Lipoic Acid Synthesis and Attachment in Yeast Mitochondria

Abstract

Lipoic acid is a sulfur-containing cofactor required for the function of several multienzyme complexes involved in the oxidative decarboxylation of ␣-keto acids and glycine. Mechanistic details of lipoic acid metabolism are unclear in eukaryotes, despite two well defined pathways for synthesis and covalent attachment of lipoic acid in prokaryotes. We report here the involvement of four genes in the synthesis and attachment of lipoic acid in Saccharomyces cerevisiae. LIP2 and LIP5 are required for lipoylation of all three mitochondrial target proteins: Lat1 and Kgd2, the respective E2 subunits of pyruvate dehydrogenase and ␣-ketoglutarate dehydrogenase, and Gcv3, the H protein of the glycine cleavage enzyme. LIP3, which encodes a lipoate-protein ligase homolog, is necessary for lipoylation of Lat1 and Kgd2, and the enzymatic activity of Lip3 is essential for this function. Finally, GCV3, encoding the H protein target of lipoylation, is itself absolutely required for lipoylation of Lat1 and Kgd2. We show that lipoylated Gcv3, and not glycine cleavage activity per se, is responsible for this function. Demonstration that a target of lipoylation is required for lipoylation is a novel result. Through analysis of the role of these genes in protein lipoylation, we conclude that only one pathway for de novo synthesis and attachment of lipoic acid exists in yeast. We propose a model for protein lipoylation in which Lip2, Lip3, Lip5, and Gcv3 function in a complex, which may be regulated by the availability of acetyl-CoA, and which in turn may regulate mitochondrial gene expression.Several oxidative decarboxylation reactions are carried out in prokaryotes and eukaryotes by multienzyme complexes. The function of these complexes requires the action of a sulfurcontaining cofactor, lipoic acid (6,8-thioctic acid) (1, 2). Lipoic acid is covalently attached via an amide linkage to a specific lysine residue on the surface of the conserved lipoyl domain of the E2 subunits of pyruvate dehydrogenase (PDH), 3 ␣-ketoglutarate dehydrogenase (␣-KDH), the branched chain ␣-keto acid dehydrogenase complexes, and the H protein of the glycine cleavage (GC) enzyme (3). The lipoyl moiety serves as a swinging arm that shuttles reaction intermediates between active sites within the complexes (1). Despite the well characterized function of lipoic acid as a prosthetic group, the mechanisms of its synthesis and attachment to proteins are the subject of ongoing investigations (4 -7). These reactions are best understood in Escherichia coli, which has two well defined pathways for lipoic acid synthesis and attachment: a de novo pathway and a salvage pathway (8). Octanoic acid, synthesized on the acyl carrier protein (ACP) (9), is the substrate for the de novo pathway. Lipoyl synthase (LipA) catalyzes the addition of two sulfur atoms to form lipoic acid from octanoic acid either before or after transfer to the target protein (10) by lipoyl(octanoyl)-ACP:protein transferase (LipB) (11, 12). The preferred order of these two reactions is attachment of oct...

Assembly of Lipoic Acid on Its Cognate Enzymes: an Extraordinary and Essential Biosynthetic Pathway

“…However, strains lacking LIP3 had normal levels of lipoylated H protein but no lipoylated 2-oxoacid dehydrogenases detectable either by Western blotting or by dehydrogenase activity (128). This effect was specific to the H protein in that strains lacking the genes encoding the other glycine cleavage system subunits, P and T, showed normal levels of lipoylated 2-oxoacid dehydrogenases, indicating that cleavage of glycine played no role (128). Moreover, mutant strains in which the H protein lysine targeted for lipoylation was replaced with other residues also blocked all lipoylation.…”

“…Assay of the lipoylation status of the three S. cerevisiae lipoylated-protein subunits, those of the pyruvate and 2-oxoglutarate dehydrogenases and the H protein of the glycine cleavage system, showed that strains lacking the LIP5 or LIP2 gene had no detectable lipoylated proteins (128). However, strains lacking LIP3 had normal levels of lipoylated H protein but no lipoylated 2-oxoacid dehydrogenases detectable either by Western blotting or by dehydrogenase activity (128). This effect was specific to the H protein in that strains lacking the genes encoding the other glycine cleavage system subunits, P and T, showed normal levels of lipoylated 2-oxoacid dehydrogenases, indicating that cleavage of glycine played no role (128).…”

“…Echoing the LIP5 case, the LIP3 lipoate metabolism gene was isolated while screening for strains defective in mitochondrial RNA processing (128). LIP3 encoded a protein having strong sequence similarity to the E. coli LplA ligase (128). Assay of the lipoylation status of the three S. cerevisiae lipoylated-protein subunits, those of the pyruvate and 2-oxoglutarate dehydrogenases and the H protein of the glycine cleavage system, showed that strains lacking the LIP5 or LIP2 gene had no detectable lipoylated proteins (128).…”

“…Moreover, both strains were unable to use glycine as the sole ammonia source, indicating a glycine cleavage defect. Echoing the LIP5 case, the LIP3 lipoate metabolism gene was isolated while screening for strains defective in mitochondrial RNA processing (128). LIP3 encoded a protein having strong sequence similarity to the E. coli LplA ligase (128).…”

“…LIP3 encoded a protein having strong sequence similarity to the E. coli LplA ligase (128). Assay of the lipoylation status of the three S. cerevisiae lipoylated-protein subunits, those of the pyruvate and 2-oxoglutarate dehydrogenases and the H protein of the glycine cleavage system, showed that strains lacking the LIP5 or LIP2 gene had no detectable lipoylated proteins (128). However, strains lacking LIP3 had normal levels of lipoylated H protein but no lipoylated 2-oxoacid dehydrogenases detectable either by Western blotting or by dehydrogenase activity (128).…”