Figure 4.
Inhibition of Translation and Proposed Role of NDUFAF3 during Complex I Assembly
HEK293 cells were induced for NDUFS3-, NDUFAF3-, and NDUFAF4-GFP expression with and without prior chloramphenicol treatment (± CAP) for 3 days.
(A) Complex I in-gel activity (CI-IGA) assay.
(B) SDS-PAGE fluorscan for GFP-tagged protein.
(C) BN-PAGE immunodetection with antibody to GFP. Previously described NDUFS3-containing intermediates are indicated on the left as by Vogel et al.8 M: monomer. Accumulation is indicated with arrows, referring to subcomplexes of NDUFAF3 (AF3) and of NDUFAF4 (AF4).
(D) The conserved gene cluster in alpha-proteobacteria that links orthologs of NDUFAF3 to the bacterial preprotein translocase complex SecD/SecF/YajC, involved in targeting proteins to the membrane, and to CrtB, a phytoene/squalene synthetase of which the human ortholog C8ORF38 has been implicated in complex I assembly.13 Green: proteins only observed in bacteria. Red: proteins observed in eukaryotes and bacteria.
(E) Tentative model for the role of NDUFAF3 and NDUFAF4 during early complex I assembly. Subcomplex 2 is loosely associated to the mitochondrial inner membrane, as demonstrated by Dieteren et al.,33 by an unknown factor (X). NDUFAF3 and NDUFAF4 are codependent, membrane localized, and present in subcomplex 3, which contains at least subunits NDUFS2 and NDUFS3, and possibly NDUFS8 and NDUFA5. This complex requires mitochondrial translation for progression to higher-molecular-weight subcomplexes. Without mitochondrial translation, NDUFAF3 no longer associates with subcomplex 3 and the complex, with NDUFAF4, accumulates. With mitochondrial translation, both proteins are present until subcomplex 6, close to finalization of holo-complex I. It is yet unknown which factors aid membrane insertion of mitochondrial translation products (indicated with Y). AF3: NDUFAF3. AF4: NDUFAF4. S3: subcomplex 3. X and Y: unknown factors. ND: ND subunits. Green structure: mitochondrial ribosome.