Effects of TROL Presequence Mutagenesis on Its Import and Dual Localization in Chloroplasts

Abstract

Thylakoid rhodanase-like protein (TROL) is involved in the final step of photosynthetic electron transport from ferredoxin to ferredoxin: NADP+ oxidoreductase (FNR). TROL is located in two distinct chloroplast compartments—in the inner envelope of chloroplasts, in its precursor form; and in the thylakoid membranes, in its fully processed form. Its role in the inner envelope, as well as the determinants for its differential localization, have not been resolved yet. In this work we created six N-terminal amino acid substitutions surrounding the predicted processing site in the presequence of TROL in order to obtain a construct whose import is affected or localization limited to a single intrachloroplastic site. By using in vitro transcription and translation and subsequent protein import methods, we found that a single amino acid exchange in the presequence, Ala67 to Ile67 interferes with processing in the stroma and directs the whole pool of in vitro translated TROL to the inner envelope of chloroplasts. This result opens up the possibility of studying the role of TROL in the chloroplast inner envelope as well as possible consequence/s of its absence from the thylakoids.

Topology of TROL protein in thylakoid membranes of Arabidopsis thaliana

“…). Previously, we have shown that imported TROL could not be extracted from membranes by high salt, urea, or high pH treatments, indicating that At4g01050 is an integral thylakoid membrane protein (Jurić et al , Vojta et al ). 6 M urea denatures unprotected proteins, 1 M NaCl disrupts electrostatic interactions between proteins and charged lipids, whereas high carbonate releases membrane‐associated/peripheral proteins.…”

“…It has also been demonstrated that the chloroplast inner envelope translocon protein Tic62 anchors FNR (Küchler et al , Stengel et al , Benz et al , Alte et al , Lintala et al , Goss and Hanke 2014). TROL is synthesized on cytoplasmic ribosomes with N‐terminal chloroplast‐targeting presequence (amino acids 01‐69; Jurić et al , Vojta et al ). This sequence is cleaved prior to import into thylakoids, but not preceding the import into the inner envelope membrane of chloroplasts.…”

“…Therefore, we can distinguish two forms of TROL inside the chloroplasts: around 70‐kDa inner membrane precursor form and 66‐kDa thylakoid membrane form. Both forms remain protected after thermolysin digestion of intact chloroplasts, indicating their complete incorporation into these membranes (Vojta et al ). Several large‐scale chloroplast proteomics studies reported this dual localization.…”

“…In an attempt to better characterize and determine the localization of TROL, a series of protein import experiments into intact pea chloroplasts have been performed under various conditions (Vojta et al ). Dual localization of TROL and its stability in both chloroplastic membranes have been confirmed by these experiments.…”

“…Also, the stromal processing site at the N‐terminus of the presequence of TROL has been characterized into more detail by quick change multisite‐directed mutagenesis. A single amino acid exchange Ala67 to Ile67 leads to the arrest of the chloroplast import at the level of the inner envelope (Vojta et al ). Aside the localization, topology of TROL in the membranes has not been experimentally confirmed yet.…”