A MONOMERIC, TIGHTLY FOLDED STROMAL INTERMEDIATE ON THE DELTA-PH-DEPENDENT THYLAKOIDAL PROTEIN-TRANSPORT PATHWAY

Two distinct mechanisms have been previously identified for the transp ort of proteins across the chloroplast thylakoid membrane, one of whic h 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: th e 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 in termediate form of this protein is similarly translocation-competent. Gel filtration tests indicate that the stromal intermediate is probabl y monomeric, Protease sensitivity tests on both the initial in vitro t ranslation product and the stromal import intermediate show that the p resequence is highly susceptible to digestion whereas the mature prote in is resistant to high concentrations of trypsin, The mature protein be comes very sensitive to digestion if unfolded in urea, or after hea ting, and we therefore propose that the natural substrate for this tra nslocation system consists of a relatively unfolded presequence togeth er with a tightly folded passenger protein, The ability of thylakoids to import pre 23K is destroyed by prior treatment of the thylakoids wi th low concentrations of trypsin, demonstrating the involvement of sur face-exposed proteins in the import process, However, we can find no e vidence for the binding of pre-23K or i23K to the thylakoid surface, a nd we therefore propose that the initial interaction of these substrat es with the thylakoidal translocase is weak, reversible, and probably Delta pH-independent. In the second phase of the translocation mechani sm, the Delta pH drives either the translocation and unfolding of prot eins, or the translocation of a fully folded protein.