Native and Imported tRNAs in Tetrahymena Mitochondria: Evidence for Their Involvement in Intramitochondrial Translation

Abstract

Tetrahymena mitochondria contain many tRNAs and isoacceptors, few of which are native tRNAs; i.e., they are coded by mtDNA (Suyama 1967; Chiu et al. 1974, 1975; Suyama and Hamada 1976). Most mitochondrial tRNAs are able to hybridize to nuclear DNA, and these so-called imported tRNAs are considered to be selected species of cytoplasmic tRNA. In the present studies, Tetrahymena mitochondrial tRNAs are resolved into 36 species by two-dimensional polyacrylamide gel electrophoresis (PAGE), and some of these are identified as native and imported tRNAs. In addition, analyses of tRNAs associated with mitochondrial ribosomes establish that these mitochondrial tRNAs, whether native or imported, are engaged in intramitochondrial translation.

RESULTS
Two-dimensional PAGE
Two-dimensional gel analysis of tRNA was carried out using a modification of the procedure of Fradin et al. (1975). Tetrahymena mitochondrial tRNA developed 36 tRNA spots (Fig. 1A), whereas cytoplasmic tRNA produced over 45 major and minor spots (Fig. 1B). Mitochondrial and cytoplasmic tRNAs do not show any appreciable difference in two-dimensional gel migration rates. To compare these tRNAs further, mitochondrial tRNA from cells labeled in vivo with ³²P was coelectrophoresed with cold cytoplasmic tRNA. A comparison between the autoradiograph and the methylene-blue-stained gel spots revealed that many tRNA spots superimpose one another. Some differences in tRNA spots are apparent: (1) Major cytoplasmic species do not always appear as major species in mitochondrial tRNA spots and (2) several radioactive spots of mitochondrial tRNA are distinctly different from cytoplasmic tRNA spots.

The two-dimensional gels obtained with various mitochondrial tRNA isolates from young...