Mechanistic characterization of the late steps of mitochondrial iron-sulfur cluster protein maturation.

Eisen-Schwefel (Fe/S) Cluster gehören zu den ältesten Co-Faktoren und sind unabdingbar für die Funktion vieler Proteine. Die Synthese von Fe/S Clustern und deren Insertion in Apoproteine sind komplexe biochemische Vorgänge. In Mitochondrien wird die Biogenese von Fe/S Proteinen durch die ISC Assembl...

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Bibliographic Details
Main Author: Uzarska, Marta Agata
Contributors: Lill, Roland (Prof. Dr.) (Thesis advisor)
Format: Doctoral Thesis
Published: Philipps-Universität Marburg 2013
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Table of Contents: Iron-sulfur (Fe/S) clusters are among the oldest protein cofactors and are essential for the function of many proteins. Fe/S cluster synthesis and insertion into apo-proteins is a complex process. In mitochondria, Fe/S protein biogenesis is performed by the ISC (iron sulfur cluster) assembly machinery and can be divided into three major steps. First, the Fe/S cluster is assembled de novo on the scaffold protein Isu1 from iron and sulfide which is provided by a cysteine desulfurase. In a second step, the Isu1-bound Fe/S cluster is labilized with the help of a specialized chaperone system, and third, it is transferred to target apo-proteins by specific maturation factors. Although several factors involved in Fe/S cluster transfer to target proteins have been identified, their interplay and exact functions are not resolved. This work focusses on the role of the monothiol glutaredoxin Grx5, the BolA-like proteins Aim1 and Yal044W, and Nfu1 in the late phases of Fe/S protein biogenesis. Mutations in human Grx5 have been associated with microcytic anemia, and its deletion in zebrafish is embryonically lethal. In yeast Grx5 is not essential, yet cells lacking Grx5 display low activities of Fe/S proteins, iron accumulation in mitochondria and oxidative stress. It was shown previously that upon depletion of Grx5, Fe/S clusters accumulate on Isu1 and cannot be properly transferred to the target proteins. Here it was shown that Grx5 binds a Fe/S cluster in vivo and is required for maturation of all cellular Fe/S proteins, regardless of the type of bound Fe/S cofactor and subcellular localization. Grx5 and Isu1 simultaneously interact with the dedicated Hsp70 chaperone Ssq1 at non-overlapping binding sites. Grx5 does not stimulate the ATPase activity of Ssq1 and most tightly binds to the ADP form of Ssq1. The vicinity of Isu1 and Grx5 on the chaperone facilitates rapid Fe/S cluster transfer from Isu1 to Grx5. Hence, Grx5 functions as a late-acting component of the core ISC assembly machinery linking Fe/S cluster synthesis on Isu1 with Fe/S cluster targeting to dedicated apo-proteins. BolA-like proteins have been linked to the monothiol Grxs by both bioinformatics and experimental approaches. To examine the function of mitochondrial BolA-like proteins yeast cells deleted for Aim1 and Yal044W proteins were investigated. While the role of Yal044W in Fe/S biogenesis remains unclear, aim1Δ cells displayed a 50 % decrease in the activities of succinate dehydrogenase and lipoate-dependent enzymes (pyruvate and α-ketoglutarate dehydrogenases). Activities of latter enzymes depend on activity of Fe/S protein lipoate synthase Lip5. Aim1 seems to be needed for catalytic activation but not for de novo Fe insertion into Lip5. The phenotype of aim1Δ yeast cells is consistent with with the one observed in human patient cells where mutations in human Aim1 homolog BOLA3 were associated with fatal infantile encephalopathy and/ or pulmonary hypertension. Hence, the role of Aim1 protein as an auxiliary ISC assembly protein facilitating the maturation of a subset of mitochondrial [4Fe-4S] proteins is conserved throughout eukaryotes. The human NFU1 has been associated with fatal infantile encephalopathy and/ or pulmonary hypertension, and is needed for the maturation of Fe/S clusters of respiratory complexes I and II and lipoate synthase. Previously, Nfu-like proteins were shown to bind Fe/S clusters in vitro and hence, a role was suggested in which Nfu1 functions as a scaffold protein alternatively to Isu1. Here, it is shown that nfu1Δ yeast cells display up to 5-fold decreased activities of succinate dehydrogenase and lipoate-dependent enzymes, similarly as in NFU1 patient cells. Yeast Nfu1 carrying the mutation (Gly194Cys) corresponding to the one found in patients bound Fe/S cluster in a more stable fashion, and thus allowed for the first time the detection of Fe/S cluster association in vivo. Formation of the Nfu1-bound Fe/S cluster was dependent on the core ISC assembly machinery, including Isu1, ruling out the possibility of Nfu1 acting as an alternative scaffold protein. Due to the increased stability of the bound Fe/S cluster, Gly194Cys Nfu1 was not able to fully rescue the defects of nfu1Δ cells, giving an insight into disease development in the human patients. Taken together, this study contributed to a better understanding of how Fe/S clusters are handled in mitochondria after de novo synthesis on the scaffold Isu1. First, the joint interaction of Isu1 and Grx5 on the specialized Hsp70 chaperone Ssq1 is important for efficient Fe/S cluster transfer from Isu1 to Grx5. Second, Grx5 is a crucial factor for maturation of all cellular Fe/S proteins. Third, an auxiliary role of yeast Nfu1 and Aim1 as dedicated maturation factors was established. These findings provide a better insight into how late-acting components of the mitochondrial ISC assembly machinery cooperate to efficiently mature Fe/S target proteins.