Poster: Organelle Biogenesis

Abs # 1170: The role of Toc64 in chloroplast protein transport: a reverse genetic approach

Presenter:	Hofmann, Nancy R, nrhofmann@ucdavis.edu
Authors	Hofmann, Nancy R (A)   Theg, Steven M (A)
Affiliations:	(A): UC-Davis

Toc64 has been suggested to be a part of the chloroplast protein translocation machinery at the chloroplast envelope. This protein includes a short hydrophobic domain followed by an amidase-like region and three tetratricopeptide repeats. Tetratricopeptide repeats in other systems have been implicated in protein-protein interactions, leading Sohrt and Soll (2000. JCB 148: 1213-1221) to suggest that this region mediates precursor protein docking at the translocon of the chloroplast envelope. We have been taking a reverse genetic approach to understanding what role Toc64 plays in chloroplast protein targeting. We are analyzing Arabidopsis thaliana T-DNA insertion lines for each of Arabidopsis’ two Toc64s: AtTOC64-III and AtTOC64-V. Preliminary results show that an insertion into the second intron of AtTOC64-V is lethal. Further analysis of this and other lines is underway in our laboratory. In addition, we are using the moss, Physcomitrella patens as model system for studying these proteins. This moss undergoes homologous recombination in the nuclear genome, allowing targeted gene knockout. We have cloned two genes from moss that encode proteins similar to Toc64. We have shown that one of these, PpToc64-1, is targeted to the chloroplast in an in vitro targeting assay and that this targeting is mediated by the N-terminal hydrophobic domain. We are in the process of knocking out these two genes in moss, to determine whether either plays a role in chloroplast biogenesis.