ANALYSIS OF NUCLEAR MUTANTS OF CHLAMYDOMONAS DEFICIENT IN THE ACCUMULATION OF SPECIFIC CHLOROPLAST RNAS

Abstract

Genetic analysis has revealed that the accumulation of several chloroplast mRNAs of the green alga Chlamydomonas reinhardtii requires specific nucleus‐encoded functions. We have characteized three nuclear mutants, nac2, 222E and F24 which lack psbD, psbB and psaB mRNA, respectively. In each case we have shown that the target of the nucleus‐encoded function affected in these mutants is the 5′ untranslated region of the corresponding mRNA (3, 4, 5). We have cloned the nuclear Nac2 and Mbb1 genes by rescue of the mutants which are deficient in the accumulation of the mRNAs of psbD and the psbB/psbT/psbH chloroplast transcription unit.. Nac2 encodes a hydrophilic polypeptide of 1385 amino acids with 9 tetratricopeptide repeats (TPR) in its C‐terminal half whereas Mbb1 encodes a polypeptide of 662 amino acids which also contains 9 TPRs. Both proteins contain a putative chloroplast transit peptide at their N‐terminal end. They have been tagged with the HA epitope and localized in the stromal compartment of the chloroplast. Nac2 and Mbb1 are part of a high molecular weight complex of 500 and 250 kDa, respectively, which is associated with RNA in the case of Nac2. A change of a conserved Ala residue of the fourth TPR motif by site‐directed mutagenesis completely abrogates Nac2 function indicating that this TPR is important for psbD mRNA stability, processing and/or translation.