Minisymposium 18: Protein Targeting

Abs # 32001: Preprotein binding by the Toc159 receptor, and requirements for targeting the receptor to chloroplasts

Presenter:	Smith, Matthew D, msmith@biochem.umass.edu
Authors	Smith, Matthew D (A)   Rounds, Caleb M (A)   Wallas, Tanya R (A)   Kessler, Felix  (B)   Schnell, Danny J (A)
Affiliations:	(A): Department of Biochemistry and Molecular Biology, University of Massachusetts, Amherst (B): Institut de Botanique, Laboratoire de Physiologie Vegetale, Universite de Neuchatel

The biogenesis and maintenance of chloroplasts requires the contribution of approximately 2000 proteins that are encoded by nuclear genes and imported into the organelle after being translated in the cytoplasm. Protein import into chloroplasts is mediated by the coordinate action of multimeric translocon complexes located in the outer (Toc complex) and inner (Tic complex) envelope membranes. The Toc complex of Arabidopsis is composed of three core components named atToc33, atToc75 and atToc159, and is responsible for initiating the import of nuclear-encoded preproteins. atToc33 and atToc159 are homologous GTPases whose GTPase activities are required for preprotein import into chloroplasts. atToc159 is equally distributed between a soluble cytoplasmic form and a chloroplast membrane-bound form. The membrane-bound form of atToc159 interacts with atToc33 and atToc75 to form a functional translocon, of which atToc75 comprises at least part of the preprotein channel through the outer membrane. Our investigation focuses on the hypothesis that atToc33 and the soluble cytoplasmic form of atToc159 function together to form a receptor system that is required for preprotein targeting and initiation of preprotein translocation into chloroplasts. Our presentation will focus on defining the mechanism of targeting the cytoplasmic form of atToc159 to chloroplasts and the requirements for preprotein binding by the soluble form of the receptor. We have demonstrated that its GTPase domain, through an interaction with the homologous GTPase domain of atToc33, mediates targeting of soluble atToc159 to the chloroplast surface. The subsequent productive integration of atToc159 into the Toc complex requires its intrinsic GTPase activity and is also influenced by the nucleotide state of atToc33. We will present data showing that soluble atToc159 specifically binds to preproteins through an interaction with their transit peptides. In addition, we will present our progress on defining the role of GTP in preprotein binding and on identifying the transit peptide binding site on atToc159. Collectively, our data are consistent with the hypothesis that soluble cytoplasmic atToc159 directly binds preproteins and that atToc159 and atToc33 together comprise a GTP-regulated receptor system that facilitates preprotein targeting and the initiation of preprotein translocation into chloroplasts.