Direct targeting of Arabidopsis cysteine synthase complexes with synthetic polypeptides to selectively deregulate cysteine synthesis

摘要

Biosynthesis of cysteine is one of the fundamental processes in plants providing the reduced sulfur forudcell metabolism. It is accomplished by the sequential action of two enzymes, serine acetyltransferaseud(SAT) and O-acetylserine (thiol) lyase (OAS-TL). Together they constitute the hetero-oligomeric cysteineudsynthase (CS) complex through specific protein–protein interactions influencing the rate of cysteineudproduction. The aim of our studies was to deregulate the CS complex formation in order to investigate itsudfunction in the control of sulfur homeostasis and optimize cysteine synthesis. Computational modelingudwas used to build a model of the Arabidopsis thaliana mitochondrial CS complex. Several polypeptidesudbased on OAS-TL C amino-acid sequence found at SAT-OASTL interaction sites were designed as probableudcompetitors for SAT3 binding. After verification of the binding in a yeast two-hybrid assay, the mostudstrongly interacting polypeptide was introduced to different cellular compartments of Arabidopsis cell viaudgenetic transformation. Moderate increase in total SAT and OAS-TL activities, but not thiols content, wasudobserved dependent on the transgenic line and sulfur availability in the hydroponic medium. Though ourudstudies demonstrate the proof of principle, they also suggest more complex interaction of both enzymesudunderlying the mechanism of their reciprocal regulation.