Larger Precursors of Mitochondria1 Translation Products in Neurospora crassa

Abstract

Specific labeling in vivo of the formylated N termini of mitochondrial translation products revealed that some mitochondrially synthesized proteins were not labeled this way. As a consequence, it was worthwhile considering that larger precursor proteins of mitochondrial translation products exist. Although we used a rapid isolation procedure, only after 2-h of labeling in the presence of cycloheximide, could three additional mitochondrial translation products (molecular mass 45, 36 and 25 kilodaltons) be detected. Preincubation with cycloheximide indicated that these proteins might be larger precursors which were no longer processed due to the prolonged presence of cycloheximide. To prevent processing of the precursors during isolation, cells of the slime mutant were directly lysed in boiling sodium dodecyl sulphate solution. In this way, the same three additional mitochondrial translation products were detected after a pulse-labeling of 1 min. These proteins behave in a precursor-like fashion. Labeling at 9 °C resulted in a partial accumulation of the three additional proteins. Finally protein blots treated with antibodies and ²⁵¹-labeled protein A, support the idea that the 45-kDa protein is a precursor of subunit 1 of cytochrome c oxidase; 50–80% of this precursor could be detected in the post-mitochondria1 supernatant, indicating that this polypeptide is not tightly bound to the membrane.