Differences between mitochondrial and cytoplasmic transfer RNA and aminoacyl transfer RNA synthetases from rat liver.

The mitochondria of yeast,1 Neurospora,2 and Tetrahymena3 have been shown to contain transfer RNA (tRNA). Barnett et al.4 have found, in addition, that Neurospora mitochondria contain 15 aminoacyl-tRNA synthetases, at least three of which acylate only mitochondrial tRNA. The purpose of this communication is twofold. First we will present evidence showing that, as with eukaryotic microbial systems, the mitochondria of mammalian cells contain species of tRNA which are distinct from those of the cytoplasm. Second, we will show that cytoplasmic leucyland tyrosyl-tRNA synthetases cannot acylate tRNA's which are exclusively mitochondrial. These observations add support to the suggestion that the protein-synthesizing machinery of the mitochondrion may be of different genetic origin than that of the cytoplasm. Materials and Methos.-Isolation of mitochondria: Rat liver mitochondria were prepared as described previously,5 with the following modifications to assure maximal sedimentation of nuclear and microsomal fragments. Homogenization was performed in a filter-sterilized solution of 0.3 M sucrose, 0.002 M ethylenediaminetetraacetate (EDTA), 0.025 M tris(hydroxymethyl)aminomethane (tris)-HCl buffer pH 7.3 (0.3M SET), which contained 0.006 M mercaptoethanol when mitochondria were prepared as a source of synthetases (0.3 M SET-SH). Two successive low-speed centrifugations at 700 X g were performed. The twice-washed mitochondrial pellets (8,000 X g) were sedimented successively on two linear gradients of filter-sterilized sucrose solutions (1.03 M-1.91 M SET or SET-SH) at 22,000 rpm (Spinco rotor SW 25.1) for 90 min each. Mitochondria were collected strictly from the center of the main band; this sacrificed a less dense fraction of mitochondria that possibly contained microsomal fragments. The final mitochondrial pellets were suspended and resedimented either in 0.3 M sucrose, 0.001 M MgCl2, 0.025 M tris-HCl pH 7.4 (for extraction of RNA) or in 0.01 M KCl, 0.01 M MgCl2, 0.1 11 tris-HCl 7.8, 0.006 M mercaptoethanol (for extraction of synthetases). The yield of mitochondrial protein from the livers of ten rats was 200-300 mg. Bacterial counts: Aliquots of the final mitochondrial preparations were plated on Difco brain-heart infusion agar (for counts of viable bacteria) and added in suspension to Difco antibiotic medium to grow bacteria for subsequent isolation. Colony counts after 24-48 hr revealed 3-50 viable bacteria/mg mitochondrial protein. Preparation of tRNA: Cytoplasmic tRNA was prepared by phenol extraction of rat liver homogenates from which the mitochondria had been removed by centrifugation. RNA was precipitated by the addition of 2 vol of 95% ethanol. The precipitates were dissolved in 0.02 M tris-HCl buffer pH 7.5 containing 0.15M NaCl (TBS). The solution was made 1 M with respect to NaCl and ribosomal RNA precipitated overnight at 4VC. The tRNA was precipitated from the supernate with ethanol and redissolved in TBS. Following 5 cycles of precipitation and re-solution, the tRNA was stored at a concentration of 1 mg/ml at -80'C. Mitochondria were suspended in TBS containing 1% sodium dodecyl sulfate, and the mitochondrial nucleic acids were extracted with phenol until no protein was found at the phenol-TBS interphase. RNA and DNA were precipitated with ethanol, as described above, and redissolved in TB3S. MgCI, was added to the final RNA