Mitochondrial-specific aminoacyl-RNA synthetases.

Abstract

Numerous lines of evidencel-'4 indicate that protein synthesis occurs within mitochondria, and tUNA8 1 16 and aminoacyl-RNA synthetases8 have been prepared from these organelles. More recently, two very interesting aspects of mitochondrial protein synthesis have been described: (a) Although amino acid incorporation by yeast cytoplasmic ribosomal systems is not inhibited by chloramphenicol,'7 protein synthesis by yeast mitochondria in vivo and in vitro is sensitive8' 18 to this antibiotic. (b) MVlitochondrial protein synthesis in vitro, and therefor presumably in vivo, is apparently restricted to the assembly of a very limited number of proteins; that is, the amino acids are incorporated preferentially into mitochondrial structural protein(s) 11, 1?, 19-21 Thus, mitochondrial protein synthesis differs from cytoplasmic protein synthesis in response to specific inhibitors and in the spectrum of proteins synthesized. In this report, evidence will be presented that the translational macromolecules, i.e., tRNA's and aminoacyl-RNA synthetases, found in Neurospora mitochondria are different from those isolated from the cytoplasm. In earlier reports,, 16 we demonstrated that the mitoclhondria of Neurospora contain a full complement of tRNA's and that the two whichi were examined in detail (aspartic acid and phenylalanine tRNA) were distinct from their cytoplasmic counterparts in acylation specificities, The experiments reported here indicate that Neurospora mitochondria contain a full complement of aminoacyl-RNA synthetases and that at least three of these, the aspartyl-, phenylalanyl-, and leucyl-RNA synthetases, together with their respective tRNA's, are exclusively associated with the mitochondria. Experimental Procedures.-Strairas: Neurospora crassa wild-type strain OR23-la was used. Preparation of mitochondrial and cytoplasmic fractions: Hyphae in the exponential phase of growth were collected from enriched Vogel's22 synthetic medium. Mitochondria were prepared as described previously16 by the methods of Hall and Greenawalt23 followed by zonal sucrose-gradient centrifugation. After removal of the nuclei and debris from the disrupted hyphae by low-speed centrifugation (1500 X g), the mitochondria were pelleted by centrifugation at 8000 X g. The supernatant from the latter centrifugation was recentrifuged at 30,000 X g for 30 mill to remove residual mitochondria; the restilting supernatant is considered as the cytoplasmic fractioni. The mitochondria were resuspended and reisolated by zonal ceiltrifugation prior to use. Preparation of tRNA: tRNA was prepared from the cytoplasmic and mitochondrial fractions as described previously'6 (essentially by the method of Holley et al.24). Preparation of enzymes: (a) Cytoplasmic: The cytoplasmic fraction was centrifuged at 78,000 X g for 2 hr and the supernatant adjusted to 0.2 M KPO4 (p11 7.5) and 0.01 M 3-mercaptoethanol; the material was then passed over a D)EAE-cellulose column (equilibrated with the same buffer) to remove nucleic acids,25 and the protein precipitated by the addition of (NH4)2SO4 crystals to a final concentration of 3.0 M. After standing overnight at -3?C, the precipitate was collected by centrifugation at 10,000 X g for 30 min. The ammonium sulfate precipitates may be stored at 0?C for at least several months without loss of enzyme activity. Ammonium sulfate and phosphate were removed prior to use by passage through Sephadex G-25 which had been equilibrated with the appropriate buffer. For use as an unfractionated synthetase preparationi, the equilibrating buffer consisted of 0.01 M Tris-HCl buffer (pH 7.5), 0.1 M KCl, 0.01 M ,-mercaptoethanol, and 20% glycerol, in which aliquots may be frozen and stored at -20?C without loss of activity.