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.
Highlights
The mitochondrial fatty acid biosynthetic system have been identified and characterized in fungi and animals; all are nuclear-encoded proteins that are transported to the matrix compartment of mitochondria
These observations indicate that the mitochondrial FAS may serve to provide the octanoyl precursor required for the biosynthesis of lipoyl moieties de novo, as well as providing fatty acids that are utilized in remodeling of mitochondrial membrane phospholipids [14]
The human mitochondrial fatty acid biosynthetic system appears uniquely adapted for the synthesis of octanoyl-acyl carrier protein (ACP) as the major product and our previous studies had shown that these octanoyl moieties can be directly translocated to apoproteins, where they presumably are substrates for the insertion of sulfur atoms by lipoic acid synthase [9, 15]
Summary
Culturing and Transfection of HEK293T Cells—Human embryonic kidney (HEK) 293T cells were maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum at 37 °C in a 5% CO2 atmosphere. Cells were collected, counted, and assayed at various times after transfection. SiRNA transfection was done at the time cells were seeded. Formazan produced in the cells was dissolved in 50 l of lysis buffer (20% SDS, 0.2 M HCl), and the mixture incubated overnight in a humidified atmosphere. Fluorescence Microscopy—Loss of mitochondrial transmembrane potential and accumulation of ROS in cultured HEK293T cells were assessed at various times following siRNA treatment using the JC-1 mitochondrial potential detection kit (Cell Technology, Inc.) and CM-H2DCFDA dye (Invitrogen), respectively, according to the manufacturer’s instructions. Mitochondria were extracted by incubation for 30 min, with occasional mixing, in a solution (20 l/21 mg wet cells) containing 2% lauryl maltoside, 50 mM sodium chloride, 50 mM imidazole-HCl, pH 7, 2 mM 6-aminohexanoic acid, 1 mM EDTA and protease inhibitors. 0.3 oxoacid dehydrogenase was purchased from Abcam (Cambridge, MA) and those recognizing -actin from Sigma
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