The translocation of plastocyanin across the thylakoid membrane in Pisum sativum has been studied in reconstitution assays and using chimeric constructs. The reconstitution assays demonstrate that plastocyanin translocation is absolutely dependent on the presence of a stromal factor(s) and nucleotide triphosphates (NTPs), whereas neither element is required for the translocation of the 23 or 16 kDa proteins of the oxygen-evolving complex. Previous studies had revealed that the transthylakoidal delta pH is essential for translocation of the 23 and 16 kDa proteins but unnecessary for plastocyanin translocation. The basis for these mechanistic differences has been tested by analysing the translocation of a chimeric construct consistng of the presequence of the 23 kDa protein linked to the mature plastocyanin sequence. This construct is efficiently imported into thylakoids in the absence of stromal extracts or NTPs and translocation across the thylakoid membrane within intact chloroplasts is totally inhibited by the uncoupler nigericin: the translocation requirements are thus identical to those of the pre-23 kDa protein and diametrically opposite to those of preplastocyanin. Transport across the thylakoid membrane of a second fusion protein, consisting of the presequence of the 16 kDa protein linked to mature plastocyanin, is also dependent on a delta pH. The data suggest that two distinct systems are involved in the translocation of proteins across the thylakoid membrane, with each system recognizing specific signals within the presequences of a subset of lumenal protein precursors.

Two distinct mechanisms have been previously identified for the transport of proteins across the chloroplast thylakoid membrane, one of which is unusual in that neither soluble factors nor ATP are required; the system requires only the transthylakoidal delta pH. We have examined this mechanism by studying the properties of one of its substrates: the extrinsic 23-kDa protein (23K) of photosystem II. Previous work has shown that this protein can be transported into isolated thylakoids as the full-length precursor protein; we show that the stromal import intermediate form of this protein is similarly translocation-competent. Gel filtration tests indicate that the stromal intermediate is probably monomeric. Protease sensitivity tests on both the initial in vitro translation product and the stromal import intermediate show that the presequence is highly susceptible to digestion whereas the mature protein is resistant to high concentrations of trypsin. The mature protein becomes very sensitive to digestion if unfolded in urea, or after heating, and we therefore propose that the natural substrate for this translocation system consists of a relatively unfolded presequence together with a tightly folded passenger protein. The ability of thylakoids to import pre-23K is destroyed by prior treatment of the thylakoids with low concentrations of trypsin, demonstrating the involvement of surface-exposed proteins in the import process. However, we can find no evidence for the binding of pre-23K or i23K to the thylakoid surface, and we therefore propose that the initial interaction of these substrates with the thylakoidal translocase is weak, reversible, and probably delta pH-independent. In the second phase of the translocation mechanism, the delta pH drives either the translocation and unfolding of proteins, or the translocation of a fully folded protein.

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The light-driven oxidation of water in the thylakoid membrane is carried out by photosystem II, a multisubunit complex containing both integral and extrinsic components. The wateroxidation reaction takes place on the lumenal side of the membrane and is catalysed by a group of proteins, often termed the oxygen-evolving complex, of which three proteins of 33, 23 and 16 kDa are particularly prominent [2,5]. The 33 kDa protein is believed to stabilise an essential manganese centre, whereas the 23 kDa and 16kDa proteins probably assume regulatory functions [ 1,6]. All three proteins are synthesised in the cytosol as larger precursors containing aminoterminal pre-sequences [11] and import into the thylakoid lumen is believed to take place by a two-step mechanism. Precursor proteins are initially transported into the stroma and cleaved to intermediate forms by a stromal processing peptidase, after which these intermediates are transferred across the thylakoid membrane and cleaved to the mature sizes by a second, thylakoid proces sing peptidase [ 3 ].We have determined the N-terminal amino acid sequence of the wheat 33 kDa protein, and have used specific antisera to isolate a full-length cDNA encoding the precursor protein from a wheat leaf library in 2gtll. The nucleotide and deduced amino acid sequences of this cDNA, p33K-2, are shown in Fig. 1. Amino acid residues 80-102 precisely match the sequence determined by N-terminal Edman degradation of the purified protein; the precursor protein therefore consists of a mature protein (246 residues) preceded by a pre-sequence of 79 amino acids. The mature wheat protein shows a high degree of conservation with the corresponding pea [9, 10] and spinach [7] proteins, with 82~o and 81~o of the residues being identical, respectively.The pre-sequence of the wheat 33 kDa protein is shorter than those of the corresponding spinach and pea proteins, by 5 and 2 residues, respectively. The intermediate cleavage site has yet to be determined, but it is believed that the envelope transfer and thylakoid transfer domains are of approximately equal length [3]. A comparison of the wheat and spinach pre-sequences is shown in Fig. 2; overall, these sequences exhibit considerably less homology (54 ~o residue identity) than do the mature sequences, but it is notable that the second, thylakoid transfer domains are more highly conserved than the envelope transfer domains. Only the amino-terminal sections of the envelope transfer domains are highly conserved, although it may be significant that the positions of basic residues are also conserved. The thylakoidThe nucleotide sequence data reported will appear in the EMBL GenBank and DDBJ Nucleotide Sequence Databases under the accession number X57408.

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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