Analysis of protein translocation across the chloroplast inner envelope membrane

概要

Chloroplasts are unique sub-cellular organelles that perform specific functions in photosynthesis and development. They are conserved in almost all plant lineages, arising from an ancient endosymbiotic event approximately one billion years ago. Most chloroplast proteins are nuclear-encoded and synthesized in the cytosol, before being transported into the organelle. Protein translocons located at the outer and inner envelope membranes of chloroplast, termed TOC and TIC, respectively, are vital for translocation of chloroplast proteins.

Previous studies have examined the main components of two translocons in Arabidopsis thaliana. TOC consists of Toc159/Toc34/Toc75, while TIC is composed of Tic214(Ycf1)/Tic100/Tic56/Tic20-I. TOC and TIC are highly conserved in green lineages. While TOC components have been extensively characterized, the components of TIC which are directly involved in preprotein translocation still require further investigation.

Previous studies from our laboratory identified an as-yet-characterized small protein was found associated with preprotein translocation intermediates in Arabidopsis thaliana as well as in Chlamydomonas reinhardtii. This study aims to investigate whether this small protein exists within the TIC complex and plays an essential role in protein translocation across the inner envelope membrane.

This tiny protein, tentatively named Tic9k, consists of 98 amino acids and is encoded by At3g09860 of the nuclear genome in Arabidopsis. This protein is highly conserved in green linages including plants and green algae. A null mutant of tic9k and a double knock-out mutant of tic9k tic20-I exhibited very similar albino seedling lethal phenotype to that of the tic20-I mutant. This result suggests that Tic9k functions similarly to Tic20-I during chloroplast protein import. Actually, by pull-down assay, Tic9k was found associated with Tic20-I-containing TIC complex. Trypsin protease treatment of chloroplasts further provided an evidence supporting its localization at the inner envelope membrane. Furthermore, two-dimensional BN/SDS-PAGE analysis showed that Tic9k was detected together with other TIC complex components, around 1-MDa size. Tic9k was copurified with various translocation intermediates, and the UV crosslinking experiments indicated that this protein directly interacted with a transit peptide of preprotein at the early stages of protein translocation across the inner envelope membrane.