期刊
JOURNAL OF BIOLOGICAL CHEMISTRY
卷 295, 期 52, 页码 18367-18378出版社
ELSEVIER
DOI: 10.1074/jbc.RA120.015726
关键词
mitochondria; iron-sulfur protein; Arabidopsis thaliana; protein-protein interaction; circular dichroism; Raman spectroscopy; iron-sulfur cluster trafficking; ISCA proteins; NFU proteins
资金
- National Institutes of Health [R37GM62524]
- Agence Nationale de la Recherche as part of the Investissements d'Avenir program [ANR-11-LABX-0002-01]
- Laboratory of Excellence ARBRE [ANR-15-IDEX-04-LUE]
- Lorraine Universite d'Excellence
- [ANR-2013-BSV6-0002-01]
Numerous iron-sulfur (Fe-S) proteins with diverse functions are present in the matrix and respiratory chain complexes of mitochondria. Although [4Fe-4S] clusters are the most common type of Fe-S cluster in mitochondria, the molecular mechanism of [4Fe-4S] cluster assembly and insertion into target proteins by the mitochondrial iron-sulfur cluster (ISC) maturation system is not well-understood. Here we report a detailed characterization of two late-acting Fe-S cluster-carrier proteins from Arabidopsis thaliana, NFU4 and NFU5. Yeast two-hybrid and bimolecular fluorescence complementation studies demonstrated interaction of both the NFU4 and NFU5 proteins with the ISCA class of Fe-S carrier proteins. Recombinant NFU4 and NFU5 were purified as apo-proteins after expression in Escherichia coli. In vitro Fe-S cluster reconstitution led to the insertion of one [4Fe-4S](2+) cluster per homodimer as determined by UV-visible absorption/CD, resonance Raman and EPR spectroscopy, and analytical studies. Cluster transfer reactions, monitored by UV-visible absorption and CD spectroscopy, showed that a [4Fe-4S](2+) cluster-bound ISCA1a/2 heterodimer is effective in transferring [4Fe-4S](2+) clusters to both NFU4 and NFU5 with negligible back reaction. In addition, [4Fe-4S](2+) cluster-bound ISCA1a/2, NFU4, and NFU5 were all found to be effective [4Fe-4S](2+) cluster donors for maturation of the mitochondrial apo-aconitase 2 as assessed by enzyme activity measurements. The results demonstrate rapid, unidirectional, and quantitative [4Fe-4S](2+) cluster transfer from ISCA1a/2 to NFU4 or NFU5 that further delineates their respective positions in the plant ISC machinery and their contributions to the maturation of client [4Fe-4S] cluster-containing proteins.
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