Sensing of Internal Sulfur Supply by the Cysteine Synthase Complex
Fig. 1: Biosynthesis of Cysteine in higher plants.
Fig. 2: Regulation of SAT activity by the cysteine synthase complex model.
Fig. 3: Analysis of SAT and OAS-TL interaction by fluorescence resonance energy transfer (FRET).
Fig. 4: Analysis of stochiomtery and affinity of the cysteine synthase complex by isothermal titration calorimetry.
Plants are primary producers and carry out assimilatory sulfate reduction to synthesize cysteine that is the precursor for all metabolites containing reduced sulfur. The position of cysteine biosynthesis between assimilation of inorganic sulfate and metabolization of organic sulfide makes it a prime target for coordination of both complex processes. It is thus a mediator between supply and demand in sulfur metabolism of a cell. Biosynthesis of Cys is a tripartite process, which can be subdivided into (1) synthesis of O-acetylserine (OAS) by serine acetyltransferase (SAT), (2) generation of sulfide by ASRP and (3) the combination of OAS and sulfide by O-acetylserine (thiol) lyase (OAS-TL) to produce Cys (Fig 1). Interestingly SAT and OAS-TL are able to interact with each other to form the decameric cysteine synthase complex (CSC). The association of CSC is accompanied by (1) activation of SAT to form OAS and (2) strong inactivation of OAS-TL which rules out any significant transfer of the reaction intermediate OAS from SAT to OAS-TL. Thus, in contrast to any other know complex of proteins, which catalyze subsequent steps in a reaction pathway, the function of CSC formation is not substrate channeling. The association of CSC is reversible and strongly dependant of cellular concentrations of the key effector metabolites sulfide and OAS. A regulatory circuit with the CSC as essential component has been proposed that adjusts the rate of cysteine biosynthesis by sensing the availability of sulfide and controlling the expression of genes encoding plasmalemma sulfate transporters and plastidic enzymes of sulfate reduction (Fig 2). In this project the reversible association of CSC is analyzed in vivo by fluoresence resonance energy transfer (FRET) between YFP and CFP-fusion proteins of SAT and OAS-TL (Fig 3). The stoichiometry and kinetics of CSC formation in response to the effectors are further characterized in vitro by using isothermal titration calorimetry (ITC, Fig 4), analytical ultracentrifugation (AUC) and surface plasmon resonance (SPR).
Compartmentation of Cysteine Biosynthesis
Fig. 1: Schematic overview of the compartimentalization of cysteine biosynthesis in higher plants
Fig. 2: Sequence Comparison of the OAS-TL gene family in Arabidopsis
Fig. 3: Phenotype of OAS-TL T-DNA knock-out mutants
Fig.4: Growth Curve and Phenotype of plants with impaired sufate reduction capacity in plastids.
Cysteine (Cys) is a proteinogenic amino acid and thus indispensable for any living cell. It must be provided from outside or synthesized inside the cell. Animal cells either take up Cys with their diet or synthesize it from methionine via trans-sulfurylation of cystathionine in the cytosol. Yeast cells, in addition to uptake, are able to synthesize Cys in the cytosol from sulfide and O-acetylhomoserine and by trans-sulfurylation. Mitochondria in such organisms must receive Cys for their protein translation machinery from the cytosol, although little is known about such amino acid permeases. In plants the situation is entirely different: not only are three compartments with independent protein biosynthesis present in the cell, but Cys can be synthesized in plastids, mitochondria and the cytosol (Fig 1). O-acetylserine(thiol)lyase (OAS-TL) catalyses the formation Cys by replacing the activated acetyl-moiety in O-acetylserine (OAS) with sulfide. In the model plant Arabidopsis thaliana OAS-TL is encoded by a small gene family that consists of nine members, which are distributed in the resepctive sub-cellular compartments (Fig 2). The exclusive location of sulfite reduction by sulfite reductase (SIR) in plastids suggests that sulfide is able to reach Cys synthesis in cytosol and mitochondria, possibly by diffusion of sulfide through membranes. In contrast OAS is produced by serine acetyltransferase (SAT) in all sub-cellular compartments with own Cys biosynthesis. Why Cys biosynthesis is organized in this way in higher plants remained unexplained so far nor is anything known about special or redundant functions or communication between the three cellular locations. In this project the impact of cysteine synthesis in a particular sub-cellular compartment will be analyzed by using knock out mutant and ami-RNAi approaches for SAT and OAS-TL (Fig 3). A knock down mutant of SIR will be used to elucidate the overall importance of plastid localized sulfite reduction for Cys biosynthesis (Fig 4).