Subcomplexes of ancestral respiratory complex I subunits rapidly turn over in vivo as productive assembly intermediates in Arabidopsis

Abstract

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 Arabidopsis 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 subcomplex, containing the ancestral γ-carbonic anhydrase (γ-CA), γ-carbonic anhydrase-like, and 20.9-kDa subunits, had a significantly higher turnover rate than intact CI or CI+CIII(2). In vitro import of precursors for these CI subunits demonstrated rapid generation of subcomplexes and revealed that their specific abundance varied when different ancestral subunits were imported. Time course studies of precursor import showed the further assembly of these subcomplexes into CI and CI+CIII(2), indicating that the subcomplexes are productive intermediates of assembly. The strong transient incorporation of new subunits into the 200-kDa subcomplex in a γ-CA mutant is consistent with this subcomplex being a key initiator of CI assembly in plants. This evidence alongside the pattern of coincident occurrence 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.

FIGURE 1.

Coomassie staining of BN and BN/SDS-PAGE separations of mitochondrial membrane protein subunits and identification of complex I subunits. Positions of subunits from CI and CI+CIII₂ and the corresponding 650- and 200-kDa subcomplexes are shown. Ellipses and numbers, identified CI subunits (evidence provided in supplemental Data Set S1); red indicates the position of the three noted CI subunits across complexes.

FIGURE 2.

Isotope incorporation ratios of subunits in CI-containing complexes after 24 and 120 h of ¹⁵N labeling. Shown is the ¹⁵N incorporation ratio (NA/(NA + H)) in peptides of CA2, CAL1, and 20.9-kDa subunit at 24 and 120 h. Error bars, S.E. values of biological replicates (n = 3 or else marked with a caret). Different colors show the 200-kDa subcomplex (red), 650-kDa subcomplex (dark red), CI (yellow-green), and CI+CIII₂ (green). *, statistically significant lower NA/(NA + H) ratio in the subcomplexes compared with intact CI (24 h CAL1 is compared with CI+CIII₂) (p < 0.05, t test). For calculations, see supplemental Data Set S2.

FIGURE 3.

Isotope incorporation ratios for CI subunits within mature CI and CI+CIII₂ after 120 and 168 h of ¹⁵N incubation. A–D, the NA/(NA + H) ratio for each subunit is shown for peptides isolated from CI and CI+CIII₂ following 120 and 168 h of incubation with ¹⁵N. Error bars, S.E. values among biological replicates (n = 3 or else marked with a caret). *, statistical significance (p < 0.05) compared with CAL1. CI subunits are divided into four groups based on colors. Red, matrix group (18 kDa, B17.2, 39 kDa, 51 kDa, and 75 kDa); yellow, membrane and matrix interaction group (B16.6, B13, PDSW, NDU10, and NDU12); blue, mitochondrion-encoded group (ND1, ND7, and ND9); green, γ-CA domain group (CA1, CA2, CAL1, and 20.9 kDa). Error bars, S.E. values among biological replicates (n = 3, or else marked with a caret). *, statistical significance (Student's t test, p < 0.05) compared with the CAL1 subunit. E and F, statistical comparison of the four groups in CI and CI+CIII₂ after 168 h of ¹⁵N incubation. Error bars, S.D. among measurements of different subunits within a group (n = 6–11). One-way analysis of variance and Tukey's post hoc multiple comparisons among four groups were used; **, p < 0.01; *, p < 0.05. For calculations, see supplemental Data Set S3.

FIGURE 4.

Import of CA domain subunits and 20.9-kDa subunit into wild type Arabidopsis mitochondria. A, [³⁵S]Met-labeled precursors (P) were incubated with mitochondria (M) for 60 min with and without PK treatment and were separated by SDS-PAGE. B, [³⁵S]Met-labeled precursors were incubated with fresh mitochondria, following which proteins from PK-treated mitochondria were separated by BN-PAGE. Radiolabeled bands aligned to a Coomassie-stained BN-polyacrylamide gel of the same material and the positions of complexes and subcomplexes are shown. Molecular masses of complexes are reported against standardized masses (23). *, location of 200–300-kDa complex I subcomplexes. The control lane is an equivalent import assay with the ϵ subunit of ATP synthase (At1g51650), which does not readily get incorporated into complexes and serves as a reference for radiolabeling patterns on SDS-polyacrylamide gel images.

FIGURE 5.

Import of CA2 into ca2 mutant mitochondria. Mitochondria were prepared from 2-week-old hydroponically grown wild type and ca2 knock-out Arabidopsis. A, CA2 precursors were incubated with wild type and ca2 knock-out mitochondria for 5, 10, 20, 30, and 60 min; mitochondria were then treated with PK; and mitochondrial proteins were separated by SDS-PAGE. B, mitochondria were treated with PK, and solubilized components were then separated by BN-PAGE. Radiolabeled bands aligned to Coomassie-stained BN-polyacrylamide gels of the same samples, and the positions of complexes and subcomplexes are indicated. *, location of 200–300-kDa complex I subcomplexes.