Human mitochondrial complex I assembly is mediated by NDUFAF1.

Area of Science:

• Cellular Biology
• Biochemistry
• Mitochondrial Function

Background:

• Complex I (NADH:ubiquinone oxidoreductase) is the largest enzyme in oxidative phosphorylation.
• The assembly process of Complex I in human cells is not well understood.
• No accessory proteins for Complex I assembly have been identified.

Purpose of the Study:

• To investigate the role of NDUFAF1, a potential Complex I assembly chaperone, in human cells.
• To determine if NDUFAF1 is involved in the assembly and stability of Complex I.
• To characterize the mitochondrial complexes associated with NDUFAF1.

Main Methods:

• RNA interference (RNAi) to knockdown NDUFAF1 expression.
• Analysis of Complex I amount and activity.
• Mitochondrial protein complex analysis using size exclusion chromatography.
• Study of NDUFAF1 expression in a conditional Complex I assembly system.
• Analysis of patient samples with Complex I deficiency.

Main Results:

• NDUFAF1 is localized to mitochondria.
• Knockdown of NDUFAF1 reduces Complex I amount and activity.
• NDUFAF1 associates with 600 kDa and 700 kDa mitochondrial complexes.
• The distribution of these NDUFAF1-associated complexes is altered in Complex I deficient patients.
• The 700 kDa complex appears to be a key intermediate in Complex I assembly.

Conclusions:

• NDUFAF1 is a critical protein for the assembly and stability of Complex I.
• NDUFAF1 functions as a mitochondrial chaperone in the Complex I assembly pathway.
• The 700 kDa complex involving NDUFAF1 represents a significant step in Complex I biogenesis.