Biogenesis of a 35-kilodalton protein associated with outer mitochondrial membrane in rat liver.

Biochemical analyses following subcellular fractionation of rat liver indicated that the outer mitochondrial membrane contains a number of membrane-specific proteins of which a 35-kilodalton species (OMM-35) is a prominent component. These results were confirmed and extended by electron microscope immunocytochemical tests based on the protein A-gold technique. OMM-35 is tightly bound to the outer mitochondrial membrane, e.g. it was not released by sonication in the presence of 1.5 M KCl and 0.1% sodium deoxycholate. However, it did not react with the photoaffinity probe azidopyrene, which indicates that OM-35 is located peripherally on the membrane rather than buried deep in the lipid bilayer as an intrinsic protein. Since low levels of detergent were required for OMM-35 in intact mitochondria to react with exogenous antibodies, OMM-35 is probably located on the side of the outer membrane which faces the interior of the organelle. When rat liver mRNA was translated in a messenger-dependent cell-free system derived from rabbit reticulocytes, antiserum against OMM-35 precipitated a single polypeptide product which migrated on sodium dodecyl sulfate-polyacrylamide gels with molecular weight characteristics of a protein slightly larger (by Mr = 500) than OMM-35 obtained from isolated outer mitochondrial membrane. The mRNA coding for OMM-35 was recovered exclusively from membrane-free polysomes. Thus, the route followed for synthesis and subsequent insertion of OMM-35 into the outer membrane of mitochondria is the post-translational pathway which has been previously described for proteins destined for the interior compartments of this organelle.