Cytoplasmic-type 80 S Ribosomes Associated with Yeast Mitochondria

Abstract

Abstract A class of 80 S ribosomes has been isolated from purified yeast mitochondria. These ribosomes are distinguished from cytoplasmic ribosomes of the postmitochondrial supernatant fraction by having a greater stability against the dissociating effect of 0.4 m KCl. These results confirm observations of Schmitt ((1969) Fed. Eur. Biochem. Soc. Lett. 4, 234–238). Ribosome dissociability has been examined as a function of the stage of cell growth. Free and membranebound cytoplasmic ribosomes of the postmitochondrial supernatant fraction and the 80 S ribosomes associated with mitochondria are all more resistant to the dissociating effect of 0.4 m KCl when isolated from log phase as compared with stationary phase cells. In all cases, however, 80 S ribosomes associated with mitochondria show resistance to KCl dissociation to a significantly larger degree than do the other classes of ribosomes. Three lines of evidence support the conclusion that the 80 S ribosomes (bound 80 S) we isolate from purified mitochondria are cytoplasmic-type ribosomes: (a) incorporation of 32Pi into rRNA of these 80 S ribosomes is insensitive to 15 µg per ml of ethidium bromide, (b) this class of 80 S ribosomes is found in the mitochondrial fraction from a cytoplasmic petite which lacks mtDNA, and, (c) poly(U)-directed phenylalanine incorporation catalyzed by purified preparations of 80 S ribosomes associated with mitochondria is sensitive to inhibition by cycloheximide but not by chloramphenicol. Evidence is presented that purified mitochondria have binding sites for the bound 80 S ribosomes; additional binding sites can be exposed by washing mitochondria with 2 mm EDTA. We observe at least two classes of binding sites with EDTA-washed mitochondria. Low affinity sites bind 260 µg of ribosomes per mg of mitochondrial protein and high affinity sites bind 116 µg of ribosomes per mg of mitochondrial protein. Ribosome binding sites on EDTA-washed mitochondria are labile; storage of mitochondria at 0° reduces their ability to bind exogenously added bound 80 S ribosomes in an apparent first order process with a half-life of 33 hours.