engleski

Determinants for import and dual localization of TROL

TROL (thylakoid rhodanese-like protein, At4g01050) is an integral membrane component of non-appressed thylakoid membranes and 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, where it is processed completely. Its role in the inner envelope, as well as the determinants for its different localization, have not been resolved yet. N-terminus of TROL consists of the presequence that directs the protein to the chloroplasts. We created six N-terminal amino acid substitutions surrounding the predicted processing site in the presequence of TROL in order to obtain the construct whose import is affected or localization limited to a single intrachloroplastic site. Wild type precursor of TROL and its e1-e6 presequence mutants were labelled with 35S-methionine during in vitro translation and imported into isolated intact pea chloroplasts under various conditions. Import has been performed for different times, under increasing ATP concentrations, and by using different extraction methods for determination of the strength of protein to membrane association. 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 a possibility to study the role of TROL in the chloroplast inner envelope as well as possible consequence/s of its absence from the thylakoids. We also confirmed that TROL is strongly associated with the respective membranes and that its two forms – of around 70 kDa in the inner envelope and 66 kDa in the thylakoids have distinct energy demands for reaching their final destination.

TROL ; protein import ; chloroplasts ; dual localization ; ATP ; inner envelope membrane ; thylakoids

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