The CFoII subunit of the ATP synthase is an integral component of the thylakoid membrane which is synthesized in the cytosol with a bipartite, lumen‐targeting presequence similar in structural terms to those of imported lumenal proteins such as plastocyanin. This presequence is shown to possess a terminal cleavage site for the thylakoidal processing peptidase, but no intermediate site for the stromal processing peptidase. The integration of CFoII into the thylakoid membrane of Pisum sativum has been analysed using in vitro assays for the import of proteins into intact chloroplasts or isolated thylakoids. Efficient integration into thylakoids is observed in the light and dark, and the integration process does not require the presence of either stromal extracts or nucleoside triphosphates. The uncoupler nigericin inhibits integration only very slightly, indicating that the thylakoidal delta pH does not play a significant role in the integration mechanism. In each of these respects, the requirements for CFoII integration differ notably from those determined for integration of the light‐harvesting chlorophyll‐binding protein of photosystem II. The integration mechanism also differs significantly from the two mechanisms involved in the translocation of lumenal proteins across the thylakoid membrane, since one of these processes requires the presence of stromal protein factors and ATP, and the other mechanism is dependent on the thylakoidal delta pH. This conclusion is reinforced by the finding that saturation of the translocation system for the precursor to the lumenal 23 kDa oxygen‐evolving complex protein does not affect integration of CFoII into thylakoids.(ABSTRACT TRUNCATED AT 250 WORDS)

The 33 kd protein of the photosynthetic oxygen‐evolving complex is synthesized in the cytoplasm as a larger precursor and transported into the thylakoid lumen via a stromal intermediate form. In this report we describe a reconstituted system in which the later stages of this import pathway can be studied in isolation. We demonstrate import of the 33 kd protein, probably as the intermediate form, into isolated pea thylakoids by a mechanism which is stimulated by the addition of ATP. The imported protein is processed to the mature size and is resistant to digestion by proteases. The thylakoidal protein transport system is specific in that non‐chloroplast proteins and precursors of stromal proteins are not imported.

In vitro reconstitution assays have been used in recent years to probe the mechanisms by which a variety of cytosolically synthesised proteins are transported across the thylakoid membrane within higher plant chloroplasts. The emerging data suggest that two distinct mechanisms operate. Translocation of a subset of lumenal proteins, namely the 23 kDa and 16 kDa proteins of the oxygen-evolving complex, and of the Cl?02 protein (an integral membrane protein), requires only the presence of the thylakoidal dpH. In contrast, two other lumenal proteins, the 33 kDa oxygenevolving complex protein and plastocyanin, require also the presence of ATP and at least one stromal factor for efficient transport into isolated thylakoids to take place.

A chimaeric gene was constructed which encodes the pre-sequence of the 33 kDa protein of the oxygen-evolving complex, a thylakoid lumen protein, linked to dihydrofolate reductase, a cytoplasmic protein. The encoded fusion protein is transported into the thylakoid lumen of isolated pea chloroplasts, with concomitant removal of the pre-sequence.

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