Mitochondrial Serine Acetyltransferase Functions as a Pacemaker of Cysteine Synthesis in Plant Cells    

Abstract

(R.Q., A.B.) Cysteine (Cys) synthesis in plants is carried out by two sequential reactions catalyzed by the rate-limiting enzyme serine acetyltransferase (SAT) and excess amounts of O-acetylserine(thiol)lyase. Why these reactions occur in plastids, mitochondria, and cytosol of plants remained unclear. Expression of artificial microRNA (amiRNA) against Sat3 encoding mitochondrial SAT3 in transgenic Arabidopsis (Arabidopsis thaliana) plants demonstrates that mitochondria are the most important compartment for the synthesis of O-acetylserine (OAS), the precursor of Cys. Reduction of RNA levels, protein contents, SAT enzymatic activity, and phenotype strongly correlate in independent amiSAT3 lines and cause significantly retarded growth. The expression of the other four Sat genes in the Arabidopsis genome are not affected by amiRNA-SAT3 according to quantitative real-time polymerase chain reaction and microarray analyses. Application of radiolabeled serine to leaf pieces revealed severely reduced incorporation rates into Cys and even more so into glutathione. Accordingly, steady-state levels of OAS are 4-fold reduced. Decrease of sulfate reduction-related genes is accompanied by an accumulation of sulfate in amiSAT3 lines. These results unequivocally show that mitochondria provide the bulk of OAS in the plant cell and are the likely site of flux regulation. Together with recent data, the cytosol appears to be a major site of Cys synthesis, while plastids contribute reduced sulfur as sulfide. Thus, Cys synthesis in plants is significantly different from that in nonphotosynthetic eukaryotes at the cellular level.

Mitochondrial Cysteine Synthase Complex Regulates O-Acetylserine Biosynthesis in Plants

“…Whether the cysteine-dependent regulation of the mCSC also applies to cytosolic and plastidic CSCs in Arabidopsis is questionable because cytosolic, plastidic, and mitochondrial SATs are subject to different cysteine feedback inhibition sensitivities (37). The 50% decrease in the incorporation capacity of serine into OAS in oastl-C and oastl-AC is in agreement with the expected predominant role of the mCSC in regulation of total SAT activity (2,11,13) and provides a molecular explanation for the observed growth phenotype of the oastl-C mutant (12). The decrease in the total serine incorporation capacity in the oastl-A mutant after 25 min points to a role for the cytosolic CSC in regulation of cellular SAT activity, although to a minor extent compared with the mCSC.…”

“…Reverse genetic approaches indicated significant export of OAS from mitochondria to support synthesis of cysteine in the cytosol and plastids (2,11,12). In both compartments, sulfide and OAS encounter a high excess of the free catalytically active OAS-TL dimer (12) that is necessary for full conversion of OAS to cysteine (28,29).…”

“…T-DNA insertion mutants for each of the SAT genes are viable, demonstrating that individually OAS synthesis in the cytosol, plastid, or mitochondria is not essential (11,13). Knockdown of mitochondrial SAT3 causes significant growth retardation, suggesting a major role for the mitochondria in supplying OAS for cysteine synthesis (2). A prominent role of the mitochondrial CSC (mCSC) in regulation of SAT3 activity and thereby cellular cysteine is also indicated by the growth retardation phenotype of oastl-C, whereas oastl-A and oastl-B mutants are unaffected (12).…”

“…Subsequently, OAS (thiol) lyase (OAS-TL; EC 2.5.1.47) replaces the acetyl group of OAS with sulfide and releases cysteine (1). Reverse genetic approaches and biochemical studies of Arabidopsis OAS-TL isoforms demonstrated that cysteine biosynthesis in plants is limited by OAS supply (2)(3)(4). The transcript abundance, protein level, and extractable activity of SAT and OAS-TLs are not significantly altered by sulfur limitation or genetic manipulation of the sulfur assimilation pathway.…”

The γ-Carbonic Anhydrase Subcomplex of Mitochondrial Complex I Is Essential for Development and Important for Photomorphogenesis of Arabidopsis

“…S11B). The left-handed b-helix structure is widely found in members of the acyltransferase superfamily (Raetz and Roderick, 1995 Pye et al, 2004;Haas et al, 2008). The left-handed b-helix structure constitutes the active site of the acetyltransferases (Pye et al, 2004).…”