000201:enzymology* / Q000648:ultrastructure; D046911:Macromolecular Substances; D008929:Mitochondria, Heart / Q000201:enzymology* / Q000648:ultrastructure; D008970:Molecular Weight; D013367:Submitochondrial Particles / Q000201:enzymology* / Q000648:ultrastructure; D013385:Succinate Dehydrogenase / Q000032:analysis*; D014461:Ultracentrifugation
D046911:Macromolecular Substances; D013385:Succinate Dehydrogenase; D010450:Endopeptidases