huInd1 depletion results in severely decreased levels of constituents of the peripheral arm of complex I. (A) HeLa cells, triply transfected with vectors as described in the legend to Fig. 2A, were fractionated using digitonin, and the mitochondrial fractions were resolved by SDS-PAGE. Immunostaining was with the polyclonal antibody against complex I (α-complex I) or an antibody that recognizes the α and β subunits of ATP synthase F₁ (α-F₁ ATP synthase α+β). (B) Blots prepared as described in the legend to panel A were probed with antibodies against NDUFS1 (α-NDUFS1), NDUFV1, NDUFA9, NDUFS3, NDUFA13 (GRIM-19), or NDUFB6 or an antibody that recognizes the α and β subunits of ATP synthase F₁. (C) The decreases in NDUFS1 and NDUFV1 upon huInd1 depletion are posttranscriptional. mRNAs were extracted from HeLa cells transfected as described in the legend to panel A and were analyzed by Northern blotting using probes generated from cDNA encoding NDUFS1 or NDUFV1 or an oligonucleotide complementary to 18S rRNA.

Appearance of a subcomplex of complex I upon depletion of huInd1. (A) HeLa cells were transfected as described in the legend to Fig. 2A. Mitochondrial preparations were resolved by hrCNE and immunostained with the polyclonal antibody against complex I (α-complex I). Two major immunoreactive bands are apparent near the top of the gel (arrows); the higher of the two (specific [S]) significantly decreased after RNAi treatment. The lower band (nonspecific [NS]) does not correspond to a positive band in an in-gel complex I activity assay (data not shown). The amount of a subcomplex of complex I migrating at approximately 450 kDa (bracket) is largely increased upon the depletion of huInd1. (B, top panels) Immunostaining of complex I proteins in samples resolved first by hrCNE, as described in the legend to panel A, and then by SDS-PAGE. The white arrowheads denote a complex I subunit (approximately 15 kDa) which is present in the 450-kDa subcomplex and increases following huInd1 depletion. The subcomplex does not contain the NDUFS1 and NDUFV1 Fe/S proteins. (Middle and bottom panels) Immunostaining of a 2D gel blot (prepared as described for the top panels) with antibodies against NDUFA9 and NDUFB6 (α-NDUFA9 and α-NDUFB6). The level of NDUFA9 in fully assembled complex I decreases upon huInd1 depletion, but NDUFA9 does not appear in a subcomplex under these conditions. In contrast, NDUFB6 is enriched in a subcomplex when huInd1 is depleted.

huInd1 is a mitochondrial Fe/S protein. (A) HeLa cells cotransfected with constructs encoding a huInd1-EGFP fusion protein and a mitochondrion-targeted Dicosoma sp. red protein (dsRed-mito) show virtually complete colocalization of the two fluorescent labels. (B) HeLa cells were permeabilized by digitonin treatment. The total cell lysate (L) was fractionated into crude mitochondria (M) and the cytosolic fraction (C) by centrifugation at 15,000 × g. Immunostaining of the samples with antibodies raised against huInd1 revealed a specific band in the mitochondrial fraction that represents endogenous huInd1, while a cross-reacting (nonspecific [NS]) protein band with greater electrophoretic mobility was present in the cytosol and cell lysate. Immunostaining of the same membranes for alpha-tubulin (localized in the cytosol) and mitochondrial aconitase (mAco; localized in the mitochondria) was used to verify the purity of the fractions. (C) UV-visible spectrum of chemically reconstituted, recombinant huInd1. The protein was produced in E. coli as a truncated version which does not include the predicted mitochondrial presequence but contains an N-terminal His₆ tag. The purified protein was chemically reconstituted under anaerobic conditions with sulfide and ferrous iron. The resulting UV-visible spectrum of 26 μM protein contained a dithionite-sensitive (2 mM) absorption shoulder at approximately 400 nm, which is indicative of a [4Fe-4S] cluster.

huInd1 depletion results in abnormal mitochondrial morphology and lactate production. (A) HeLa cells were transfected with an empty vector (pSup) or the RNAi vector siIND1. After growth for 3 days, cells were harvested and a fraction was retransfected. This procedure was repeated twice. Three days after each transfection with siIND1, samples (designated 1 to 3) were collected and fractionated using digitonin and the mitochondrial fractions were analyzed by SDS-PAGE and immunostaining for huInd1. Loading of equal amounts was verified by probing the membrane with an antibody that recognizes the α and β subunits of ATP synthase F₁ (F₁ α/β). (B) HeLa cells were transfected as described in the legend to panel A except that an additional sample in which cells were cotransfected with vectors siIND1 and smIND1 was generated. The latter vector contains silent mutations which render its mRNA resistant to RNAi depletion and thus allows RNAi-resistant synthesis of huInd1. Three days after the third transfection, cells were fixed directly in culture dishes and examined by electron microscopy (5). Two representative images are shown for each condition. Scale bars, 1 μm. (C) Lactate levels in culture medium conditioned for 3 days by HeLa cells transfected three times with either the empty vector (pSup), the RNAi vector (siIND1), or the RNAi and rescue vectors (siIND1+smIND1). Values were adjusted with respect to the total cell number and then normalized according to the lactate level in medium from cells transfected with the empty vector (pSup).

RNAi-mediated depletion of huInd1 specifically affects complex I. HeLa cells were transfected up to three times (as indicated by numbers 1, 2, and 3) with the indicated vectors as described in the legend to Fig. 2 and further analyzed. (A) Digitonin-solubilized crude mitochondrial fractions were assayed spectrophotometrically for complex I activity (i.e., DQA-sensitive deamino-NADH oxidation). Samples treated with RNAi (siIND1) show a three- to fourfold decrease in complex I activity after the third transfection. This decreased activity was recovered upon coexpression of an RNAi-resistant version of huInd1 (from smIND1). Error bars for the control samples (transfected with the empty vector pSup) reflect the standard deviations in a representative set of transfections. For the RNAi-treated samples, data from each transfection set were normalized to the control values within that set and the normalized values from three sets of transfections were then averaged. Error bars for these values thus represent the standard deviations across three transfection sets. (B) In-gel complex I enzymatic activity of HeLa mitochondrial preparations after resolution of complex I by BN-PAGE. S₁ and S₀ are the supercomplexes containing complex I-complex III₂-complex IV and complex I-complex III₂, respectively (74). (C) The indicated enzyme activities were assayed spectrophotometrically using digitonin-solubilized mitochondrial fractions prepared identically to those used for BN-PAGE. The activities of mitochondrial enzymes were normalized to that of CS, while those of cytosolic fractions were normalized to LDH activity. The data are shown as fractions of the control values (i.e., those for cells transfected with the empty vector in each set of transfections). SDH, complex II; CIV, cytochrome c oxidase. (D) Total cellular lysate samples described above were analyzed by immunostaining of the cytosolic Fe/S protein GPAT and of tubulin after SDS-PAGE.

Depletion of huInd1 interferes with the assembly of complex I and leads to respiratory chain remodeling. (A) HeLa cells were transfected up to three times (1 to 3) with the indicated vectors as described in the legend to Fig. 2. Mitochondrial preparations were solubilized with digitonin, resolved by BN-PAGE, and immunostained with a polyclonal anti-complex I antibody (α-complex I). S₁ is the supercomplex containing complex I-complex III₂-complex IV (74). (B) BN-PAGE-resolved mitochondrial samples from triply transfected cells described in the legend to panel A were run in a second dimension (SDS-PAGE) and were subsequently silver stained. Whereas levels of complex I-containing supercomplex protein (S₁; black arrows) decreased in RNAi-treated samples, the levels of complex III dimer (III₂; white arrowheads) increased. V, respiratory complex V. (C) Samples were treated as described in the legend to panel B. Instead of being silver stained, the gels were blotted onto PVDF membranes and probed with the polyclonal antibody against complex I. The lanes corresponding to complex I subunits are indicated by black arrows, and the positions of complex I Fe/S proteins NDUFS1 and NDUFV1 are indicated. The cross-reacting spots corresponding to the abundant ATP synthase F₁ α and β subunits (white arrowheads) show the equal degrees of exposure of the two membranes. (D) ⁵⁵Fe autoradiography analysis of BN-PAGE-resolved mitochondrial samples from HeLa cells transfected three times as described in the legend to panel A. In this experiment, growth after the third transfection was in the presence of 1 μM ⁵⁵Fe-transferrin. After BN-PAGE, the radioactivity was evaluated by phosphorimaging. The positions of ⁵⁵Fe binding proteins or complexes are indicated. (E) Immunostained BN-PAGE-resolved mitochondrial preparations. Samples were probed with an antibody raised against complex III₂ (top) or an antibody that recognizes the α and β subunits of ATP synthase F₁ (bottom). The positions of respiratory complexes or supercomplexes are indicated.

The conserved cysteine residues of the CXXC motif are essential for huInd1 function. (A to D) HeLa cells were transfected with vectors as described in the legend to Fig. 2A. Where indicated, an additional, smIND1-based construct encoding a form of huInd1 with both conserved cysteine residues (C244 and C247) mutated to alanine (smIND1CC) was used. Mitochondrial fractions from the cells were then resolved on BN-PAGE gels and assessed for complex I activity (A) or assayed spectrophotometrically for complex I activity (B). The BN-PAGE-separated samples were immunostained for complex I protein (C) or run in a second dimension (SDS-PAGE) and silver stained (D). The complex I-containing supercomplex lanes are labeled S. ATP synthase F₁F_o (V) served as an internal control. The samples in panels A and C were run in the same respective gels, but irrelevant lanes were removed. α-complex I, anti-complex I antibody. (E and F) Analyses of the fluorescence of wild-type and mutated huInd1-EGFP fusion proteins in HeLa cells indicate that both proteins are present in situ. (E) Confocal micrographs of HeLa cells transfected thrice with either the vector encoding the rescue mutant (smIND1), which contains silent mutations that render the mRNA resistant to RNAi treatment, or a version of this rescue construct with C244 and C247 mutated to alanine residues (smIND1CC). (F) Flow cytometry analysis of HeLa cells transfected as described the legend to Fig. 2A. In the panels on the left, cells were analyzed already after the first transfection. (G) Western blot analysis of cells transfected three times with the empty vector (pSup), the huInd1 RNAi construct (siIND1), the RNAi construct together with the silent-mutation rescue construct (siIND1+smIND1), or the RNAi construct together with the C244A/C247A mutant rescue construct (siIND1+smIND1CC). The blot was probed with anti-huInd1 antiserum (α-huInd1). Because the rescue constructs also encode C-terminal EGFP, the rescue proteins (huInd1-EGFP) are detected at a position of slower mobility in the gel. Note that the endogenous level of huInd1 is only slightly lower than the level of huInd1 from the rescue mutant (smIND1).