Inner envelope protein 32 is imported into chloroplasts by a novel pathway

The 32 kDa chloroplast inner envelope protein (IEP32) is imported into the organelle in the absence of a cleavable N-terminal pre-sequence. The ten N-terminal amino acids form an essential portion of this targeting information as deduced from deletion mutants. Recognition and translocation of IEP32 is not catalysed by the general chloroplast outer envelope translocon subunits Toc159, Toc75III and Toc34, because IEP32 import is neither inhibited by proteolytic removal of Toc34 and Toc159 nor by inhibition of the Toc75 import channel by CuCl2 or spermine. Import of IEP32 only requires ATP concentrations of below 20 muM indicating that stromal chaperones are not involved in the process, but that IEP32 might be directly inserted from the intermembrane space into the inner envelope by a so far unidentified pathway. IEP32 may require the assistance of Tic22, an intermembrane space translocon subunit for import as indicated by the presence of a chemical crosslinked product between both polypeptides.