Abstract
BackgroundLipoylation of 2-ketoacid dehydrogenases is essential for mitochondrial function in eukaryotes. While the basic principles of the lipoylation processes have been worked out, we still lack a thorough understanding of the details of this important post-translational modification pathway. Here we used yeast as a model organism to characterize substrate usage by the highly conserved eukaryotic octanoyl/lipoyl transferases in vivo and queried how amenable the lipoylation system is to supplementation with exogenous substrate.ResultsWe show that the requirement for mitochondrial fatty acid synthesis to provide substrates for lipoylation of the 2-ketoacid dehydrogenases can be bypassed by supplying the cells with free lipoic acid (LA) or octanoic acid (C8) and a mitochondrially targeted fatty acyl/lipoyl activating enzyme. We also provide evidence that the S. cerevisiae lipoyl transferase Lip3, in addition to transferring LA from the glycine cleavage system H protein to the pyruvate dehydrogenase (PDH) and α-ketoglutarate dehydrogenase (KGD) E2 subunits, can transfer this cofactor from the PDH complex to the KGD complex. In support of yeast as a model system for human metabolism, we demonstrate that the human octanoyl/lipoyl transferases can substitute for their counterparts in yeast to support respiratory growth and protein lipoylation. Like the wild-type yeast enzyme, the human lipoyl transferase LIPT1 responds to LA supplementation in the presence of the activating enzyme LplA.ConclusionsIn the yeast model system, the eukaryotic lipoylation pathway can use free LA and C8 as substrates when fatty/lipoic acid activating enzymes are targeted to mitochondria. Lip3 LA transferase has a wider substrate specificity than previously recognized. We show that these features of the lipoylation mechanism in yeast are conserved in mammalian mitochondria. Our findings have important implications for the development of effective therapies for the treatment of LA or mtFAS deficiency-related disorders.
Highlights
Lipoylation of 2-ketoacid dehydrogenases is essential for mitochondrial function in eukaryotes
There is no evidence for the existence of a native S. cerevisiae lipoic acid (LA)-scavenging pathway analogous to the E. coli LplA–dependent route, since LA supplementation does not improve growth of lipoylation- or mtFASdeficient yeast strains on non-fermentable carbon sources, and Mitochondrial fatty acid synthesis (mtFAS) appears to be the sole producer of the C8 used as a precursor for LA synthesis in mitochondria [6, 9, 14]
To investigate whether fatty acid substrates fed to yeast can be transported into mitochondria and activated by a purposefully mislocalized enzyme, we took advantage of the FAM1-1 suppressor allele which encodes a mitochondrially mislocalized fatty acyl-CoA ligase that restores the growth of mtFAS mutant strains on nonfermentable carbon sources [26]
Summary
Lipoylation of 2-ketoacid dehydrogenases is essential for mitochondrial function in eukaryotes. Decades have passed since the discovery and characterization of LA, and the essentials of synthesis and attachment of LA in Escherichia coli and Bacillus subtilis have been worked out, our understanding of the basic mechanism of lipoylation in eukaryotes (Fig. 1) and the substrates used in these processes has improved only recently [3, 4]. There is no evidence for the existence of a native S. cerevisiae LA-scavenging pathway analogous to the E. coli LplA–dependent route, since LA supplementation does not improve growth of lipoylation- or mtFASdeficient yeast strains on non-fermentable carbon sources, and mtFAS appears to be the sole producer of the C8 used as a precursor for LA synthesis in mitochondria [6, 9, 14].
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