ArabidopsisTic62 and Ferredoxin-NADP(H) Oxidoreductase Form Light-Regulated Complexes That Are Integrated into the Chloroplast Redox Poise  

Abstract

Translocation of nuclear-encoded preproteins across the inner envelope of chloroplasts is catalyzed by the Tic translocon, consisting of Tic110, Tic40, Tic62, Tic55, Tic32, Tic20, and Tic22. Tic62 was proposed to act as a redox sensor of the complex because of its redox-dependent shuttling between envelope and stroma and its specific interaction with the photosynthetic protein ferredoxin-NADP(H) oxidoreductase (FNR). However, the nature of this close relationship so far remained enigmatic. A putative additional localization of Tic62 at the thylakoids mandated further studies examining how this feature might be involved in the respective redox sensing pathway and the interaction with its partner protein. Therefore, both the association with FNR and the physiological role of the third, thylakoid-bound pool of Tic62 were investigated in detail. Coexpression analysis indicates that Tic62 has similar expression patterns as genes involved in photosynthetic functions and protein turnover. At the thylakoids, Tic62 and FNR form high molecular weight complexes that are not involved in photosynthetic electron transfer but are dynamically regulated by light signals and the stromal pH. Structural analyses reveal that Tic62 binds to FNR in a novel binding mode for flavoproteins, with a major contribution from hydrophobic interactions. Moreover, in absence of Tic62, membrane binding and stability of FNR are drastically reduced. We conclude that Tic62 represents a major FNR interaction partner not only at the envelope and in the stroma, but also at the thylakoids of Arabidopsis thaliana and perhaps all flowering plants. Association with Tic62 stabilizes FNR and is involved in its dynamic and light-dependent membrane tethering.

N-Terminal Structure of Maize Ferredoxin:NADP+ Reductase Determines Recruitment into Different Thylakoid Membrane Complexes

“…Two proteins have recently been reported to bind FNR at the thylakoid membrane in Arabidopsis: TROL (Juric et al, 2009) and Tic62 (Benz et al, 2009). Blue native-PAGE analysis has shown that TROL binds FNR to complexes of ;140 to 190 kD, and Tic62 binds FNR into several complexes at higher molecular masses.…”

“…Approximate molecular masses are given in kilodaltons to the left of the blot, and FNR-containing complexes coordinated by Tic62 and TROL are indicated to the right of the blot. (Benz et al, 2009;Juric et al, 2009), there has been much speculation that FNR bound to different protein complexes, and in the soluble phase, might coordinate different electron transfer processes Joliot and Johnson, 2011). As we found Arabidopsis plants expressing maize FNR1, FNR2, and FNR3 are respectively enriched in FNR bound to TROL, Tic62, and in soluble FNR, we compared their PET capacity, to gain further insight into this question.…”

“…These roles in different electron transport pathways have led to intense research interest in the mechanism and location of FNR binding to the thylakoid membrane (Andersen et al, 1992;Jose Quiles and Cuello, 1998;Zhang et al, 2001;Peng et al, 2008;Benz et al, 2009;Juric et al, 2009;Alte et al, 2010;Baniulis et al, 2011). Two thylakoid FNR binding proteins named TROL and Tic62 have been identified in Arabidopsis thaliana.…”

“…Two thylakoid FNR binding proteins named TROL and Tic62 have been identified in Arabidopsis thaliana. TROL mutants are impaired in photosynthesis (Juric et al, 2009), and Tic62 has no direct photosynthetic role but seems rather to stabilize FNR (Benz et al, 2009). An FNR binding domain containing a Ser/Pro-rich motif has been identified in both TROL and Tic62, and its interaction with FNR has recently been thoroughly characterized (Alte et al, 2010).…”

LTD is a protein required for sorting light-harvesting chlorophyll-binding proteins to the chloroplast SRP pathway

“…Tic110, Tic20 and Tic21 have been suggested to function as the protein translocation channel of the inner envelope 1,2,7,8 . Several stromal proteins, including chaperones, proteases and redox regulators, are also involved in the import process 6,9,10 . The stromal domain of Tic110 and Hip/Hop domain of Tic40 may function as scaffolds that tether stromal chaperones, including Hsp93 and Hsp70, which facilitate protein translocation across the inner membrane 2,6,11 .…”