A CONSTITUENT OF THE CHLOROPLAST IMPORT COMPLEX REPRESENTS A NEW-TYPEOF GTP-BINDING PROTEIN

The 34 kDa polypeptide of the outer envelope membranes from pea chloro plasts (OEP 34) is a major constituent of this membrane. OEP 34 is det ected on polyacrylamide gels under non-reducing condition in associati on with OEP 75, the putative protein translocation pore. An antiserum against OEP 34 is able to co-immunoprecipitate the precursor of Rubisc o small subunit from a partially purified import complex of chloroplas t outer envelope membranes. A full-length cDNA clone coding for pea OE P 34 has been isolated. Analysis of the deduced amino acid sequence re vealed typical and conserved sequence motifs found in GTP-binding prot eins, making it a new and unique member of this superfamily. OEP 34 be haves as an integral constituent of the outer chloroplast envelope, wh ich is anchored by its C-terminus into the membrane, while the majorit y of the protein projects into the cytoplasm. OEP 34 does not possess a cleavable N-terminal transit sequence but it is targeted to the chlo roplasts and integrated into the outer membranes by internal sequence information which seems to be present in the C-terminal membrane ancho r region. Productive integration of OEP 34 into the outer envelope req uires, in contrast to other OEPs, protease-sensitive chloroplast surfa ce components and is stimulated by ATP. The GTP binding specificity of OEP 34 is demonstrated by photo-affinity labelling in the presence of [alpha-P-32]GTP. Overexpressed and purified OEP 34 possesses endogeno us GTPase activity. These results indicate a possible regulatory funct ion of OEP 34 in protein translocation into chloroplasts.