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Biogenesis of a 35-kilodalton protein associated with outer mitochondrial membrane in rat liver
Abstract:
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.
Insights
The outer mitochondrial membrane protein OMM-35 is synthesized post-translationally. Its mRNA is found on membrane-free polysomes, indicating a specific pathway for mitochondrial outer membrane protein insertion.
Area of Science:
- Mitochondrial biology
- Cellular biochemistry
- Protein localization
Background:
- The outer mitochondrial membrane (OMM) harbors specific proteins crucial for organelle function.
- Understanding the biogenesis and localization of these OMM proteins is essential for comprehending mitochondrial dynamics.
Purpose of the Study:
- To investigate the synthesis, localization, and insertion pathway of a prominent OMM protein, OMM-35.
- To determine if OMM-35 is an intrinsic or peripheral membrane protein and its orientation within the OMM.
Main Methods:
- Subcellular fractionation of rat liver followed by biochemical analysis.
- Electron microscope immunocytochemistry using the protein A-gold technique.
- Photoaffinity labeling with azidopyrene.
- In vitro translation of rat liver mRNA and immunoprecipitation with OMM-35 antiserum.
Main Results:
- OMM-35 (35 kDa) is a prominent, tightly bound protein of the OMM.
- OMM-35 is peripherally located, not embedded in the lipid bilayer, and faces the mitochondrial interior.
- mRNA for OMM-35 originates from membrane-free polysomes.
- In vitro translation produced a slightly larger precursor, suggesting post-translational modification.
Conclusions:
- OMM-35 is synthesized on free ribosomes and inserted into the OMM post-translationally.
- This pathway is similar to that of proteins targeted to the mitochondrial interior compartments.
- OMM-35's peripheral localization and orientation provide insights into OMM protein assembly.