Genetic dissection of the mitochondrial lipoylation pathway in yeast

Pietikäinen et al. BMC Biology (2021) 19:14 https://doi.org/10.1186/s12915-021-00951-3 RESEARCH ARTICLE Open Access Genetic dissection of the mitochondrial lipoylation pathway in yeast Laura P. Pietikäinen1, M. Tanvir Rahman1, J. Kalervo Hiltunen1, Carol L. Dieckmann2 and Alexander J. Kastaniotis1* Abstract Background: Lipoylation 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. Results: We 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. Conclusions: In 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. Keywords: Lipoylation, Mitochondrial fatty acid synthesis (mtFAS), Octanoyl/lipoyl transferases, S. cerevisiae model, Supplementation studies, Lip3/LIPT1, Lip2/LIPT2, Lip3 substrate, Lipoylation disorders Background Like its functional cousin biotin, the enzyme cofactor lipoic acid (LA) acts as a “swinging arm” moiety to shuttle reaction intermediates from one enzymatic reaction center to another in multidomain protein complexes catalyzing oxidative decarboxylation of 2-ketoacids [1]. In eukaryotes, the octanoic acid (C8) precursor of endogenously * Correspondence: 1 Faculty of Biochemistry and Molecular Medicine and Biocenter Oulu, University of Oulu, PO Box 5400, FI-90014 Oulu, Finland Full list of author information is available at the end of the article synthesized LA is generated by mitochondrial fatty acid synthesis (mtFAS). In humans, all LA-dependent enzyme complexes are mitochondrial: pyruvate dehydrogenase (PDH), α-ketoglutarate dehydrogenase (KGD), the glycine cleavage system (GCS), branched chain dehydrogenase (BCD), and α-ketoadipate dehydrogenase (OAD) [2]. With the exceptions of BCD and OAD, the LAdependent complexes are found also in mitochondria of the yeast Saccharomyces cerevisiae. Invariantly, LA is attached to the N6-amino group of specific lysine residues of the E2 (PDH, KGD, BCD) or H (GCS) protein © The Author(s). 2021 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Pietikäinen et al. BMC Biology (2021) 19:14 subunits of these complexes in a stable amide linkage. Because of the central role for KGD in the tricarboxylic acid cycle, LA is essential for cells and organisms that cannot satisfy their energy demands solely by glycolysis. Although 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]. A basic set of three enzymes appears to be required for lipoylation: lipoyl synthase LipA/Lip5/LIAS (E. coli/S. cerevisiae/humans) [5–7] and the lipoyl/octanoyl transferases LipB/Lip2/LIPT2 [7–9] and LplA/Lip3/LIPT1 [10–13]. In E. coli, the LipB transferase in combination with the LA synthase LipA is sufficient for lipoylation of all LA-requiring enzymes. In an alternative route for protein lipoylation in E. coli, the lipoyl transferase LplA activates exogenously available free LA by adenylation, and then transfers it to the target proteins. In yeast, Lip2 can modify only the Gcv3 H-subunit of the GCS. Lipoylated Gcv3 is then the substrate for subsequent lipoylation of the E2 subunits by Lip3-catalyzed transamidation [11]. 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]. The schematic depicted in Fig. 1 summarizes the current model of the lipoylation pathway in eukaryotes. Page 2 of 15 Several groups of disorders caused by of inborn errors leading in humans that affect lipoylation in mitochondria have been described [15]. The first group encompasses defects due to failures in synthesis of Fe-S clusters [16–19]. A second group of mitochondrial diseases is defined by mutations in lipoic acid synthase, Lip5/LIAS (S. cerevisiae/ humans), and pro (...truncated)