plant growth, organelle isolation, in vitro import, protein localization
Chloroplasts are plant-specific organelles that perform many essential metabolic functions including oxygenic photosynthesis and biosynthesis of amino acids, lipids, nucleotides, hormones, pigments and vitamins. As a consequence of their endosymbiotic origin, most of their genes have been transferred into the host nucleus in the course of evolution. Thus, today the vast majority of the chloroplast proteome is synthesized on cytosolic ribosomes and subsequently transported to and imported into chloroplasts. Even though proteomic approaches have proved to be powerful in describing the content of organelles, the localization of proteins has to be confirmed on the case basis. For this purpose the in vitro protein translocation into chloroplasts is very important tool. Here we describe a protocol for chloroplast isolation with focus on the model plant Pisum sativum, in vitro protein translocation into chloroplasts as well as the subsequent analysis of the protein localization within the organelle for bench side use.