Subcomplexes of Ancestral Respiratory Complex I Subunits Rapidly Turn Over in Vivo as Productive Assembly

Subcomplexes of mitochondrial respiratory complex I (CI; EC 1.6.5.3) are shown to turn over in vivo, and we propose a role in an ancestral assembly pathway. By progressively labeling Arabi- dopsis cell cultures with 15 N and isolating mitochondria, we have identified CI subcomplexes through differences in 15 N incorporation into their protein subunits. The 200-kDa sub- complex, containing the ancestral -carbonic anhydrase (-CA), -carbonic anhydrase-like, and 20.9-kDa subunits, had a significantly higher turnover rate than intact CI or CICIII2. In vitro import of precursors for these CI subunits demonstrated rapid generation of subcomplexes and revealed that their spe- cific abundance varied when different ancestral subunits were imported. Time course studies of precursor import showed the further assembly of these subcomplexes into CI and CICIII2, indicating that the subcomplexes are productive intermediates of assembly. The strong transient incorporation of new subunits into the 200-kDa subcomplex in a -CA mutant is consistent with this subcomplex being a key initiator of CI assembly in plants. This evidence alongside the pattern of coincident occur- rence of genes encoding these particular proteins broadly in eukaryotes, except for opisthokonts, provides a framework for the evolutionary conservation of these accessory subunits and evidence of their function in ancestral CI assembly.