PARENT SESSION

Symposium S3D Genomic, proteomic and related technologies
Tuesday August 31st, 2004 10:20 AM-12:20 PM Room 510B
Chair: Arthur Grossman
Co-Chair: Jacques Joyard

The thylakoid and stromal proteome of chloroplasts of a. thaliana; Expression profiling, protein interactions, properties and functions. Jean-Benoit Peltier¹, Jimmy Ytterberg¹, Giulia Friso¹, Yang Cai¹, Lisa Giacomelli¹, Andrea Rudella¹, Qi Sun, Klaas van Wijk^*,1, ¹ Department of Pant Biology, Emerson Hall 332, Ithaca, NY, USA

ABSTRACT- Chloroplasts are predicted to contain several thousands of proteins, forming an interacting network of proteins. A comprehensive identification and quantification of these proteins and their interactions is critical in understanding chloroplast function. This study provides a semi-quantitative overview of stromal, as well as thylakoid-associated proteins and protein complexes in Arabidopsis thaliana chloroplasts and their relative distributions. Thylakoid-associated and stromal proteins were extracted from purified intact chloroplasts under native conditions from Arabidopsis thaliana leaves. The two soluble proteomes were then separated by native separation techniques, followed by SDS-PAGE. Protein spots were analyzed by mass spectrometry. Much emphasis was placed on distinguishing between different members of gene families, since different paralogues are often expressed at subcellular localizations, playing a critical role in regulation of metabolic flux and development. Proteins were assigned to molecular functions and metabolic pathways based on functional domain prediction, information from metabolic databases (KEGG and Aracyc), as well as published literature. Identification and expression profiling of membrane proteomes remains challenging. To facilitate an in-depth analysis of the thylakoid membrane proteome of Arabidopsis chloroplasts we developed a fast and scalable procedure, based on three-phase partitioning with butanol (TPP), followed by either in-gel and in-solution digestion and Tandem Mass Spectrometry. About identified 85 proteins had not been observed experimentally in thylakoids. Our data were cross-correlated to our previous published analyses of the thylakoid proteome, as well as chloroplast membrane proteome analyses by other laboratories. A bioinformatics infrastructure was created to visualize the native protein gel images and associated protein and mass spectrometry information - these and other plastid proteome data will become publicly available via the Plastid Proteome Database, PPDB (http://pdb.tc.cornell.edu/).

KEY WORDS: protein-protein interactions, thylakoid membrane proteome, plastid proteome, Arabidopsis thaliana