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Synthesis and transport of mitochondrial proteins were followed in a cell-free homogenate of Neurospora crassa in which mitochondrial translation was inhibited. Proteins synthesized on cytoplasmic ribosomes are transferred into the mitochondrial fraction. The relative amounts of proteins which are transferred in vitro are comparable to those transferred in whole cells.

Cycloheximide and puromycin inhibit the synthesis of mitochondrial proteins but not their transfer into mitochondria.

The transfer of immunoprecipitable mitochondrial proteins was demonstrated for matrix proteins, carboxyatractyloside-binding protein and cytochrome c.

Import of proteins into mitochondria exhibits a degree of specificity. The transport mechanism differentiates between newly synthesized proteins and preexistent mitochondrial proteins, at least in the case of matrix proteins.

In the cell-free homogenate membrane-bound ribosomes are more active in the synthesis of mitochondrial proteins than are free ribosomes. The finished translation products appear to be released from the membrane-bound ribosomes into the cytosol rather than into the membrane vesicles.

The results suggest that the transport of cytoplasmically synthesized mitochondrial proteins is essentially independent of cytoplasmic translation; that cytoplasmically synthesized mitochondrial proteins exist in an extramitochondrial pool prior to import; that the site of this pool is the cytosol for at least some of the mitochondrial proteins; and that the precursors in the extramitochondrial pool differ in structure or conformation from the functional proteins in the mitochondria.