In-vitro transport of chloroplast proteins in a homologousEuglena system with particular reference to plastid leucyl-tRNA synthetase

Abstract

In-vitro translations of total or polyadenylated RNA from chemoorganotrophic and photoautotrophicEuglena gracilis showed no substantial differences in the polypeptide patterns of the two cell types. By contrast, the corresponding patterns of in-vivo labelling indicated that posttranscriptional control of abundant cellular proteins occurred in illuminated cells. This type of control was confirmed forEuglena chloroplast proteins of cytoplasmic origin. The posttranslational transport of in-vitro-formed polypeptides into homologous chloroplasts allowed the plastid-targeted proteins to be recognized. Estimations of the amounts of in-vitro-translated polypeptides showed that the mRNA levels for nuclearencoded chloroplast proteins were almost constant throughout chloroplast development inEuglena. The import of the in-vitro-translation products into the chloroplasts was demonstrated (i) by kinetic determination of radioactivity increasing inside and decreasing outside the organelles, (ii) by autoradiographic analysis of the transported translation products among two-dimensionally separated chloroplast proteins, and (iii) by autoradiographic estimation of the decrease in labelled polypeptides in the incubation medium. After 60 min of incubation in the presence of chloroplasts, about 20% of the in-vitro-labelled translation products, corresponding to 30 protein spots, were found to be redistributed into the organelles. Low-abundance chloroplast leucyl-tRNA synthetase (LeuRS) was detected immunologically among the in-vitro translation products as a precursor protein (pre-LeuRS) of 112 kilodaltons (kDa), which was transported into chloroplasts where it was present in the form of the 105-kDa mature enzyme. Processing of pre-LeuRS also seemed to occur using wheat-germ translation system; however, the mature enzyme was not sequestered intoEuglena chloroplasts. Our results indicate that a nuclear-encoded chloroplastic aminoacyl-tRNA synthetase, the product of a low-abundance mRNA, is transported into and processed in chloroplasts in vitro.