DOE OSTI2018
Plsp1 is a type I signal peptidase that is responsible for removing the targeting signal peptide from passenger proteins upon their delivery to the thylakoid lumen. Plsp1 is itself a resident of the thylakoid membrane, with a single membrane anchor and the catalytic domain facing the lumen. Work to uncover the targeting mechanism for this protein has been underway in the Inoue lab from the previous DOE funding period has established that Plsp1 is targeted at the thylakoid by the Sec1 translocase, and that it passes through a high molecular weight intermediate in the stroma en route to the thylakoid membrane. During this funding period we have demonstrated by MS/MS of immunoprecipitated samples that the high molecular weight complex containing Plsp1 in the stroma contains primarily the α1 and β1 forms of Cpn60 in approximately equal amounts. Mixing experiments with overexpressed purified proteins have reproduced the Cpn60/Plsp1 complex. Surprisingly, the co-chaperonins Cpn10 and Cpn20 were not detected in the immunoprecipitated sample, even though they are able to bind to the Cpn60/Plsp1 complex formed in mixing experiments. We have further confirmed that the addition of purified stromal cpSecA1 results in a decrease in the amount of Plsp1 successfully incorporated into the thylakoid membrane. We are currently working on experiments to describe the conditions for the handoff between Cpn60 and cpSec1 in the stroma prior to membrane targeting. The structure of Plsp1 can be inferred by threading its sequence onto the structure of its E. coli homologue LepB to contain a single membrane anchor followed by a soluble structure consisting of ten β strands. In the last DOE funding period it was determined that the transmembrane domain not only acts as the membrane anchor, but probably mediates the interaction with cpSecA1. We have now determined that the binding to Cpn60 is likely mediated by β strands 1 and 2. What is the mechanism and significance of oxidative folding of Plsp1? It was found during the last DOE funding period that Plsp1 purified after overexpression in E. coli loses its peptidase activity upon reduction of its conserved cysteines. We have now determined that Plsp1 undergoes a measurable conformational change upon reduction, both by monitoring susceptibility to protease and by native tryptophan fluorescence. We have also shown that Plsp1 is at least partly, and likely completely, oxidized in the thylakoid lumen. While these results were not unexpected, experiments on Plsp1 in vivo were completely surprising. We have known for years (from work conducted with prior DOE funding) that a Plsp1 knockout is embryo lethal. Accordingly, given that reduction of the isolated protein inactivates it, we expected that plants in which one or both of the cysteine pair were mutated would also lead to a severe mutant phenotype. This was not observed, and in fact, these mutants complemented fully the plsp1 plants. This lead to the hypothesis that either association with an interacting protein or with the membrane preserves Plsp1structure even when the disulfides are reduced. Work performed in the last DOE funding cycle revealed that Pgrl1, a protein reportedly involved in cyclic electron flow, interacts stably with Plsp1. However, the Plsp1 cysteine-less mutants are still able to compliment the plsp1 mutant even in the pgrl1 knockout mutant background. We have also found that in vitro activity of Plsl1 is not abolished by reduction in thylakoids that have been permeabilized by low concentrations of detergent, whereas the activity of fully extracted protein is. This points to membrane association as the condition important to activity, and by extension, structure preservation. We are presently working to isolate Plsp1 in a lipid nanodisk to test this hypothesis.
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