 of a number of cellular enzymes, including pyruvate dehydrogenase, pyruvate carboxylase, carnitine acetyltransferase, and citrate synthase (13)(14)(15).
The analysis of CoA metabolic intermediates in bacteria and mammalian cells demonstrated that both pantothenate and 4ЈPP could accumulate in significant amounts, indicating the existence of two rate-limiting steps in CoA biosynthesis (16,17). Studies from several laboratories provided evidence that pantothenate kinase is the main regulatory enzyme in CoA biosynthesis (3,17). The activity of pantothenate kinase is strongly inhibited in a feedback regulatory mode by CoA and all of its acyl esters (6,18,19). So far, very little is known about the regulation of 4ЈPP adenylyltransferase activity of CoA synthase, which seems to be another control point in maintaining CoA homeostasis. No physiological regulators of CoA synthase have been reported so far. However, the regulation of this step may control the re-utilization of 4ЈPP arising from the turnover of the acyl carrier protein or the degradation of CoA by a phosphodiesterase.
The compartmentalization of the CoA biosynthetic pathway is very important but is not well understood. In baker's yeast, a multienzymatic CoA-synthesizing complex has been isolated and shown to contain all enzymatic activities involved in CoA biosynthesis (20,21). However, such a complex has not been observed in other organisms. In mammalian cells, the first three enzymes in the pathway of CoA biosynthesis are cytosolic proteins (17,22,23). The data on the localization of CoA synthase are somewhat controversial. The 4ЈPP adenylyltransferase and dephospho-CoA kinase activities of CoA synthase have been reported in the cytosol (3), the mitochondrial outer membrane (25) and in the mitochondrial matrix (26). Molecular cloning of CoA synthase cDNA has facilitated the task of analyzing subcellular localization of the enzyme and its regulation in response to various extracellular stimuli and metabolic changes (27)(28)(29).
In this study, we demonstrate by confocal microscopy and subcellular fractionation that CoA synthase is localized on mitochondria. Furthermore, CoA synthase was mapped more precisely to the mitochondrial outer membrane. Mitochondrial localization of CoA synthase was shown to be mediated by the N-terminal sequence, which targets green fluorescent protein solely to mitochondria. We also provide evidence for the first time that both activities of CoA synthase are activated by phosphatidylcholine and phosphatidylethanolamine. This study demonstrates that the last two steps of CoA biosynthesis occur on the outer mitochondrial membrane and that phospholipids can modulate the activity of CoA synthase.

EXPERIMENTAL PROCEDURES
Materials-Tissue culture media and reagents were purchased from Invitrogen; insulin-like growth factor, epidermal growth factor (EGF), phorbol 12-myristate 13-acetate, dephospho-CoA, phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, and phosphatidylinositol were from Sigma; mitochondrial marker MitoTracker Orange CMT-MRos was from Molecular Probes. All chemicals used in this study were of analytical reagent grade.
Cell Cultures and Antibodies-MCF7 and human embryonic kidney 293 cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum. NIH3T3 cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% donor calf serum. The anti-VDAC1, anti-Mn-SOD and anti-Myc monoclonal antibodies (9E-10) were purchased from Santa Cruz Biotechnology. Fluorescein isothiocyanate-conjugated goat anti-mouse antibodies were from Jackson ImmunoResearch Laboratories. Generation of rabbit polyclonal anti-CoAsy antibodies was described previously (27).
Plasmid Construction, Expression and Purification of Recombinant Proteins-Full-length coding sequence of CoAsy was PCR-amplified by Vent polymerase and cloned into pFastBacHTb vector in frame with the His-tag epitope at the N terminus (Invitrogen). Generation of recombinant baculovirus was carried out using Bac-to-Bac baculovirus expression system (Invitrogen). Amplification of recombinant baculovirus, infection of Sf9 cells, and affinity purification of His-CoAsy were performed according to manufacturer's recommendations. To generate mammalian expression constructs, PCR-amplified sequences of fulllength CoAsy (amino acids 1-563), ⌬TMCoAsy (amino acids 29 -563), ⌬NCoAsy (amino acids 187-563), and dPCoAK (amino acids 349 -563) were cloned into pcDNA3.1 vector (Invitrogen) in frame with the Cterminal Myc-tag epitope. An 87-bp cDNA fragment coding for the first 29 amino acids of CoAsy was amplified by PCR and cloned in frame with the 5Ј-end of the green fluorescent protein (EGFP) cDNA into pEGFP-N1 vector (Clontech). All constructs were verified by DNA sequencing using an ABI 373A automated sequencer.
Transfected cells were cultured for 24 h and then treated with 100 nM MitoTracker Orange CMTMRos (Molecular Probes) for 15 min before fixation with 4% paraformaldehyde and permeabilization with 0.2% Triton X-100. Primary anti-Myc antibodies (1:1000 dilution) were incubated with fixed cells for 1 h followed by incubation with secondary fluorescein isothiocyanate-anti-mouse antibodies. Transfection of human embryonic kidney 293 cells with pEGFP-N1 and pEGFP-N1-CoAsyN plasmids was carried out using LipofectAMINE according to manufacturer's recommendations (Invitrogen). Fluorescently labeled cells were viewed with a Zeiss LSM510 confocal microscope, and the images were analyzed using the LSM510 image browser software.
Isolation of Mitochondria-Crude heavy and light mitochondrial fractions were obtained by differential centrifugation as recommended in OptiPrep Application Sheets (Axis-Shield PoC AS; www.axis-shieldpoc.com/density/dtech.htm). Briefly, MCF-7 cells were collected from two 150-mm plates and resuspended in 2 ml of buffer A (0.25 M sucrose, 1 mM EDTA, and 10 mM HEPES-NaOH, pH 7.4). Cell suspension was homogenized with 15-20 passages through a 25-gauge needle. The homogenate was then centrifuged at 1,000 ϫ g for 10 min. The supernatant was subsequently centrifuged at 3,000 ϫ g for 10 min. The pellet was retained and the supernatant further centrifuged at 17,000 ϫ g for 10 min. The last two pellets were termed heavy and light mitochondrial fractions and used for isopycnic fractionation in an OptiPrep density gradient.
Fractionation of Organelles with Preformed Density Gradient-Fractionation of the light and heavy mitochondrial pellets was performed in a continuous 10 -30% OptiPrep (iodixanol) gradient according to manufacturer's recommendations (Axis-Shield PoC AS). Briefly, the gradient was formed by diffusion of 10/20/25/30% steps for 60 min. Heavy and light mitochondrial fractions were re-suspended in 5% OptiPrep, layered on the top of the gradient, and centrifuged in a swinging-bucket rotor at 80,000 ϫ g for 2 h. After unloading the gradient, obtained fractions were frozen in an ethanol/dry ice bath and stored at Ϫ80°C.
Limited Proteolysis, Alkaline and Urea Extractions-To determine the sensitivity of CoAsy to protease treatment, a light mitochondrial fraction (100 g of proteins) was incubated with 1 g of proteinase K for 30 min at room temperature in buffer containing 50 mM Tris-HCl, pH 7.4, 0.25 M sucrose, and 2 mM CaCl 2. The reaction was stopped by adding 2 mM phenylmethylsulfonyl fluoride and boiling in gel loading buffer. The samples were separated by SDS-PAGE and immunoblotted for detection of CoAsy, VDAC1, and Mn-SOD.
The same quantity of a light mitochondrial fraction was extracted with 3 M urea and 0.1 M Na 2 CO 3, pH 11.0, or under neutral conditions as described previously (30). The pellets and soluble fractions obtained in all types of extraction were separated by SDS-PAGE and probed with anti-CoAsy, anti-VDAC1, and anti-Mn-SOD antibodies.
Enzymatic Assays-PPAT and dPCoAK activities were measured as described previously (27). Enzymatic assays were performed in the presence or absence of freshly prepared phospholipid vesicles. All phospholipids were dissolved in chloroform and stored at Ϫ80°C in small aliquots. Appropriate amounts of phospholipid solutions were mixed and solvent was vaporized in a SpeedVac at room temperature for 1 h. Lipid vesicles were freshly prepared in 1ϫ assay buffer by vigorous vortexing of mixed phospholipids followed by sonication for 1 min at room temperature. The quality of prepared vesicles was analyzed by phase-contrast microscopy.

The N-terminal Region of CoAsy Possesses a Conserved
Hydrophobic Stretch-We and others have recently cloned cDNAs encoding mammalian CoAsy (27)(28)(29). Sequence analysis and biochemical studies revealed that CoAsy is a multidomain protein, possessing two enzymatic domains: 4ЈPP adenylyltransferase in the middle and dephospho-CoA kinase at the C terminus. No significant homology to known protein domains or motifs was reported for the N-terminal region of CoAsy in previous studies. However, further inspection of CoAsy revealed the presence of a hydrophobic segment, extending from residues 7