Two independent activities define Ccm1p as a moonlighting protein in Saccharomyces cerevisiae

Ccm1p is a nuclear-encoded PPR (pentatricopeptide repeat) protein that localizes intomitochondria of Saccharomyces cerevisiae. It was first defined as an essentialfactor to remove the bI4 [COB (cytochrome b) fourth intron)] andaI4 [COX1 (cytochrome c oxidase subunit 1) fourth intron] ofpre-mRNAs, along with bI4 maturase, a protein encoded by part of bI4 and preceding exons thatremoves the intronic RNA sequence that codes for it. Later on, Ccm1p was described as key tomaintain the steady-state levels of the mitoribosome small subunit RNA (15S rRNA). bI4 maturase isproduced inside the mitochondria and therefore its activity depends on the functionality ofmitochondrial translation. This report addresses the dilemma of whether Ccm1p supports bI4 maturaseactivity by keeping steady-state levels of 15S rRNA or separately and directly supports bI4 maturaseactivity per se. Experiments involving loss of Ccm1p, SMDC (sudden mitochondrialdeprivation of Ccm1p) and mutations in one of the PPR (pentatricopeptide repeat) motifs revealedthat the failure of bI4 maturase activity in CCM1 deletion mutants was not due to amalfunction of the translational machinery. Both functions were found to be independent, definingCcm1p as a moonlighting protein. bI4 maturase activity was significantly more dependent on Ccm1plevels than the maintenance of 15S rRNA. The novel strategy of SMDC described here allowed the studyof immediate short-term effects, before the mutant phenotype was definitively established. Thisapproach can be also applied for further studies on 15S rRNA stability and mitoribosomeassembly.