Reconstitution of Co-Translational Targeting of Polytopic Membrane Proteins to the Thylakoids in a Homologous Chloroplast Translation System

Abstract

Chloroplasts in higher plants posses circular DNA containing 35 genes encoding for essential proteins located in the thylakoid membranes. The mechanisms of targeting and insertion of these proteins are unknown, despite their importance. Based on a number of observations in chloroplasts, it can be postulated that targeting and insertion of the polytopic chloroplast-encoded membrane proteins occurs co-translationally (1–3). Thus in order to reconstitute this targeting and insertion process, a homologous chloroplast in vitro initiation/translation system is required in which plasmid derived transcripts can be faithfully translated. The recent discovery of a translation system isolated from tobacco chloroplasts has opened up novel possibilities to address these important processes at a molecular level (4). In this paper, we have set out to evaluate the interaction of soluble stromal components cpSRP54 (5), cpSRP43 (6) and SecA (7) with the chloroplast encoded D1 protein, using this translation system. We show that ribosome D1 nascent chain complexes (D1 rncs) can be targeted very efficiently to the membrane and make functional interactions. In addition we show that cpRP54 interacts specifically with D1 rncs of defined length, implying a role for cpSRP54 in D1 biogenesis. To study the extent of conservation and mechanisms of the chloroplast targeting mechanisms with prokaryotes, we translated E.coli Leader peptidase (Lep) in the chloroplast system and attempted targeting Lep to the thylakoid membrane. Lep has two membrane spans and has served as a model protein to study targeting and insertion to the E. coli inner membrane (8–10) as well as to ER membranes (e.g.11). Lep is made without a cleavable signal sequence and its N- and C-terminal domains face the periplasmic side of the inner membrane (Fig. 4). In E.coli, insertion of Lep is SecA, SecY and SRP dependent (8–10).