Plant Science 207 (2013) 148–157 Contents lists available at SciVerse ScienceDirect Plant Science jou rn al hom epa ge: www.elsevier.com/locate/plantsci Direct targeting of Arabidopsis cysteine synthase complexes with synthetic polypeptides to selectively deregulate cysteine synthesis Anna Wawrzy ´ nska ∗ , Agata Kurzyk, Monika Mierzwi ´ nska, Danuta Płochocka, Grzegorz Wieczorek, Agnieszka Sirko Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Pawi´ nskiego 5A St, 02-106 Warsaw, Poland a r t i c l e i n f o Article history: Received 27 November 2012 Received in revised form 22 February 2013 Accepted 24 February 2013 Available online 14 March 2013 Keywords: Serine acetyltransferase Cysteine Cysteine synthase complex Synthetic polypeptide Protein–protein interaction a b s t r a c t Biosynthesis of cysteine is one of the fundamental processes in plants providing the reduced sulfur for cell metabolism. It is accomplished by the sequential action of two enzymes, serine acetyltransferase (SAT) and O-acetylserine (thiol) lyase (OAS-TL). Together they constitute the hetero-oligomeric cysteine synthase (CS) complex through specific protein–protein interactions influencing the rate of cysteine production. The aim of our studies was to deregulate the CS complex formation in order to investigate its function in the control of sulfur homeostasis and optimize cysteine synthesis. Computational modeling was used to build a model of the Arabidopsis thaliana mitochondrial CS complex. Several polypeptides based on OAS-TL C amino-acid sequence found at SAT-OASTL interaction sites were designed as probable competitors for SAT3 binding. After verification of the binding in a yeast two-hybrid assay, the most strongly interacting polypeptide was introduced to different cellular compartments of Arabidopsis cell via genetic transformation. Moderate increase in total SAT and OAS-TL activities, but not thiols content, was observed dependent on the transgenic line and sulfur availability in the hydroponic medium. Though our studies demonstrate the proof of principle, they also suggest more complex interaction of both enzymes underlying the mechanism of their reciprocal regulation. © 2013 Elsevier Ireland Ltd. All rights reserved. 1. Introduction Cysteine is the first organic compound, biosynthesized in plant cells after sulfate assimilation and reduction. It is the main donor for all subsequent compounds with reduced sulfur. Together with methionine it is crucial for proper protein structure and function but it also constitutes the tripeptide glutathione, the first shield of defense against free radicals and the major storage compound for soluble reduced sulfur [1]. Besides, it is the precursor of iron–sulfur clusters which are among the oldest and most versatile cofactors participating in many regulatory processes [2]. Biosynthesis of cysteine in plants, but also in archea and eubacteria, is carried out by a two-step pathway [3]. In the first step serine acetyltransferase (SAT; acetyl-CoA:L-serine O- acetyltransferase; EC 2.3.1.30) transfers an acetyl-moiety from Abbreviations: AD, transcription activation domain of GAL4; BD, DNA-binding domain of GAL4; CS, cysteine synthase; Cys, cysteine; FW, fresh weight; GAL4, transcriptional activator of galactose regulon in yeast; OAS, O-acetylserine; OAS-TL, O-acetylserine (thiol) lyase (EC 2.5.1.47); PEP, polypeptide; rbcS1, small subunit of ribulose-1,5-bisphosphate carboxylase (EC 4.1.1.39); SAT, serine acetyltransferase (EC 2.3.1.30); Y2H, yeast two-hybrid. ∗ Corresponding author. Tel.: +48 22 5925749; fax: +48 22 6584804. E-mail address: blaszczyk@ibb.waw.pl (A. Wawrzy ´ nska). acetyl coenzyme A to serine to form O-acetylserine (OAS). Subsequently, O-acetylserine (thiol) lyase (OAS-TL; O3-acetyl- l-serine:hydrogen-sulfide 2-amino-2-carboxyethyltransferase; EC 2.5.1.47) exchanges the activated acetyl group with sulfide by a -replacement reaction to produce cysteine. It is well established, that OAS-TL and SAT physically interact to form multimeric com- plex known as cysteine synthase (CS) complex. Interestingly, the function of the complex formation is not metabolic channeling, but sensing the sulfur status of the cell to properly adjust the sulfur homeostasis. Whereas OAS-TL is only active outside the CS complex, SAT is strongly activated by association with OAS- TL [3]. The stability of the CS complex is reciprocally regulated by free sulfide and OAS. When sulfur is not limiting, sulfide pro- duced by assimilatory sulfate reduction pathway stabilizes the CS complex. However, when sulfide concentrations decrease due to sulfate deprivation, excess OAS promotes dissociation of CS com- plex, resulting in the formation of less active free SAT. When sulfide becomes available the CS complex associates again. In this way, the CS complex effectively senses both OAS and sulfur avail- ability, and self-regulates further OAS production accordingly to supply and demand. The situation is additionally complicated by uneven distribution of both enzymes between cytosol, plastids and mitochondria. This subcellular compartmentalization is in con- trast to provision of sulfide that takes place exclusively in plastids, 0168-9452/$ – see front matter © 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.plantsci.2013.02.016