Poster: Protein Targeting & Vesicular Trafficking

Abs # 1183: Mapping the Topology of the Pea Chloroplastic Protein Import Channel Toc75

Presenter:	Froehlich, John E., froehli5@msu.edu
Authors	Froehlich, John E. (A)   Reumann, Sigrun  (C) (D)  Ray, W. Keith  (B)   Phinney, Brett S (B)   Wilkerson, Curtis G (A)   Keegstra, Kenneth  (A)
Affiliations:	(A): MSU-DOE Plant Research Lab (B): Michigan State University Proteomics Facility (C): Department of Plant Biochemistry (D): University of Goettingen

The majority of proteins destined for the chloroplasts are nuclear encoded, synthesized in the cytosol and post-translationally imported into the organelle by a translocation complex located in the envelope membranes. Toc75, the most abundant protein in the outer envelope membrane, is postulated to be the channel whereby precursors are transported across the outer membrane. In order to gain further insight into the structure and function of this component, we are investigating the topology of Toc75 using two different approaches. In the first, intact pea chloroplasts were used to examine the sensitivity of endogenous Toc75 to digestion with various proteases. Proteolytic fragments of Toc75 derived from these various protease treatments were resolved by SDS-PAGE and analyzed by western blotting to identify regions of Toc75 that are exposed to the cytosol. To complement the first approach, our second strategy involved incubating intact pea chloroplasts with membrane impermeable nonradioactive labeling reagents to tag surface exposed domains of Toc75. Labeled intact chloroplasts were recovered, lysed and a total membrane fraction was isolated. Membrane fractions containing tagged Toc75 were resolved by SDS-gel electrophoresis and a gel slice containing tagged Toc75 was isolated and subjected to an in-gel digestion with the protease, Trypsin. After protease digestion, tagged peptide fragments were extracted and analyzed by LC/MS/MS to identify regions of Toc75 that were orientated towards the cytosol. Mapping the topology of Toc75 by these two different strategies should allow us to identify potential domains that may be involved in interacting with precursor proteins during the translocation of protein import into chloroplasts.