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Dissecting the molecular interactions of mitochondrial glutaredoxin S15 in plants

Subject Area Plant Physiology
Term from 2018 to 2023
Project identifier Deutsche Forschungsgemeinschaft (DFG) - Project number 406708156
 
Final Report Year 2023

Final Report Abstract

Several cellular processes in plants depend on iron-sulfur (Fe–S) proteins, whose cofactors are assembled through dedicated assembly machineries present in the cytosol, plastids and mitochondria. To cite only a few examples, Fe–S proteins are present in the photosynthetic and respiratory electron transfer chains (ETCs), they are needed for sulfur and nitrogen assimilation, and also for synthesis of other cofactors such as biotin and lipoic acid. The incorporation of Fe–S clusters (ISCs) into proteins requires first the de novo assembly of ISCs onto scaffold proteins and their transfer to acceptor proteins via the action of several maturation factors, and among those class II glutaredoxins (GRXs). Glutaredoxin S15 (GRXS15) is an essential player in the mitochondrial ISC assembly where it (i) transfers [2Fe–2S] clusters from the first assembly machinery to the second assembly system responsible for building [4Fe–4S] clusters and (ii) possibly also mediates direct supply of [2Fe–2S] clusters to the respective recipient apo-proteins. Interestingly, however, deficiencies in GRXS15-mediated cluster transfer almost exclusively causes defects in protein lipoylation with a very distinct metabolite signature. The reason for this selective manifestation of deficiencies in cluster trafficking probably lies in the extremely low copy number of lipoyl synthase (LIP1) and the fact that LIP1 needs a new [4Fe–4S] cluster after each catalytic cycle. The combination of low copy number and high catalytic demand suggests that under limiting conditions LIP1 is not capable of competing for sufficient clusters. Interestingly, this bottleneck discovered in grxs15 mutants can be overcome by deleting aconitase 3 as one of the most abundant [4Fe– 4S] cluster proteins or by overexpressing LIP1 to render it more competitive against other apo-proteins waiting for supply of [4Fe–4S]. Rather unexpectedly, it was then discovered that overexpression of LIP1 in WT plants or weak grxs15 mutants does become deleterious. The apparent toxicity of LIP1 is assumed to result from the release of sulfide, which inhibits cytochrome c oxidase (COX). This intoxication can be shown indirectly through measurement of respiratory O2 consumption and through increased expression of alternative oxidase 1a to prevent a complete block of the ETC. Genetically, the release of sulfide and intoxication of COX was confirmed through partial suppression of the resulting dwarf phenotype by overexpressing O-acetylserine(thiol)lyase C, which binds free sulfide for cysteine biosynthesis. These results show the need for sulfide fixation capacity in the mitochondrial matrix. Monitoring sulfide release by LIP1 dependent on the efficiency of upstream cluster supply on a phenotypic basis will allow investigating defects in the mitochondrial ISC and alterations caused by overexpression of distinct proteins and engineered variants. In this way, putative interactions of GRXS15 can be explored in future work.

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