Abstract
Mitochondrial respiratory complex I (NADH:ubiquinone oxidoreductase) is essential for energy production. Human NADH dehydrogenase (ubiquinone) Fe-S protein 7 (NDUFS7) is a well-conserved subunit and contains an iron-sulfur cluster N2 (4Fe-4S tetranuclear) as the finial electron transport center in complex I. As a nuclear-encoded protein and translated in cytosol, we previously demonstrated NDUFS7 is imported into mitochondria by a mitochondrial targeting sequence (MTS). In addition, we also identified that NDUFS7 possesses a nuclear export signal (NES) and a nuclear localization signal (NLS) near the C-terminus. SUMOylation, the process of protein conjugation with a small ubiquitin-like modifier (SUMO), is one type of reversible modification and has been considered to play a key role in modulating many important cellular processes. In previous studies, we have shown that NDUFS7 could be modified by SUMO1 and the importance of this modification has been discussed. In this study, we tried to explore other involvement of SUMO3, another SUMO paralogue, in NDUFS7 modification and function. To map the major SUMOylation sites, we first mutated the lysine residue at position 11 on SUMO3 to arginine, which was used to study the mono-SUMOylation and abolished the poly-SUMOylated chain. We also constructed a series of NDUFS7 mutants with mutation(s) at seven lysine residues or removing the predicated SUMO-interacting motif (SIM). According to the result of mutation-scanning analyses, SUMOylation of NDUFS7 couldn’t be fully abolished in any of the mutant construct. Therefore, multiple lysine residues and SIMs might be involved in the SUMOylation of NDUFS7. In addition, the change of NDUFS7 translocation was observed in NDUFS7_SUMO3AA fusion protein. In contrast to SUMO1 modification, NDUFS7 conjugation with SUMO3 facilitated the import of NDUFS7 into mitochondria and nearly not in the nucleus. The biological meaning of NDUFS7 modification with SUMO3 was investigated by introducing various treatments such as dissipation of mitochondrial membrane potential, etoposide-induced III apoptosis, hypoxia, oxidative stress and deprivation of nutrition, respectively. Among them, we uncovered that SUMOylation of NDUFS7 with SUMO3 was up-regulated in the CoCl2-induced hypoxia condition but down-regulated under H2O2-triggered oxidative stress. The detailed mechanism about how SUMOylation influences NDUFS7 subcellular localization and its contribution to physiologic consequences deserves further exploration.