Growing yeast spheroplasts were shown to have, on the average, four times the number of cytoplasmic ribosomes in contact with the outer mitochondrial membrane compared to starved spheroplasts. Ribosomes in contact with mitochondria in the growing spheroplast preparation, like free cytoplasmic ribosomes, exist primarily as polysome structures. In the starved spheroplast preparation, both mitochondria-bound and free cytoplasmic ribosomes exist primarily as monosomes. Mitochondria isolated from growing spheroplasts in a medium containing lmM Mg++ have cytoplasmic ribosomes bound directly to the outer membrane. These ribosomes can be quantitatively removed by washing the mitochondria with 2 mM EDTA. Mitochondria from starved spheroplasts are capable of accepting either free cytoplasmic polysomes or cytoplasmic polysomes extracted from mitochondria. However, the extent of polysome binding to mitochondria was shown to be a direct function of the Mg++ concentration; a smaller percentage of the input polysomes bind as the Mg++ concentration is lowered. At 1 mM Mg++, neither free cytoplasmic nor mitochondria-bound polysomes bind to mitochondria. Nevertheless, when growing spheroplasts are broken and mitochondria isolated in medium containing 1 mM Mg++, the mitochondria are seen to have cytoplasmic ribosomes firmly attached to the outer membrane. This result, in addition to our earlier data (Kellems, R. E., and R. A. Butow. 1974. J. Biol. Chem. 249:3304-3310), support the view that cytoplasmic ribosomes attached to the outer membrane of purified mitochondria were attached in vivo. In preparations of mitochondria isolated from growing spheroplasts, ribosomes appear to be found to specific regions of the outer membrane, namely those regions which are in close association or in contact with the inner mitochondrial membrane. This is particularly evident with mitochondria in a condensed configuration. This finding suggests a mechanism whereby cytoplasmically synthesized mitochondrial protein could be transferred by a process of vectorial translation across both membranes of the organelle.

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