Localization and integration of thylakoid protein translocase subunit cpTatC

Abstract

SUMMARYThylakoid membranes have a unique complement of proteins, most of which are nuclear encoded synthesized in the cytosol, imported into the stroma and translocated into thylakoid membranes by specific thylakoid translocases. Known thylakoid translocases contain core multi-spanning, membrane-integrated subunits that are also nuclear-encoded and imported into chloroplasts before being integrated into thylakoid membranes. Thylakoid translocases play a central role in determining the composition of thylakoids, yet the manner by which the core translocase subunits are integrated into the membrane is not known. We used biochemical and genetic approaches to investigate the integration of the core subunit of the chloroplast Tat translocase, cpTatC, into thylakoid membranes. In vitro import assays show that cpTatC correctly localizes to thylakoids if imported into intact chloroplasts, but that it does not integrate into isolated thylakoids. In vitro transit peptide processing and chimeric precursor import experiments suggest that cpTatC possesses a stroma-targeting transit peptide. Import time-course and chase assays confirmed that cpTatC targets to thylakoids via a stromal intermediate, suggesting that it might integrate through one of the known thylakoid translocation pathways. However, chemical inhibitors to the cpSecA-cpSecY and cpTat pathways did not impede cpTatC localization to thylakoids when used in import assays. Analysis of membranes isolated from Arabidopsis thaliana mutants lacking cpSecY or Alb3 showed that neither is necessary for cpTatC membrane integration or assembly into the cpTat receptor complex. These data suggest the existence of another translocase, possibly one dedicated to the integration of chloroplast translocases.

Identification of Putative Substrates of SEC2, a Chloroplast Inner Envelope Translocase

“…Only slight reductions of the outer envelope protein TOC75 were observed. In contrast, the abundance of most of these proteins, with the exception of SCY1, was unaffected in SCY1-RNAi seedlings, consistent in part with previous results (Martin et al, 2009). These results suggest that the loss of SCY2 creates a different and more global effect on chloroplast biogenesis in developing seedlings than loss of SCY1.…”

“…This appears to be due to a breakdown of RNAi, as SCY1 null mutants are stably and uniformly chlorotic (Martin et al, 2009;Skalitzky et al, 2011). qRT-PCR analysis confirmed that both SCY1 and SCY2 transcripts had returned to wildtype levels in the green true leaves (Martin, 2010).…”

“…Analysis of endogenous SCY1, ALB3, and TATC from pea and FTSH12 from Arabidopsis (Supplemental Fig. S3A; Martin et al, 2009) as well as imported proteins showed that their integrated forms are resistant to 0.1 M NaOH extraction (Supplemental Fig. S3, B-D).…”

Plastids Contain a Second Sec Translocase System with Essential Functions

“…Thylakoids are thought to initiate through the budding or invagination of the inner envelope membrane (for discussion, see von Wettstein, 2001;Vothknecht and Westhoff, 2001), and translocases that mediate the expansion and population of proteins of the system may themselves need to be initially inserted in the envelope (for discussion, see Cline, 2003). This has been proposed for the cpTatC subunit based on experimental observations that are inconsistent with integration by any of the known thylakoid translocase systems (Martin et al, 2009). The cpTat complex assembles in scy1 and alb3 mutants, and biochemical inhibitors of cpTat and SRP were without effect on cpTatC localization.…”

“…In bacteria, the homologous subunits are cotranslationally integrated by the Sec machinery (for discussion, see Martin et al, 2009). A recent study of the biogenesis of cpTatC ruled out involvement of the thylakoid Tat, Sec, and SRP systems as well as the YidC homolog Alb3 (Martin et al, 2009). Because cpTatC is not integrated into isolated thylakoids, the spontaneous pathway is also not involved.…”