The mitochondrial disease associated protein Ndufaf2 is dispensable for Complex-1 assembly but critical for the regulation of oxidative stress

Abstract

Deficiency in human mitochondrial Complex-1 has been linked to a wide variety of neurological disorders. Homozygous deletion of the Complex-1 associated protein, Ndufaf2, leads to a severe juvenile onset encephalopathy involving degeneration of the substantia nigra and other sub-cortical regions resulting in adolescent lethality. To understand the precise role of Ndufaf2 in Complex-1 function and its links to neurologic disease, we studied the effects on Complex-1 assembly and function, as well as pathological consequences at the cellular level, in multiple in vitro models of Ndufaf2 deficiency. Using both Ndufaf2-deficient human neuroblastoma cells and primary fibroblasts cultured from Ndufaf2 knock-out mice we found that Ndufaf2-deficiency selectively reduces Complex-1 activity. While Ndufaf2 is traditionally referred to as an assembly factor of Complex-1, surprisingly, however, Ndufaf2-deficient cells were able to assemble a fully mature Complex-1 enzyme, albeit with reduced kinetics. Importantly, no evidence of intermediate or incomplete assembly was observed. Ndufaf2 deficiency resulted in significant increases in oxidative stress and mitochondrial DNA deletion, consistent with contemporary hypotheses regarding the pathophysiology of inherited mutations in Complex-1 disorders. These data suggest that Ndufaf2, unlike other Complex-1 assembly factors, may be more accurately described as a chaperone involved in proper folding during Complex-1 assembly, since it is dispensable for Complex-1 maturation but not its proper function.

Respiratory supercomplexes act as a platform for complex III ‐mediated maturation of human mitochondrial complexes I and IV

“…Also, in D4-CYB cells, increased amounts of NDUFV1 and NDUFV2 were accumulated at sizes ranging from 25 to 49 kDa, further demonstrating impaired incorporation of the N-module. In addition, downregulation of NDUFAF2 expression had no drastic effects on cI assembly or activity in the WT cells, as previously described (Schlehe et al, 2013), and did not promote cI maturation in the D4-CYB cells (Fig EV5E-G). This is supported by the detection of large amounts of NDUFAF2 in the 991 kDa peak in D4-CYB, while it was absent in WT cells (Figs 5B and EV4).…”

“…However, NDUFAF2 overexpression did not prompt the accumulation of pre-cI in the WT cells, and the amount of mature active cI was the same as in the cells transfected with an empty vector ( Fig EV5B-D). In addition, downregulation of NDUFAF2 expression had no drastic effects on cI assembly or activity in the WT cells, as previously described (Schlehe et al, 2013), and did not promote cI maturation in the D4-CYB cells (Fig EV5E-G). These results clearly indicate the occurrence of stalled assembly of cI in the absence of MT-CYB.…”

Accessory NUMM (NDUFS6) subunit harbors a Zn-binding site and is essential for biogenesis of mitochondrial complex I

“…In line with previous studies, the deletion of the N7BML gene did not prevent assembly of complete and active complex I (23,28). However, in Y. lipolytica mitochondria we found a marked reduction of complex I content suggesting that assembly efficiency was decreased.…”

Exome sequencing of patients with histiocytoid cardiomyopathy reveals a de novo NDUFB11 mutation that plays a role in the pathogenesis of histiocytoid cardiomyopathy

“…Although activity at each locus is presumably provided by the alternate allele, we postulate (in light of the NDUFB11 results) that compound loss of function of these two components of the same electron transport chain is sufficient to promote Histiocytoid CM. Furthermore, NDUFAF2 and NDUFB9 have both been implicated previously in mitochondrial complex I deficiency [Schlehe et al, 2013; Haack et al, 2012]. …”