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.

Homologous and heterologous reconstitution of Golgi to chloroplast transport and protein import into the complex chloroplasts of Euglena

“…The pre-sequence region C-terminal to the hydrophobic membrane anchor that remains on the cytoplasmic membrane face of the transport vesicle was not required for import into pea chloroplasts suggesting it contains the Golgi to chloroplast targeting information. In contrast to previous reports (Reinbothe et al, 1990;Shashidhara et al, 1992), Euglena pSSU and deletion constructs were not imported into isolated Euglena chloroplasts indicating vesicular transport is the obligate import mechanism. 1996).…”

In Situ Conversion of Protochlorophyllideb to Protochlorophyllide a in Barley

“…Preparation and Solubilization of Prolamellar Bodies-Etioplasts were prepared from etiolated barley plants by Percoll density gradient centrifugation as described previously (35,36). For low temperature analyses at 77 K (see below) and pigment measurements (see above), the etioplast suspension was directly used.…”

A plastid enzyme arrested in the step of precursor translocation in vivo.

“…Plastid Isolation. Plastids were isolated from surfacesterilized leaves by differential centrifugation, followed by Percoll (Pharmacia LKB) density gradient centrifugation (19). After a final purification step on Percoll cushions (20), the plastids were diluted to a final concentration of 50 ,g chlorophyll per ml (21) with the import buffer described by della-Cioppa et al (22) (22).…”

“…If required, doubledistilled water was added to adjust the final reaction volume. After a 15-min incubation, the plastids were sedimented by centrifugation (19) and treated with thermolysin (26), but in those cases indicated in the text.…”