Puromycin sensitivity of ribosomal label after incorporation of tiC-labelled amino acids into isolated mitochondria from Neurospora ¢rassa. Eur. 3

Radioactive amino acids were incorporated into isolated mitochondria from Neurospora crassa. Then the mitochondrial ribosomes were isolated and submitted to density gradient centrifugation. A preferential labelling of polysomes was observed. However, when the mito-chondrial suspension was treated with puromycin after amino acid incorporation, no radio-activity could be detected in either the monosomes or the polysomes. The conclusion is drawn that isolated mitochondria under these conditions do not incorporate significant amounts of amino acids into proteins of their ribosomes. Mitochondrial ribosomes from Neurospora crassa have been isolated and characterized by Kuntzel and No11 [I] and by Rifkin et al. [2]. Radioactivity has been found in these ribosomes after labelling of isolated mitochondria by incorporation of [l4CC]-leucine [l]. However, no distinction was made be-tween labelling of the proteins of the ribosomes and the peptide chains being actually synthesized. Puro-mycin, which is known to release the growing peptide chain from the ribosome, was used to examine this question. MATERIALS AND METHODS Hyphae of Neurospora crassa (wild type 74A) were grown for 18 h on a reciprocal shaker from an inoculum of 2 x 106 conidia/ml in 50 ml portions of Vogel's minimal medium supplemented with 2 O l i o sucrose. They were harvested by filtration. Mitochondria were prepared by a slight modi-fication of the method described by Luck [3]. Cells were mixed with 3 volumes (with respect to wet weight) of isolation fluid (0.44 M sucrose, 10 mM Tris, 2 mM EDTA, pH 7.2) and ground with sand (0.7 g per g of wet weight) for 8 min. The suspension, diluted with 6 additional volumes of isolation fluid, was passed through a filter cloth. The residue was ground again under the same conditions for 5min. For isolation of mitochondria the combined filtrates were successively centrifuged in the following way