Physiological function of plastidic type I signal peptidase 1 in two membranes of Arabidopsis thaliana chloroplasts

Chloroplast development is essential to autotrophy in plants. Previously, we found that Plastidic type 1 signal peptidase 1 (Plsp1) is essential for this process; its lack causes seedling lethality and aberrant thylakoid development in Arabidopsis thaliana. We also showed that Plsp1 is required for removing targeting information from the mature domains of at least one protein each in the chloroplast envelope and thylakoids, Toc75 and OE33 respectively. This result suggested that Plsp1 may be located in two sub-compartments of the chloroplast. In this work, we aimed to elucidate the mechanism by which Plsp1 is involved in chloroplast biogenesis by cytological and genetic approaches. First, we used electron microscope immunolocalization to show that the sub-organellar location of Plsp1 depends on chloroplast development. In developing chloroplasts from meristematic tissues, Plsp1 was found to be evenly distributed between the envelope and thylakoids, whereas in developed chloroplasts from leaf mesophyll cells, it was mainly located in thylakoids. This novel developmental localization pattern was corroborated by expression patterns and maturation efficiencies of the putative substrates of Plsp1. Second, we addressed the nature of aberrant chloroplast development in plsp1 knockout lines. Immunoblotting and transcriptional studies show that accumulation of a subset of thylakoidal proteins is post-transcriptionally downregulated, and identify two additional potential substrates of Plsp1. We also investigated the levels of the two Plsp1 homologues (Plsp2 and TPP), and concluded that Plsp1 may be the major peptidase at both the thylakoid and envelope membranes in wildtype chloroplasts. Finally, we disproved the possibility that improper processing of Toc75, a major chloroplast protein import channel, is responsible for the plsp1 phenotype by complementing toc75 knockout A. thaliana with unprocessable Toc75 mutants and analyzing resultant plant phenotypes. Together, these findings support the biological significance of multiple localizations of Plsp1, and also provide a novel tool to elucidate the mechanism of thylakoid development.