Structural studies on the organization of proteins in mitochondrial membranes using proteolytic enzymes

Abstract

Proteolytic enzymes, pronase and trypsin, digest protein in ETP and in SU-particles (devoid of the soluble ATPase) at similar rates and to the same extents for intact and lipid-depleted membranes, showing that lipids do not constitute a barrier to the action of the proteases. The rates and extents of hydrolysis are slightly depressed when membranes are reconstituted from lipid-depleted particles and phospholipids. The hydrolysis rates for the various particles are not greatly enhanced by detergent solubilization nor by other denaturing treatments, indicating that the rates measured in absence of treatments are maximal under the conditions used. The circular dichroism spectra of pronase treated ETP are noticeably altered showing modification of the original conformation. Moreover, enzymic activities of mitochondria and submitochondrial particles are progressively affected by proteases according to their localization at, or near to, a given surface of the membrane. The matrix enzyme, malate dehydrogenase, is not apparently reIeased from mitochondria during the initial incubation period. The results are tentatively discussed in terms of organization of lipids and proteins in the mitochondrial membrane.Previous investigations from our laboratory (1-3) have shown that the proteins of the inner mitochondrial membrane bind phospholipids hydrophobically in reconstitution experiments. Indirect reasons have suggested that the proteins which are bound hydrophobically in the intact membrane are those which may be considered "intrinsic" and are water-insoluble when separated from the lipids (4). Superficial "extrinsic" proteins (4) which are water-soluble when detached, may be bound by polar forces t o the membrane continuum (5).The type of binding alone may not be a sufficient tool t o evaluate the organization of proteins and lipids in the inner mitochondrial membrane. For example, hydrophobic binding between lipids and proteins is compatible with different membrane models, although the macromolecular organization is quite different in these models. Membrane proteins may be hydrophobically bound t o phospholipids according to any one of the following possibilities: (a) penetration of phospholipid fatty acyl chains from the lipid bilayer into superficial protein layers (6); (b) partial penetration of protein side residues between lipid molecules in the bilayer (7); (c) extensive penetration of protein molecules into interstices of the lipid bilayer, like in some mosaic models recently proposed (8-10);