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.
Cytosol-synthesised chloroplast and mitochondrial precursor proteins are proteolytically processed after import by highly specific, metal-dependent soluble enzymes : the stromal processing peptidase (SPP) and the matrix processing peptidase (MPP), respectively. We have used in vitro processing assays to compare the reaction specificities of highly purified preparations of pea SPP and Neurospora crassa MPP, both of which are unable to cleave a variety of 'foreign' proteins. We show that SPP can cleave all five mitochondrial precursor proteins tested, namely cyclophilin, the p subunit of the F,-ATPase complex, the Rieske FeS protein, the a-MPP subunit and cytochrome 6,. In contrast, MPP is unable to cleave any chloroplast precursor proteins tested. Several of the mitochondrial precursor proteins are cleaved more efficiently by SPP than are many authentic chloroplast precursor proteins but, in each case, cleavage takes place at a site or sites which are Nterminal to the authentic MPP site; pre-cyclophilin is cleaved 5 residues upstream of the MPP site and the precursor of the p subunit of the F,-ATPase complex is cleaved at sites 5 and 12 residues upstream. We discuss the implications of these data for the SPP reaction mechanism.
We have partially purified the stromal processing peptidase from Chlamydomonas reinhardtii and compared the properties of this activity with those of the pea counterpart. Whereas previous studies have suggested that the two enzymes may have significantly different reaction specificities, we find that they are in fact very similar. Both enzymes process precursors of two higher-plant thylakoid lumen proteins, and one C. reinhardtii lumenal protein, to similar intermediate-size forms. However, whereas the algal enzyme processes the precursor of C. reinhardtii Rubisco small subunit to the correct mature size, this precursor is cleaved only to an intermediate size by the pea enzyme. The small subunit precursor from pea appears to be cleaved by both enzymes in a similar manner. In terms of sensitivity to inhibitors, the two activities are notably different; the pea enzyme has previously been shown to be inhibited by several types of heavy-metal chelator, but we have found that none of these compounds affect the algal activity.
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