Import of proteins into mitochondria. Translatable mRNAs for imported mitochondrial proteins are present in free as well as mitochondria-bound cytoplasmic polysomes

Abstract

In order to assess the role of different polysomal populations in mitochondrial protein import, yeast spheroplasts were treated with cycloheximide to prevent polysome "run-off" and fractionated into free polysomes, polysomes bound to the mitochondrial outer surface, and polysomes bound to the endoplasmic reticulum. These polysomes were analyzed for translatable mRNAs coding for 8 cytosolic and 12 imported mitochondrial proteins. The mitochondrial proteins included 7 proteins of the inner membrane, 2 proteins of the matrix, 2 proteins of the intermembrane space, and 1 protein of the outer membrane. Of the mRNAs for imported mitochondrial proteins, 8 were enriched in mitochondria-bound polysomes, 3 were enriched in free polysomes, and 1 was enriched in neither. All mRNAs for cytosolic proteins were enriched in free polysomes. Polysomes bound to the endoplasmic reticulum lacked significant levels of translatable mRNAs for either cytosolic or mitochondrial proteins. Even though mRNAs for imported mitochondrial proteins were enriched in mitochondria-bound polysomes, these polysomes represented only 12-18% of the total cytoplasmic polysomes. As a consequence, none of the translatable mRNAs for imported mitochondrial proteins tested was predominantly associated with mitochondria-bound polysomes. While mitochondria-bound polysomes may contribute to mitochondrial protein import, they do not appear to be obligatory for this process.