Methods for studying protein targeting to and within the chloroplast

Distinct protein complements impart each of the chloroplast's three membranes and three aqueous spaces with specific functions essential for plant growth and development. Chloroplasts capture light energy, synthesize macromolecular building blocks and specialized metabolites, and communicate environmental signals to the nucleus. Establishing and maintaining these processes requires approximately 3000 proteins derived from nuclear genes, constituting approximately 95% of the chloroplast proteome. These proteins are imported into chloroplasts from the cytosol, sorted to the correct subcompartment, and assembled into functioning complexes. In vitro import assays can reconstitute these processes in isolated chloroplasts. We describe methods for monitoring in vitro protein import using Pisum sativum chloroplasts and for protease protection, fractionation, and native protein electrophoresis that are commonly combined with the import assay. These techniques facilitate investigation of the import and sorting processes, of where a protein resides, and of how that protein functions.