Down-regulation of mitochondrial acyl carrier protein in mammalian cells compromises protein lipoylation and respiratory complex I and results in cell death

Abstract

The objective of this study was to evaluate the physiological importance of the mitochondrial fatty acid synthesis pathway in mammalian cells using the RNA interference strategy. Transfection of HEK293T cells with small interfering RNAs targeting the acyl carrier protein (ACP) component reduced ACP mRNA and protein levels by >85% within 24 h. The earliest phenotypic changes observed were a marked decrease in the proportion of post-translationally lipoylated mitochondrial proteins recognized by anti-lipoate antibodies and a reduction in their catalytic activity, and a slowing of the cell growth rate. Later effects observed included a reduction in the specific activity of respiratory complex I, lowered mitochondrial membrane potential, the development of cytoplasmic membrane blebs containing high levels of reactive oxygen species and ultimately, cell death. Supplementation of the culture medium with lipoic acid offered some protection against oxidative damage but did not reverse the protein lipoylation defect. These observations are consistent with a dual role for ACP in mammalian mitochondrial function. First, as a key component of the mitochondrial fatty acid biosynthetic pathway, ACP plays an essential role in providing the octanoyl-ACP precursor required for the protein lipoylation pathway. Second, as one of the subunits of complex I, ACP is required for the efficient functioning of the electron transport chain and maintenance of normal mitochondrial membrane potential.

FIGURE 1.

Effect of mitochondrial ACP down-regulation on growth of HEK293T cells. HEK293T cells were treated with 100 nm mitochondrial ACP siRNA (diamonds), nonspecific siRNA (circles, dotted lines), or no siRNA (squares). A, ACP mRNA level, normalized to β-actin mRNA. B, cell number. C, MTT assay.

FIGURE 2.

Effect of siRNA treatment on the lipoylation profile of HEK293T cells. Cells were treated with either vehicle alone (lanes 1), 100 nm nonspecific siRNA (lanes 2), or 100 nm ACP siRNA (lanes 3) and cultured for up to 96 h. Whole cell extracts were prepared and the proteins from 30,000 cells were subjected to SDS-PAGE/Western blotting using antibodies specific for lipoic acid, the E2 subunit of pyruvate dehydrogenase (PDH) or β-actin. The electrophoretic mobilities of PDH-E2 and branched chain oxoacid dehydrogenase E2 (BCDH-E2) corresponded to molecular masses of ∼60 and 47 kDa, respectively.

FIGURE 3.

Effect of siRNA treatment on the integrity of respiratory complex I. HEK293T cells were treated with 10 nm siRNA targeting either the ACP or NDUFB8 subunits of complex I and cultured up to 48 h. Mitochondria were isolated and a 2% lauryl maltoside extract prepared. A, the content of three complex I subunits was assessed by SDS-PAGE and Western blotting with specific antibody probes for ACP, NDUFB8, and NDUFS3. In the control experiment (c) no siRNA treatment was performed; si, siRNA-treated. B, respiratory complexes were fractionated by Blue Native gel electrophoresis and complex I activity was assayed directly in the gel. The location of complex I in the gel (arrow) was confirmed by blotting onto a polyvinylidene fluoride membrane and probing with antibodies specific for antibodies of the NDUFA9 subunit (far right lane). C and D, two-dimensional electrophoretic analysis of complex I. After Blue Native gel electrophoresis in the direction indicated by the arrows, SDS-PAGE was performed in the second dimension and selected subunits of complex I were detected by Western blotting using a mixture of five specific antibodies. Integrated density measurements for all detected subunits indicate that the amount of complex I in ACP and NDUFB8 siRNA-treated cells was 122 ± 41 and 40 ± 19%, respectively, of the control values.

FIGURE 4.

Effect of down-regulation of NDUFB8 on cell growth and lipoylation profile. HEK293T cells were treated with various concentrations of siRNAs targeting the NDUFB8 subunit of respiratory complex I for up to 96 h. A, cell growth. B, lipoylation profile, monitored 96 h post-transfection. Control cells were treated with either vehicle alone or nonspecific siRNAs. The electrophoretic mobility of NDUFB8 corresponds to a molecular mass of ∼19 kDa.

FIGURE 5.

Effect of siRNA treatment on cell morphology, mitochondrial membrane potential, and ROS. HEK293T cells were treated with nonspecific control siRNA or ACP siRNA (10 nm), cultured for 80 h, and exposed to the fluorescent dye JC-1 to monitor mitochondrial membrane potential. Healthy cells with normal mitochondrial membrane potential translocate the green fluorescent dye from the cytosol into mitochondria, where it forms red aggregates. A, E, and I, phase-contrast images. Bar in panel A, 20 μm. B, F, and J, green fluorescence. C, G, and K, red fluorescence. D, H, and L, superimposed green and red fluorescence. Panels I–L are higher magnification images illustrating cytoplasmic membrane blebs (arrows). In M–R, HEK293T cells, cultured with either nonspecific control siRNA or ACP siRNA for 96 h, were exposed to CM-H₂DCFDA, which is taken up by mitochondria and converted to a green fluorescent product in the presence of ROS. M–O, phase-contrast images. P—R, green fluorescence. Panels O and R are higher magnification images illustrating ROS accumulation in cytoplasmic membrane blebs (arrows).

FIGURE 6.

Effect of exogenous lipoate on siRNA-treated HEK293T cells. A, cells were treated with either ACP siRNA or nonspecific siRNA (10 nm), cultured in medium supplemented with various concentrations of Tris-lipoate for 96 h, and cell number monitored. B, morphology of cells treated with ACP siRNA supplemented with lipoate. C, lipoylation profile of cells grown in medium supplemented with 1 μm lipoate. Lipoylation was monitored by Western blotting using anti-lipoyl antibodies as probe. Ns, nonspecific siRNA. Similar results were obtained using the sodium salt of lipoic acid.