Mitochondrial Protein Lipoylation Does Not Exclusively Depend on the mtKAS Pathway of de Novo Fatty Acid Synthesis in Arabidopsis

Abstract

The photorespiratory Arabidopsis (Arabidopsis thaliana) mutant gld1 (now designated mtkas-1) is deficient in glycine decarboxylase (GDC) activity, but the exact nature of the genetic defect was not known. We have identified the mtkas-1 locus as gene At2g04540, which encodes b-ketoacyl-[acyl carrier protein (ACP)] synthase (mtKAS), a key enzyme of the mitochondrial fatty acid synthetic system. One of its major products, octanoyl-ACP, is regarded as essential for the intramitochondrial lipoylation of several proteins including the H-protein subunit of GDC and the dihydrolipoamide acyltransferase (E2) subunits of two other essential multienzyme complexes, pyruvate dehydrogenase and a-ketoglutarate dehydrogenase. This view is in conflict with the fact that the mtkas-1 mutant and two allelic T-DNA knockout mutants grow well under nonphotorespiratory conditions. Although on a very low level, the mutants show residual lipoylation of H protein, indicating that the mutation does not lead to a full functional knockout of GDC. Lipoylation of the pyruvate dehydrogenase and a-ketoglutarate dehydrogenase E2 subunits is distinctly less reduced than that of H protein in leaves and remains unaffected from the mtKAS knockout in roots. These data suggest that mitochondrial protein lipoylation does not exclusively depend on the mtKAS pathway of lipoate biosynthesis in leaves and may occur independently of this pathway in roots.

Discovery and Characterization of the 3-Hydroxyacyl-ACP Dehydratase Component of the Plant Mitochondrial Fatty Acid Synthase System

“…Since then, this FAS system has been characterized in yeast and humans (Hiltunen et al, 2010), confirming its multicomponent, type II character. In plant cells, to date only the mtACP isoforms (Shintani and Ohlrogge, 1994;Meyer et al, 2007), mtKAS (Olsen et al, 2004;Yasuno et al, 2004;Ewald et al, 2007), mtPPT (Guan et al, 2015), and mtMCS (Guan and Nikolau, 2016) have been experimentally identified and characterized as components that support mitochondrial fatty acid biosynthesis. In this study, we identified and characterized the Arabidopsis genetic locus AT5G60335 as encoding the mtHD component of the Arabidopsis mtFAS system, which catalyzes the third iterative reaction of the FAS cycle, the dehydration of the 3-hydroxyacyl-ACP intermediate.…”

“…The phenotypic reversal in the elevated CO 2 atmosphere is typical of photorespiratory deficiency (Somerville and Ogren, 1979), with the elevated CO 2 levels inhibiting the oxygenation reaction of Rubisco and, thus, reducing the levels of 2-phosphoglycolate, the starter metabolite of photorespiration. Furthermore, this phenotype also is conferred by mutations in other mtFAS components, such as mtkas (Ewald et al, 2007), mtppt-rnai (Guan et al, 2015), and aae13 (Guan and Nikolau, 2016).…”

“…Mitochondrial lipoic acid biosynthesis is primed with octanoyl-ACP generated by the mtFAS system (Ewald et al, 2007). Therefore, we examined the protein lipoylation status of different enzymes in the mtFAS mutants (i.e.…”

“…mthd-rnai, mtkas-2, and mtkas-2-mthd-rnai mutants). Using a western-blot procedure with antilipoic acid antibodies, we examined the lipoylation status of the H subunit of GDC, E2 subunits of mitochondrial PDH and KGDH, and plastidial PDH (Ewald et al, 2007;Guan et al, 2015;Fig. 8).…”

“…Based on the characterization of three Arabidopsis (Arabidopsis thaliana) mtFAS enzymatic components, mitochondrial b-ketoacyl-ACP synthase (mtKAS; Olsen et al, 2004;Yasuno et al, 2004;Ewald et al, 2007), phosphopantetheinyl transferase (mtPPT; Guan et al, 2015), and malonyl-CoA synthetase (mtMCS; Guan and Nikolau, 2016), it appears that the plant mtFAS system resembles the type II FAS system that occurs in bacteria and plant plastids (Ohlrogge and Jaworski, 1997;White et al, 2005). Type II FAS systems recruit ACP as the carrier of the intermediates of the process and utilize dissociated, monofunctional enzymes to catalyze the iterative reactions that produce fatty acids (Hiltunen et al, 2010).…”