Puromycin Sensitivity of Ribosomal Label after Incorporation of 14C-Labelled Amino Acids into Isolated Mitochondria from Neurospora crassa

Abstract

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 mitochondrial suspension was treated with puromycin after amino acid incorporation, no radioactivity 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 between labelling of the proteins of the ribosomes and the peptide chains being actually synthesized. Puromycin, which is known to release the growing peptide chain from the ribosome, was used to examine this question. MATERIALS AND METHODSHyphae 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 Olio sucrose. They were harvested by filtration.Mitochondria were prepared by a slight modification 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, p H 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 : 10 rnin at 1500 xg, the resulting supernatant 30 rnin a t 11 000 x g, the resuspended pellet 10 min a t 550 x g, the resulting supernatant 20 min a t 17000 xg, and the resulting supernatant again 20 rnin a t 17000 x g.Unusual Abbreviation. ATeo unit, the quantity of material contained in 1 ml of a solution which has an absorbance of 1 at 260 nm when measured in a 1 cm path length cell. One portion served as a control. It was immediately cooled to 0". To the other portion puromycin hydrochloride (Nutritional Biochemicals) was added to a final concentration of 0.4 mM. The incubation was continued for additional 7 min. This part of the suspension was then also cooled to 0". From this point on control and puromycin treated mitochondria were handled in the same way. 3 volumes of isolation fluid containing 5 mM each of unlabelled L-leucine, L-isoleucine and L-phenylalanine were added. The mitochondria were centrifuged for 20 rnin a t I7 000 x g.The pellet was resuspended in isolation fluid containing amino acids. The suspension was centrifuged again for 20 min a t 17 000 x g.For further purification mitochondria were resuspended in isolation fl...

The ribosomal RNA genes on Neurospora crassa mitochrondrial DNA are adjacent

“…Taking into account the distance between the two genes, we estimate a precursor length of at least 6700 nucleotides. This value is in good agreement with the length estimate of 6900 nucleotides for the ribosomal precursor RNA [6], which was calibrated against the same molecular weights for the rRNAs [16] as used in our calculations.…”

“…Cytoplasmic rRNA does not hybridize with mtDNA [2,14]. To calculate the size of the rRNA genes, we have taken the molecular weight estimates for the 24-S and 17-S rRNAs given by Neupert et al [16] Hybridization of 24 S rRNA to a stripfilter containing Bam HI and Eco RI restriction fragments of mtDN:A, reveals that only fragment B1 and E1 are labelled ( Fig. 2A and 2B resp.).…”

The 73 S ribosome of Neurospora crassa is the native mitochondrial ribosome

“…The resulting ribosomal pellet was resuspended in 0.1 M NH4C1, 0.01 M MgCl~, 0.01 M Tris • HC1, (pH 7.5), and the suspension layered onto a sucrose gradient. Gradient density centrifugation was carried out as described previously [10] and the gradients were monitored with a Zeiss PMQ II photometer, using a flow cell (50 pl, 1 cm path-length).…”

Mitochondrial Translation Products before and after Integration into the Mitochondrial Membrane in Neurospora crassa

“…J. Biochem. taining 0.1 M NH,CI, 0.01 M MgCl, and 0.01 M TrisHCl pH 7.5 and lo/o Triton X-100 as described [14], with the modification that the lysate was placed on a layer of 1.5 ml 1. 4 M sucrose in buffer A and centrifuged for 2 h at 144000 x g.…”

“…Sucrose-density-gradient centrifugation of mitochondrial and cytoplasmic ribosomes was carried out as described previously [14]. Gradients were monitored with a Zeiss PMQ I1 photometer, using a flow cell (50-pl volume, I-cm path-length).…”