Topology of TROL protein in thylakoid membranes of Arabidopsis thaliana

Abstract

Thylakoid rhodanase‐like protein (TROL) is a nuclear‐encoded protein of thylakoid membranes required for tethering of ferredoxin:nicotinamide adenine dinucleotide phosphate (NADPH) oxydoreductase (FNR). It has been proposed that the dynamic interaction of TROL with flavoenzyme FNR, influenced by environmental light conditions, regulates the fate of photosynthetic electrons, directing them either to NADPH synthesis or to other acceptors, including reactive oxygen species detoxification pathways. Inside the chloroplasts, TROL has a dual localization: an inner membrane precursor form and a thylakoid membrane mature form, which has been confirmed by several large‐scale chloroplast proteomics studies, as well as protein import experiments. Unlike the localization, the topology of TROL in the membranes, which is a prerequisite for further studies of its properties and function, has not been experimentally confirmed yet. Thermolysin was proven to be a valuable protease to probe the surface of chloroplasts and membranes in general. By treating the total chloroplast membranes using increasing protease concentration, sequential degradation of TROL was observed, indicating protected polypeptides of TROL and possible domain orientation. To further substantiate the obtained results, TROL‐overexpressing Arabidopsis line (OX) and line in which the central rhodanase‐like domain (RHO) has been partially deleted (ΔRHO), were used as well. While OX line showed the same degradation pattern of TROL as the wild‐type, surprisingly, TROL from ΔRHO membranes was not detectable even at the lowest protease concentration applied, indicating the importance of this domain to the integrity of TROL. In conclusion, TROL is a polytopic protein with a stroma‐exposed C‐terminal FNR‐binding region, and the thylakoid lumen‐located RHO domain.

Thylakoid Rhodanese-like Protein–Ferredoxin:NADP+ Oxidoreductase Interaction Is Integrated into Plant Redox Homeostasis System

“…These results indicate that in conditions when TROL-FNR stoichiometry is altered, e.g., when TROL is overly accumulated, not only is the local thylakoid-associated ROS scavenging altered, but the oxidative balance of the entire plant cell is perturbed. This is not surprising since the TROL has dual localization, in thylakoid membranes and in the inner chloroplast envelope [3] and might therefore be involved in ROS signaling which originates from chloroplasts. It is therefore reasonable to assume that the TROL-FNR interaction and the associated dynamic FNR release could represent the source elements in the redox homeostasis cascade.…”

“…In plants and bacteria, FNR is also an important scavenger of free radicals [1]. TROL is built out of several structurally and functionally different regions: the N-terminal stromal domain including chloroplast targeting pre-sequence [2], the two transmembrane domains, the rhodanese-like domain (RHO) in thylakoid lumen, and the C-terminal domain protruding into the stroma [3]. The C-terminal stretch comprises of the proline-rich region PEPE that provides flexibility and swiveling motion, and the very terminal ITEP domain which is responsible for proteinprotein recognition and the interaction with the FNR dimer [2][3][4].…”

“…TROL is built out of several structurally and functionally different regions: the N-terminal stromal domain including chloroplast targeting pre-sequence [2], the two transmembrane domains, the rhodanese-like domain (RHO) in thylakoid lumen, and the C-terminal domain protruding into the stroma [3]. The C-terminal stretch comprises of the proline-rich region PEPE that provides flexibility and swiveling motion, and the very terminal ITEP domain which is responsible for proteinprotein recognition and the interaction with the FNR dimer [2][3][4]. It has been shown that the TROL-FNR binding is dynamic [5], light-dependent [5], and influenced by chloroplast energetic needs-prioritizing either energy production or energy dissipation [6].…”

A Ubiquitin-Based Module Directing Protein–Protein Interactions in Chloroplasts

“…CP26 is known to be tightly associated with PSII super-complexes [21]. On the other hand, TROL plays a role in tethering ferredoxin: nicotinamide adenine dinucleotide phosphate (NADPH) oxidoreductase (FNR), which is located in the acceptor side of the PSI complex [22]. Due to their different localization and functions within thylakoids, CP26 and TROL are unlikely to be physically associated in vivo without assistance.…”

Photosynthesis—European Congress on Photosynthesis Research

“…A number of articles treat regulatory aspects of photosystem II (Shevela et al ), the plastoquinone pool (Borisova‐Mubarakshina et al ), cytochrome c (Bernal‐Bayard et al ), redox regulation (Nikkanen et al ), photoacclimation (Hu et al ) and photorespiration (García‐Caledrón et al ). The growing interest in structural aspects is shown by articles on the dynamics of the thylakoid membrane (Solovchenko et al , Konert et al , Ughy et al ) and of specific proteins (Carius et al , Vojta and Fulgosi ), protein complexes (Kuthanová Trsková et al , Fedorov et al ) and the antenna (Albanese et al , Kuthanová Trsková et al 2019). An important tool for photosynthesis research is variable chlorophyll fluorescence, which is used to study photosystem II in isolated form, in thylakoid membranes as well as in vivo.…”