Cys synthesis in plants takes place in plastids, cytosol, and mitochondria. Why Cys synthesis is required in all compartments with autonomous protein biosynthesis and whether Cys is exchanged between them has remained enigmatic. This question was addressed using Arabidopsis thaliana T-DNA insertion lines deficient in the final step of Cys biosynthesis catalyzed by the enzyme O-acetylserine(thiol)lyase (OAS-TL). Null alleles of oastlA or oastlB alone showed that cytosolic OAS-TL A and plastid OAS-TL B were completely dispensable, although together they contributed 95% of total OAS-TL activity. An oastlAB double mutant, relying solely on mitochondrial OAS-TL C for Cys synthesis, showed 25% growth retardation. Although OAS-TL C alone was sufficient for full development, oastlC plants also showed retarded growth. Targeted affinity purification identified the major OAS-TL-like proteins. Two-dimensional gel electrophoresis and mass spectrometry showed no compensatory changes of OAS-TL isoforms in the four mutants. Steady state concentrations of Cys and glutathione and pulse-chase labeling with [ 35 S]sulfate indicated strong perturbation of primary sulfur metabolism. These data demonstrate that Cys and also sulfide must be sufficiently exchangeable between cytosol and organelles. Despite partial redundancy, the mitochondria and not the plastids play the most important role for Cys synthesis in Arabidopsis.

(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.

Cysteine synthesis in bacteria and plants is catalyzed by serine acetyltransferase (SAT) and O-acetylserine (thiol)-lyase (OAS-TL), which form the hetero-oligomeric cysteine synthase complex (CSC). In plants, but not in bacteria, the CSC is assumed to control cellular sulfur homeostasis by reversible association of the subunits. Application of size exclusion chromatography, analytical ultracentrifugation, and isothermal titration calorimetry revealed a hexameric structure of mitochondrial SAT from Arabidopsis thaliana (AtSATm) and a 2:1 ratio of the OAS-TL dimer to the SAT hexamer in the CSC. Comparable results were obtained for the composition of the cytosolic SAT from A. thaliana (AtSATc) and the cytosolic SAT from Glycine max (Glyma16g03080, GmSATc) and their corresponding CSCs. The hexameric SAT structure is also supported by the calculated binding energies between SAT trimers. The interaction sites of dimers of AtSATm trimers are identified using peptide arrays. A negative Gibbs free energy (⌬G ‫؍‬ ؊33 kcal mol ؊1 ) explains the spontaneous formation of the AtCSCs, whereas the measured SAT:OAS-TL affinity (K D ‫؍‬ 30 nM) is 10 times weaker than that of bacterial CSCs. Free SAT from bacteria is >100-fold more sensitive to feedback inhibition by cysteine than AtSATm/c. The sensitivity of plant SATs to cysteine is further decreased by CSC formation, whereas the feedback inhibition of bacterial SAT by cysteine is not affected by CSC formation. The data demonstrate highly similar quaternary structures of the CSCs from bacteria and plants but emphasize differences with respect to the affinity of CSC formation (K D ) and the regulation of cysteine sensitivity of SAT within the CSC.Cysteine biosynthesis in plants and bacteria is catalyzed by a two-step process. Serine acetyltransferase (SAT 2 ; EC 2.3.1.30) activates serine by transfer of the acetyl moiety from acetyl coenzyme A to form O-acetylserine (OAS). Then OAS accepts sulfide by catalysis of OAS (thiol)-lyase (OAS-TL; EC 2.5.1.47). This fixation of free sulfide from assimilatory sulfate reduction or external sulfide sources is the exclusive entry of reduced sulfur into cellular metabolism. SAT and OAS-TL form the hetero-oligomeric cysteine synthase complex (CSC). In enterobacteria and plants, the interaction of SAT and OAS-TL is stabilized by the presence of sulfide, although the addition of OAS dissociates the two enzymes (1, 2). Plant and bacterial OAS-TLs are dimers that are catalytically inactive in the CSC but become fully active upon dissociation of the complex by OAS (1, 3). However, these properties do not seem to relate to metabolic regulation of cysteine synthesis in enterobacteria. In Escherichia coli, regulation of cysteine synthesis is mainly achieved by control of the cysteine regulon that includes OAS-TL and the genes encoding for proteins catalyzing sulfate uptake and reduction but not bacterial SAT. Bacterial SAT is constitutively expressed but strongly inhibited by cysteine (K I ϭ 1.1 M cysteine). In the presence of cysteine, SAT of E. coli...

Background: Cysteine biosynthesis is the exclusive entry point for reduced sulfur in cellular metabolism. Results: The mitochondrial cysteine synthase complex (mCSC) regulates serine acetyltransferase activity in response to cysteine availability. Conclusion:The mCSC is a sensor of sulfur availability and regulates cysteine synthesis. Significance: The integration of cysteine in the regulatory model of the CSC establishes a new sensory function for the mCSC.