In this study, we used biochemical and genetic approaches to identify a function of the mitochondrial carriers Leu5p and hGP in the accumulation of CoA in the matrix. First, the CoA levels in Δleu5 mitochondria were 15-fold lower than those in wild-type or various mutant organelles. No corresponding decrease in the CoA concentration was detectable in the PMS of Δleu5 cells (see below). Second, the CoA-dependent enzyme IPMS was largely defective in intact Δleu5 mitochondria. This was solely due to a shortage in the substrate acetyl-CoA, since the enzyme was fully active when detergent extracts of Δleu5 mitochondria were supplied with acetyl-CoA. Third, the surprising interference between mitochondrial Leu5p and peroxisomal citrate synthase Cit2p is best explained on the basis of a role of Leu5p in CoA accumulation in the mitochondrial matrix. The decreased CoA levels in Δleu5 mitochondria lead to impaired synthesis of citrate by mitochondrial Cit1p. In Δleu5Δcit2 cells, citrate can no longer be supplied to mitochondria by peroxisomal Cit2p-catalyzed synthesis. This results in a mutant cell that closely mimics the double deletion of the CIT1
genes in that the cellular citrate concentration is largely decreased. As a further consequence, the flux through the citric acid cycle in Δleu5Δcit2 cells is low, and cells are impaired in generating glutamate from α-ketoglutarate. This defect may be further enhanced by the low activity of the other CoA-dependent enzymes of the citric acid cycle. In passing, our data nicely support the functional communication between mitochondria and peroxisomes by retrograde regulation (see, e.g., references 10
). A final point supporting the role of Leu5p in mitochondrial CoA accumulation is the dramatic defect in heme-containing proteins in Δleu5Δcit2 cells. The heme deficiency is a result of the lowered activity of δ-aminolevulinate synthase, the first enzyme of heme biosynthesis using succinyl-CoA as a substrate. The impairment of heme biosynthesis in Δleu5Δcit2 cells may be the major reason why these cells do not grow on the nonfermentable carbon source glycerol.
One may wonder why Δleu5 cells, as opposed to Δleu5Δcit2 cells, are not auxotrophic for glutamate or defective in heme-containing proteins. First, the Km
values for acetyl-CoA are 10-fold higher for IPMS than for citrate synthase, rendering IPMS most sensitive to reduced CoA concentrations (38
). A second obvious reason may be the relatively high amounts of leucine required for cellular protein biosynthesis.
To our knowledge, Leu5p and hGP are the first proteins for which a function in the cellular distribution of CoA has been demonstrated. The cellular compartmentation of CoA is an important but still poorly understood aspect of metabolism (47
). In wild-type cells, the highest CoA concentrations are found in mitochondria and the peroxisomes (46
), where the coenzyme participates in numerous pathways, such as the citric acid cycle, the biosynthesis of heme, the β-oxidation of fatty acids, and the glyoxylate cycle. About 5- to 10-fold lower levels of CoA as compared to mitochondria are found in the cytosol of wild-type cells (Fig. ) (46
). Strikingly, in a mutant lacking Leu5p the relative concentrations of CoA in mitochondria and the cytosol were reversed. These data suggest that either CoA or a precursor of CoA is the substrate of the Leu5p carrier protein. A decision between these possibilities cannot be made presently, since little is known about the compartmentation and the molecular identity of the five enzymes participating in CoA biosynthesis. Only for the first enzymatic step has the gene been identified. Pantothenate kinase (gene YDR531w
in S. cerevisiae
]) catalyzes the committed step of biosynthesis and is tightly regulated in its activity by acetyl-CoA (47
). While pantothenate kinase is known to reside in the cytosol, the location of the enzymes completing biosynthesis of CoA has not been determined with certainty. Dephospho-CoA kinase mediating the final reaction of CoA biosynthesis has been reported to be associated with mitochondria but is believed to be located outside the inner membrane (53
). These findings render it likely that CoA is synthesized externally to the mitochondrial inner membrane and must be transported into the matrix space. In support of this view, an in vitro transport system for CoA uptake into isolated mitochondria of rat liver has been reported (59
). Mitochondrial import of CoA required an electrical gradient (56
), but the transporter has not been identified (57
). Using isolated yeast mitochondria, we were unable to apply these findings for setting up a transport assay for CoA. This was mainly due to the fact that radiolabeled CoA was rapidly metabolized when added to isolated yeast mitochondria, presumably by cleavage of CoA to 4-phosphopantetheine, a reaction catalyzed by CoA hydrolase (5
). The data presented here fit nicely with the cytosolic synthesis of CoA, as Δleu5 cells were capable of producing normal levels of cytosolic CoA. On that basis, the most likely substrate of Leu5p is CoA. Clearly, definitive identification of the substrate of Leu5p will have to await the purification of Leu5p and the reconstitution of the transport reaction, as has been recently achieved for several mitochondrial carrier proteins (28
Mitochondria isolated from Δleu5 cells contain low yet significant amounts of CoA. Thus, an alternative pathway must exist to supply the organelles with CoA. Most likely, another member of the mitochondrial carrier family takes over the function of Leu5p in accumulating CoA in the mitochondrial matrix, even though it does so at low efficiency. The best candidates for such a supplementary task are the three ADP/ATP carrier proteins (AAC) of yeast. In support of this suggestion, Leu5p shares highest sequence similarity to the AAC subgroup of the yeast carriers (44
). Further, based on their substrate specificity, AAC proteins seem to be optimally suited for the transport of the adenine nucleotide CoA across the mitochondrial inner membrane.
Our study answers the long-standing question of the connection between the IPMS Leu4p and the membrane protein Leu5p (9
). Only Δleu4Δleu5 double mutant cells, but not the single mutants, exhibit an auxotrophy for leucine. Now, this finding can be easily understood on the basis of the low content of mitochondrial CoA upon deletion of LEU5
. In the absence of Leu4p, α-IPM is synthesized by Leu9p (Fig. A), an isoenzyme of Leu4p that exhibits 83% amino acid identity and is localized to the mitochondrial matrix (W. Pelzer, unpublished). Since Leu4p is located in both the matrix and the cytosol (3
), in cells lacking LEU5
, α-IPM can be generated by cytosolic Leu4p. In the Δleu4Δleu5 double mutant, however, synthesis of α-IPM can only be mediated by mitochondrial Leu9p, which functions poorly due to the low CoA concentration.
Graves' disease is a multifactorial autoimmune disorder in which hyperthyroidism is caused by the production of autoantibodies against the thyrotropin receptor and other thyroid proteins (reviewed in references 4
). A cDNA for hGP has been identified by expression cloning in an immunoscreen using antisera from patients with active Graves' disease (69
). The similarity of hGP to mitochondrial carrier proteins has been noted earlier, but a function has not been assigned yet. As shown here, hGP can replace the yeast carrier Leu5p, demonstrating that both proteins are functional orthologues required for the accumulation of CoA in the mitochondrial matrix. We were unable to detect hGP after functional expression in yeast by immunostaining with antisera derived from several patients with hyperthyroidism (data not shown). These findings indicate that hGP is not a major autoantigen of Graves' disease. Further, our insights into the function of hGP in CoA transport suggest no direct involvement of the mitochondrial carrier in this disorder.
The identification of the function of Leu5p and hGP in the accumulation of CoA in the mitochondrial matrix is a seminal step in the understanding of the mechanisms underlying proper subcellular distribution of CoA. For a comprehensive knowledge of CoA metabolism our studies will have to be followed up by a search for components involved in the biosynthesis of CoA. Moreover, membrane transporters facilitating supply of, e.g, peroxisomes, with this important cofactor will have to be identified.