Membrane Chaperoning of a Thylakoid Protease Whose Structural Stability Is Modified by the Protonmotive Force.

Protein folding is a complex cellular process often assisted by chaperones, but it can also be facilitated by interactions with lipids. Disulfide bond formation is a common mechanism to stabilize a protein. This can help maintain functionality amid changes in the biochemical milieu, including those relating to energy-transducing membranes. Plastidic Type I Signal Peptidase 1 (Plsp1) is an integral thylakoid membrane signal peptidase that requires an intramolecular disulfide bond for in vitro activity. We have investigated the interplay between disulfide bond formation, lipids, and pH in the folding and activity of Plsp1. By combining biochemical approaches with a genetic complementation assay using Arabidopsis thaliana plants, we provide evidence that interactions with lipids in the thylakoid membrane have reconstitutive chaperoning activity toward Plsp1. Further, the disulfide bridge appears to prevent an inhibitory conformational change resulting from proton motive force-mimicking pH conditions. Broader implications related to the folding of proteins in energy-transducing membranes are discussed.

Polyglycine Acts as a Rejection Signal for Protein Transport at the Chloroplast Envelope.

PolyGly is present in many proteins in various organisms. One example is found in a transmembrane ß-barrel protein, translocon at the outer-envelope-membrane of chloroplasts 75 (Toc75). Toc75 requires its N-terminal extension (t75) for proper localization. t75 comprises signals for chloroplast import (n75) and envelope sorting (c75) in tandem. n75 and c75 are removed by stromal processing peptidase and plastidic type I signal peptidase 1, respectively. PolyGly is present within c75 and its deletion or substitution causes mistargeting of Toc75 to the stroma. Here we have examined the properties of polyGly-dependent protein targeting using two soluble passenger proteins, the mature portion of the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (mSS) and enhanced green fluorescent protein (EGFP). Both t75-mSS and t75-EGFP were imported into isolated chloroplasts and their n75 removed. Resultant c75-mSS was associated with the envelope at the intermembrane space, whereas c75-EGFP was partially exposed outside the envelope. Deletion of polyGly or substitution of tri-Ala for the critical tri-Gly segment within polyGly caused each passenger to be targeted to the stroma. Transient expression of t75-EGFP in Nicotiana benthamiana resulted in accumulation of c75-EGFP exposed at the surface of the chloroplast, but the majority of the EGFP passenger was found free in the cytosol with most of its c75 attachment removed. Results of circular dichroism analyses suggest that polyGly within c75 may form an extended conformation, which is disrupted by tri-Ala substitution. These data suggest that polyGly is distinct from a canonical stop-transfer sequence and acts as a rejection signal at the chloroplast inner envelope.

Chaperone-assisted Post-translational Transport of Plastidic Type I Signal Peptidase 1.

Type I signal peptidase (SPase I) is an integral membrane Ser/Lys protease with one or two transmembrane domains (TMDs), cleaving transport signals off translocated precursor proteins. The catalytic domain of SPase I folds to form a hydrophobic surface and inserts into the lipid bilayers at the trans-side of the membrane. In bacteria, SPase I is targeted co-translationally, and the catalytic domain remains unfolded until it reaches the periplasm. By contrast, SPases I in eukaryotes are targeted post-translationally, requiring an alternative strategy to prevent premature folding. Here we demonstrate that two distinct stromal components are involved in post-translational transport of plastidic SPase I 1 (Plsp1) from Arabidopsis thaliana, which contains a single TMD. During import into isolated chloroplasts, Plsp1 was targeted to the membrane via a soluble intermediate in an ATP hydrolysis-dependent manner. Insertion of Plsp1 into isolated chloroplast membranes, by contrast, was found to occur by two distinct mechanisms. The first mechanism requires ATP hydrolysis and the protein conducting channel cpSecY1 and was strongly enhanced by exogenously added cpSecA1. The second mechanism was independent of nucleoside triphosphates and proteinaceous components but with a high frequency of mis-orientation. This unassisted insertion was inhibited by urea and stroma extract. During import-chase assays using intact chloroplasts, Plsp1 was incorporated into a soluble 700-kDa complex that co-migrated with the Cpn60 complex before inserting into the membrane. The TMD within Plsp1 was required for the cpSecA1-dependent insertion but was dispensable for association with the 700-kDa complex and also for unassisted membrane insertion. These results indicate cooperation of Cpn60 and cpSecA1 for proper membrane insertion of Plsp1 by cpSecY1.

Plastidic type I signal peptidase 1 is a redox-dependent thylakoidal processing peptidase.

Thylakoids are the photosynthetic membranes in chloroplasts and cyanobacteria. The aqueous phase inside the thylakoid known as the thylakoid lumen plays an essential role in the photosynthetic electron transport. The presence and significance of thiol-disulfide exchange in this compartment have been recognized but remain poorly understood. All proteins found free in the thylakoid lumen and some proteins associated to the thylakoid membrane require an N-terminal targeting signal, which is removed in the lumen by a membrane-bound serine protease called thylakoidal processing peptidase (TPP). TPP is homologous to Escherichia coli type I signal peptidase (SPI) called LepB. Genetic data indicate that plastidic SPI 1 (Plsp1) is the main TPP in Arabidopsis thaliana (Arabidopsis) although biochemical evidence had been lacking. Here we demonstrate catalytic activity of bacterially produced Arabidopsis Plsp1. Recombinant Plsp1 showed processing activity against various TPP substrates at a level comparable to that of LepB. Plsp1 and LepB were also similar in the pH optima, sensitivity to arylomycin variants and a preference for the residue at -3 to the cleavage site within a substrate. Plsp1 orthologs found in angiosperms contain two unique Cys residues located in the lumen. Results of processing assays suggested that these residues were redox active and formation of a disulfide bond between them was necessary for the activity of recombinant Arabidopsis Plsp1. Furthermore, Plsp1 in Arabidopsis and pea thylakoids migrated faster under non-reducing conditions than under reducing conditions on SDS-PAGE. These results underpin the notion that Plsp1 is a redox-dependent signal peptidase in the thylakoid lumen.

Stable complex formation of thylakoidal processing peptidase and PGRL1.

The thylakoid-transfer signal is required for energy-dependent translocation of preproteins into the thylakoid lumen and is removed by the thylakoidal processing peptidase (TPP). PGRL1 is an essential component of antimycin A-sensitive photosynthetic cyclic electron flow in chloroplasts. Here we report that one of the TPP isoforms, Plsp1, forms a stable complex with PGRL1. Genetic data demonstrate that PGRL1 is not essential for Plsp1 activity in vivo, leading to a possibility that PGRL1 may act as a regulator of TPP.

Functional diversification of thylakoidal processing peptidases in Arabidopsis thaliana.

Thylakoidal processing peptidase (TPP) is responsible for removing amino-terminal thylakoid-transfer signals from several proteins in the thylakoid lumen. Three TPP isoforms are encoded by the nuclear genome of Arabidopsis thaliana. Previous studies showed that one of them termed plastidic type I signal peptidase 1 (Plsp1) was necessary for processing three thylakoidal proteins and one protein in the chloroplast envelope in vivo. The lack of Plsp1 resulted in seedling lethality, apparently due to disruption of proper thylakoid development. The physiological roles of the other two TPP homologs remain unknown. Here we show that the three A. thaliana TPP isoforms evolved to acquire diverse functions. Phylogenetic analysis revealed that TPP may have originated before the endosymbiotic event, and that there are two groups of TPP in seed plants: one includes Plsp1 and another comprises the other two A. thaliana TPP homologs, which are named as Plsp2A and Plsp2B in this study. The duplication leading to the two groups predates the gymnosperm-angiosperm divergence, and the separation of Plsp2A and Plsp2B occurred after the Malvaceae-Brassicaceae diversification. Quantitative reverse transcription-PCR assay revealed that the two PLSP2 genes were co-expressed in both photosynthetic tissues and roots, whereas the PLSP1 transcript accumulated predominantly in photosynthetic tissues. Both PLSP2 genes were expressed in the aerial parts of the plsp1-null mutant at levels comparable to those in wild-type plants. The seedling-lethal phenotype of the plsp1-null mutant could be rescued by a constitutive expression of Plsp1 cDNA but not by that of Plsp2A or Plsp2B. These results indicate that Plsp1 and Plsp2 evolved to function differently, and that neither of the Plsp2 isoforms is necessary for proper thylakoid development in photosynthetic tissues.

Keep the balloon deflated: the significance of protein maturation for thylakoid flattening.

Thylakoidal processing peptidase (TPP) catalyzes the removal of signal peptide which leads to maturation of a subset of proteins including photosynthetic electron transport components in thylakoids. The biochemical properties of TPP were highly defined during the 1980's and 1990's, but the physiological significance of the TPP activity had remained undefined. Completion of genome sequencing revealed the presence of three TPP isoforms in the model plant Arabidopsis thaliana. A recent genetic study demonstrated that one isoform, plastidic type I signal peptidase 1 (Plsp1), is necessary for proper thylakoid assembly. Interestingly, Plsp1 was found in both the chloroplast envelope and thylakoids, being responsible for maturation of an outer membrane protein Toc75 and a lumenal protein OE33. A more recent study has shown that Plsp1 is involved in maturation of two additional lumenal proteins, OE23 and plastocyanin, and that accumulation of unprocessed Toc75 does not disrupt normal chloroplast development. The study also revealed that plsp1-null plastids accumulate balloon-like vesicles that appear to be the remnants of thylakoids as they contain unprocessed OE33 in the peripheral regions. These findings suggest that proper maturation of lumenal proteins is required for correct assembly and/or maintenance of thylakoids, but may not be necessary for initiation of membrane development. The ballooned thylakoids in plsp1-null plastids may be a useful tool to elucidate the mechanism of thylakoid flattening, which correlates with the energized state of the membranes.