NDUFAF3 is Involved in the Assembly of the Q/P Modules of Respiratory Complex I in the Green Microalga Chlamydomonas reinhardtii.

Abstract

The mitochondrial NADH:ubiquinone oxidoreductase, or complex I, is composed of a hydrophobic arm comprising the P module and a hydrophilic arm comprising the N and Q modules. The assembly of complex I is well characterized in humans and is catalyzed by a series of assembly factors that join the Q, P, and N modules sequentially. The complex I of protists and plants, however, contains additional ancestral features, namely a ferredoxin bridge that connects the matrix and the membrane arms and a γ carbonic anhydrase domain, whose mechanisms of assembly are unknown. In this work, a strain where the complex I assembly factor NDUFAF3 has been tagged with a 3×FLAG at the C-terminus is investigated in the green microalga Chlamydomonas reinhardtii. Like its human homolog, NDUFAF3 interacts strongly with the classical subunits of the Q and P modules, but also with the γ carbonic anhydrase domain and C1-FDX, a subunit of the ferredoxin bridge. The predicted structural positioning of NDUFAF3 within the Q module suggests a role in the formation of this bridge. In contrast, subunits of the N module are only loosely associated with NDUFAF3. We further demonstrate that the N module is attached at a later stage of assembly, suggesting that Chlamydomonas complex I assembles in a human-like sequence. This contrasts with what is documented in Angiosperms, where the N and Q modules are attached together before anchoring to the P module. Altogether, these results highlight a conserved and ancestral role of NDUFAF3 in complex I manufacture.