Topology studies of the chloroplast protein import channel Toc75

A major goal in understanding protein transport across membranes is the inv estigation of the structure and regulation of the translocon subunits. We a nalysed Toc75, a pore-forming subunit of the translocon of the outer envelo pe of chloroplasts. Toc75 was overexpressed and reconstituted into liposome s. Immunoprecipitation of liposome-reconstituted Toc75 indicates an N-in-C- in orientation of Toc75. Limited proteolytic digestion of Toc75 present in outer envelope vesicles with specific proteases combined with amino acid se quencing was used to study the topology of Toc75. Finally, computer modelli ng based on known protein structures indicates that Toc75 traverses the out er envelope with 16 amphiphilic beta sheets and the topology model is prese nted.