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In vitro chloroplast protein import assays have been performed since the late 1970s, initially with plant species (e.g., pea and spinach) that readily provide an abundant source of starting material and also, subsequently, a good yield of chloroplasts for import assays. However, the sequencing of the Arabidopsis genome paved the way for an additional model system that is more amenable to genetic analysis, as a complement to the more biochemically orientated models such as pea and spinach. A prerequisite for this change was an efficient and reliable protocol for the isolation of chloroplasts for use in protein import assays, enabling biochemical approaches to be combined with the genetic potential of the plant. The method described here was developed as a rapid and low-cost procedure that can be accessed by everyone due to its simplicity. Despite its rapidity and simplicity, the method yields highly pure chloroplasts, and in addition works well with mutant plants that exhibit pale or chlorotic phenotypes. The protocol is also optimized for work with material from young plants (10–14 days old), when protein import is believed to be at its peak, and so plant growth can be conducted in vitro on Murashige and Skoog medium. The isolation method has been used not only for protein import assays, but also for proteomic analysis and further subfractionation studies.
