Protease-sensitive thylakoidal import machinery for the Sec-, ΔpH- and signal recognition particle-dependent protein targeting pathways, but not for CFoII integration

Nuclear-encoded proteins are targeted into and across the thylakoid membrane by a surprising variety of mechanisms. Distinct Sec- and ΔpH-dependent mechanisms have been shown to operate for lumenal proteins, and an integral membrane protein, LHCP, has been shown to insert via a signal recognition particle-dependent route. Integration of a further membrane protein, CF_oII, requires neither soluble factors nor energy sources, prompting speculation of a spontaneous insertion mechanism. Although the requirements for soluble factors and energy sources have been determined in some detail, much less is known of the events taking place at the membrane surface. This report examines whether thylakoid proteins are involved in each of these pathways, by testing the effects of trypsin on the capacity of isolated thylakoids to import proteins. Because all of the pathways rely to some extent on the thylakoidal ΔpH, and a light-induced ΔpH is easily destroyed by proteolysis, the conditions under which reverse action of the ATP synthase in the dark generates a high ΔpH even after proteolysis of thylakoids have been established. This system is used to show that protease-sensitive thylakoidal import machinery is crucial for the ΔpH-, Sec- and signal recognition particle-dependent pathways, but not for integration of CF_oII.