Mitochondrial ribosome assembly in Neurospora. Preparation of mitochondrial ribosomal precursor particles, site of synthesis of mitochondrial ribosomal proteins and studies on the poky mutant

Abstract

Mitochondrial ribosomal RNAs in Neurospora are transcribed in tandem as a32 S precursor molecule which is subsequently cleaved and trimmed to yieldthe mature 19 S and 25 S RNA species ( Kuriyama & Luck,1973 a ). As part of a study of mitochondrial ribosome assembly and the poky (mi-1) mutant, procedures were developed for isolating mitochondrial ribosomal precursor particles which consist of 32 S RNA and 15 or more newly synthesized large and small subunit protiens.The precursor particles comprise less than 5% of the total mitochondrial ribonucleprotein and were initially identified in pulse-labeling experiments by cosedimentation (approx. 30 S)of peaks containing newly synthesized RNA and protein.Although not yet completely free of mature ribosomal subunits, the precursor particles could be studied in pulse and pulse-chase experiments combined with electrophoretic analysis of RNA and protein. Such experiments provided strong evidence that the precursor particles are formed in vivo by specific association of the RNA and protein species. The pulse-labeling experiments led to additional information about mitochondrial ribosome processing. Several small subunit proteins were identified which have exceptionally small free pools (as judged by high specific activity of pulse-label in mature small subunits), and which may thus control the rate of mitochondrial ribosome assembly. Experiments using inhibitors of protein synthesis (anisomycin and chloramphenicol) showed that one of these protiens, S-4a (apparent M r =52,000), is synthesized inside the mitochondria, whereas all other mitochondrial ribosomal proteins are synthesized in the cytosol. Finally, studies on poky , a non-Mendelian mutant with defective mitochondrial ribosome assembly( Rifkin & Luck, 1971 ) suggested that poky small subunits are deficient in several proteins, possibly including S-4a. These several lines of evidence are discussed in light of the possiblity that S-4a is the site of the primary defect in poky .