ABSTRACT
Plants have evolutionarily conserved NFU-domain proteins that are targeted to plastids or mitochondria. The ‘plastid-type’ NFU1, NFU2 and NFU3 in Arabidopsis thaliana play a role in iron-sulfur (Fe-S) cluster assembly in this organelle, whereas the type-II NFU4 and NFU5 proteins have not been subjected to mutant studies in any plant species to determine their biological role. Here we confirm that NFU4 and NFU5 are targeted to the mitochondria. The proteins are constitutively produced in all parts of the plant, suggesting a housekeeping function. Double nfu4 nfu5 knockout mutants were embryonic lethal, and depletion of the proteins led to growth arrest of young seedlings. Biochemical analyses revealed that NFU4 and NFU5 are required for lipoylation of the H proteins of the glycine decarboxylase complex and the E2 subunits of other mitochondrial dehydrogenases, with little impact on Fe-S cluster-containing respiratory complexes and aconitase. Consequently, the Gly-to-Ser ratio was increased in mutant seedlings and early growth was improved by elevated CO2. In addition, pyruvate, 2-oxoglutarate and branched-chain amino acids accumulated in the nfu4 nfu5 mutants, further supporting defects in the other three mitochondrial lipoate-dependent enzyme complexes. NFU4 and NFU5 interacted with mitochondrial lipoyl synthase (LIP1) in yeast 2-hybrid and bimolecular fluorescence complementation assays. These data indicate that NFU4 and NFU5 have a more specific function than previously thought, in providing Fe-S clusters to lipoyl synthase.
One sentence summary A pair of evolutionarily conserved proteins involved in iron-sulfur cofactor assembly have a specific role in lipoate biosynthesis for mitochondrial dehydrogenases.
Footnotes
↵1 Joint first authors
↵4 Author for contact: janneke.balk{at}jic.ac.uk
5 This work was supported by a studentship from the John Innes Foundation to A.E.M.; the Biotechnology and Biological Sciences Research Council, grant awards BB/P012523/1 and BB/P012574/1 to M.F. and J.B; funding from the T4F and the Kempe Foundations (GÖF) to O.K.; the French National Research Agency, grant awards ANR-13-BSV6-0002-01, ANR-11-LABX-0002-01 (Lab of Excellence ARBRE) and ANR-15-IDEX-04-LUE (Lorraine Université d’Excellence) to N.R. and J.P.T.
The authors responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instruction of Authors are: Janneke Balk (janneke.balk{at}jic.ac.uk) and Nicolas Rouhier (Nicolas.Rouhier{at}univ-lorraine.fr).