Chapter 15 In Vitro Reconstitution of Protein Transport into Chloroplasts

This chapter discusses various steps required for the in vitro reconstitution of protein transport into chloroplasts, the first being the production of precursor proteins. This is normally accomplished with an in vitro translation system, but other methods are also possible. The second step is isolation and purification of intact chloroplasts. Then, the precursor proteins and chloroplasts are incubated together under conditions that will allow transport to occur. The transport reaction is terminated by separating the chloroplasts, containing imported proteins, from the reaction mixture that contains residual precursors. It is possible to fractionate the recovered chloroplasts to determine which chloroplastic compartment contains the imported protein. Finally, the extent of transport is determined by measuring the amount of mature protein accumulated inside the chloroplasts. Transport has been reconstituted with many different precursors and with chloroplasts from several different species. The procedures that are used to study the transport of precursors into pea chloroplasts are discussed in the chapter. The products are then analyzed by electrophoretic separation of products and quantitative analysis of import products.