Differential Inhibition of Arabidopsis Methionine Adenosyltransferases by Protein S-Nitrosylation

Abstract

In animals, protein S-nitrosylation, the covalent attachment of NO to the thiol group of cysteine residues, is an intensively investigated posttranslational modification, which regulates many different processes. A growing body of evidence suggests that this type of redox-based regulation mechanism plays a pivotal role in plants, too. Here we report the molecular mechanism for S-nitrosylation of methionine adenosyltransferase (MAT) of Arabidopsis thaliana, thereby presenting the first detailed characterization of S-nitrosylation in plants. We cloned three MAT isoforms of Arabidopsis and tested the effect of NO on the activity of the purified, recombinant proteins. Our data showed that incubation with GSNO resulted in blunt, reversible inhibition of MAT1, and of the of inhibition of MAT1, this is in and of that is the of this is effect of GSNO of and of that of is MAT the of the and NO is to in plants, this the and NO In animals, protein S-nitrosylation, the covalent attachment of NO to the thiol group of cysteine residues, is an intensively investigated posttranslational modification, which regulates many different processes. A growing body of evidence suggests that this type of redox-based regulation mechanism plays a pivotal role in plants, too. Here we report the molecular mechanism for S-nitrosylation of methionine adenosyltransferase (MAT) of Arabidopsis thaliana, thereby presenting the first detailed characterization of S-nitrosylation in plants. We cloned three MAT isoforms of Arabidopsis and tested the effect of NO on the activity of the purified, recombinant proteins. Our data showed that incubation with GSNO resulted in blunt, reversible inhibition of MAT1, and of the of inhibition of MAT1, this is in and of that is the of this is effect of GSNO of and of that of is MAT the of the and NO is to in plants, this the and NO NO is a that plays in regulation of and and NO is in many different is that NO in NO for the different a which is in in Arabidopsis of NO and to this of NO of is the of is the of this in of the of NO a of and NO NO of protein NO is a of the and which to is for the and of is an which is of for for NO to of is for the of with and molecular to for and in NO with the thiol group of cysteine to is for methionine adenosyltransferase and of with and with the of to a protein S-nitrosylation a regulation and many the of this redox-based mechanism for different in cysteine residues, is a of protein that the to S-nitrosylation and the of a that the of the and NO the in NO methionine methionine to to and the cysteine for protein S-nitrosylation in In is evidence for three to Arabidopsis NO the of and in that and the in NO S-nitrosylation we Arabidopsis and Arabidopsis for protein S-nitrosylation and group of the methionine and adenosyltransferase (MAT) MAT the of which is the in and a for the of and the of this of is that MAT In and different MAT that an is in the is in the is that is is for the is the S-nitrosylation of the cysteine of which is a that to the that the NO the activity of MAT NO regulates the of and the of the is this we report the inhibition of MAT of Arabidopsis the NO We the of three MAT isoforms and the in with recombinant with GSNO that the activity of is to the isoforms this and showed that S-nitrosylation of the cysteine of is for the inhibition of the the first that is S-nitrosylation of a cysteine to the of MAT in the regulation of and of methionine adenosyltransferase A and for the of the of MAT1, and of of MAT the of the different MAT isoforms the Arabidopsis and for and the of for of and and for of and a of of the the and of the of the and the to the of the the of the different MAT isoforms the in and of the in of recombinant with incubation for protein the in an of and recombinant the with to the in and with and activity with of of to of a and for for and on of the in and with for to of and the of for a of the of the to a of the of of in a of and of of and a for the and with and the recombinant protein in of of with of for this to a the to a of of a of of the NO group and A of and of Arabidopsis in in the with different of GSNO and in the of the the and of the of the a with a with a and a a we S-nitrosylation which is for for NO in We Arabidopsis with different of the NO GSNO and GSNO we a inhibition of the of a incubation the inhibition of to and with GSNO to of and the is with incubation of Arabidopsis with Arabidopsis with and GSNO and the the the of mechanism for of the S-nitrosylation of the data we different in of the different and and cysteine and of the of Arabidopsis MAT cysteine and of is with an the with to the of the activity of the Arabidopsis MAT is NO we first to the of the Arabidopsis proteins. the of the of and we on the isoforms and of the of three MAT isoforms and the in on the three showed the molecular of in effect of NO on the activity of the different MAT isoforms tested the with GSNO and activity of to the activity of of activity of the of with different of GSNO showed that a of the activity to inhibition of Arabidopsis MAT isoforms recombinant MAT in and in the of GSNO for activity MAT to with activity of to the of different inhibition of Arabidopsis purified, recombinant with different of GSNO for the the of three different and activity of Arabidopsis MAT with for the activity of the with activity of the to of the mechanism of MAT we tested the of the cysteine and on the activity of the different MAT is a that and cysteine thiol is an that of a and cysteine thiol in a with proteins. in to to of MAT1, and activity to and We a with and showed a activity of and MAT in a with the and this with the Arabidopsis MAT to the of MAT a to the of the Arabidopsis MAT MAT is a of which the of the and an the of the cysteine of the MAT isoforms to cysteine for of the cysteine and for the cysteine of is to the of the of Arabidopsis MAT of the MAT isoforms with the of MAT A and of the MAT of the the of is and of is the and the this of and in this cysteine is a for S-nitrosylation, we the this cysteine to the for protein S-nitrosylation and to the for the of cysteine of the cysteine and of the of the cysteine a is of an in the the is GSNO first for in the for data and to in for protein S-nitrosylation, and In the GSNO for is the of Arabidopsis that the and of the S-nitrosylation to the group of a of the the of the thiol group of to the group of is and to the group of and is and that for GSNO and in a of and to the thiol group of of the of with the and the GSNO for protein S-nitrosylation for protein S-nitrosylation the cysteine GSNO the in a of the of Arabidopsis the of Arabidopsis with the of MAT A and is in the thiol group of and the of the and the is we and with an We a of in with the of a of of the and of the the and the with the cysteine in and the for the of resulted in the of the NO group of and of the of of S-nitrosylation of recombinant with GSNO for and with of the and MAT1, to S-nitrosylation of for the different to the and the to the in a of S-nitrosylation of recombinant with GSNO for and with of and to S-nitrosylation of of a of Arabidopsis the to that is for the inhibition of the recombinant with and the activity in GSNO the to and activity with incubation of the with the activity to for the of GSNO and on recombinant with GSNO and for and activity activity of the to for the different to in the GSNO and of plays a role in and in the the of of NO mechanism NO protein is the of protein Here we the inhibition of Arabidopsis MAT isoforms NO and the of inhibition of in we Arabidopsis MAT for protein S-nitrosylation the In Arabidopsis MAT isoforms with of for and MAT protein and for of activity suggests the of cysteine in the of MAT Arabidopsis MAT isoforms to in to the effect of NO on Arabidopsis MAT Our data showed that incubation of the purified, recombinant with GSNO resulted in inhibition of MAT1, and inhibition the to the reversible effect of NO is for protein S-nitrosylation and for the activity of and the cysteine of the three MAT isoforms we the of the of inhibition of MAT1, this is the of this and is in and and evidence for the of this for inhibition the effect of GSNO of that this cysteine is the for inhibition of of that of is GSNO and GSNO to activity for the of to NO the of we the for protein S-nitrosylation to the GSNO and residues, which S-nitrosylation of the the of the thiol group of the of the NO In the is to the thiol group of on the of the group of on the the of S-nitrosylation of GSNO in a that the NO group of GSNO is to the group of the molecular and data and to in which to to S-nitrosylation of GSNO is the of GSNO and the of the of the In the of for of and and the thiol group of in of GSNO for an NO a of the of NO to the a in a of the NO of MAT1, the NO of and the of that is the for regulation of of the with the and resulted in of activity for of cysteine of and the to in protein which a effect on the for the activity of and with the the effect is is for and inhibition of to and the of to GSNO is a of S-nitrosylation of of the that of cysteine that the for is that is the and of is for and for the activity of is the of for the regulation of S-nitrosylation of of a effect on the activity of that cysteine to redox-based in the of which protein of of animals, protein S-nitrosylation regulates many different and protein In of the of protein S-nitrosylation in is the a of S-nitrosylation is for Arabidopsis protein is to NO of and NO a role in NO is evidence that of S-nitrosylation of cysteine of in that GSNO of Arabidopsis to and in and GSNO the of in of GSNO to on the of in a of plants, NO and to for and and NO of and that of and in with a of NO and of that NO to in and A of NO and in and in and and the effect of NO in the of with that NO of Arabidopsis that NO inhibition of activity S-nitrosylation in of the and we that this protein a for NO and MAT is the NO showed that in of different NO in the of the that NO the S-nitrosylation of and methionine of the and of the In we the first detailed molecular characterization of S-nitrosylation in plants. of of of the a on NO is a that plays in regulation of and and NO is in many different is that NO in NO for the different a which is in in Arabidopsis of NO and to this of NO of is the of is the of this in of the of NO a of and NO NO of protein NO is a of the and which to is for the and of is an which is of for for NO to of is for the of with and molecular to for and in NO with the thiol group of cysteine to is for methionine adenosyltransferase and of with and with the of to a protein S-nitrosylation a regulation and many the of this redox-based mechanism for different in cysteine residues, is a of protein that the to S-nitrosylation and the of a that the of the and NO the in NO methionine methionine to to and the cysteine for protein S-nitrosylation in In is evidence for three to Arabidopsis NO the of and in that and the in NO S-nitrosylation we Arabidopsis and Arabidopsis for protein S-nitrosylation and group of the methionine and adenosyltransferase (MAT) MAT the of which is the in and a for the of and the of this of is that MAT In and different MAT that an is in the is in the is that is is for the is the S-nitrosylation of the cysteine of which is a that to the that the NO the activity of MAT NO regulates the of and the of the is In this we report the inhibition of MAT of Arabidopsis the NO We the of three MAT isoforms and the in with recombinant with GSNO that the activity of is to the isoforms this and showed that S-nitrosylation of the cysteine of is for the inhibition of the the first that is S-nitrosylation of a cysteine to the of MAT in the regulation of and of methionine adenosyltransferase A and for the of the of MAT1, and of of MAT the of the different MAT isoforms the Arabidopsis and for and the of for of and and for of and a of of the the and of the of the and the to the of the the of the different MAT isoforms the in and of the in of recombinant with incubation for protein the in an of and recombinant the with to the in and with and activity with of of to of a and for for and on of the in and with for to of and the of for a of the of the to a of the of of in a of and of of and a for the and with and the recombinant protein in of of with of for this to a the to a of of a of of the NO group and A of and of Arabidopsis in in the with different of GSNO and in the of the the and of the of the a with a with a and a and of methionine adenosyltransferase A and for the of the of MAT1, and of of MAT the of the different MAT isoforms the Arabidopsis and for and the of for of and and for of and a of of the the and of the of the and the to the of the the of the different MAT isoforms the in and of the in of recombinant with incubation for protein the in an of and recombinant the with to the in and MAT with and activity with of of to of a and for for and on of the in and with for to of and the of for a of the of the to a of the of of in a of and of of and a for the and with and the recombinant protein in of of with of for this to a the to a of of a of of the NO group and A of and of Arabidopsis in in the with different of GSNO and in the of the the and of the of the a with a with a and a a we S-nitrosylation which is for for NO in We Arabidopsis with different of the NO GSNO and GSNO we a inhibition of the of a incubation the inhibition of to and with GSNO to of and the is with incubation mechanism for of the S-nitrosylation of the data we different in of the different and and cysteine and of the of Arabidopsis MAT cysteine and of is with an the with to the of the activity of the Arabidopsis MAT is NO we first to the of the Arabidopsis proteins. the of the of and we on the isoforms and of the of three MAT isoforms and the in on the three showed the molecular of in effect of NO on the activity of the different MAT isoforms tested the with GSNO and activity of to the activity of of activity of the of with different of GSNO showed that a of the activity to inhibition of Arabidopsis MAT isoforms recombinant MAT in and in the of GSNO for activity MAT to with activity of to the of different inhibition of Arabidopsis purified, recombinant with different of GSNO for the the of three different and activity of Arabidopsis MAT with for the activity of the with activity of the to of the mechanism of MAT we tested the of the cysteine and on the activity of the different MAT is a that and cysteine thiol is an that of a and cysteine thiol in a with proteins. in to to of MAT1, and activity to and We a with and showed a activity of and MAT in a with the and this with the Arabidopsis MAT to the of MAT a to the of the Arabidopsis MAT MAT is a of which the of the and an the of the cysteine of the MAT isoforms to cysteine for of the cysteine and for the cysteine of is to the of the of Arabidopsis MAT of the MAT isoforms with the of MAT A and of the MAT of the the of is and of is the and the this of and in this cysteine is a for S-nitrosylation, we the this cysteine to the for protein S-nitrosylation and to the for the of cysteine of the cysteine and of the of the cysteine a is of an in the the is GSNO first for in the for data and to in for protein S-nitrosylation, and In the GSNO for is the of Arabidopsis that the and of the S-nitrosylation to the group of a of the the of the thiol group of to the group of is and to the group of and is and that for GSNO and in a of and to the thiol group of of the of with the and the GSNO for protein S-nitrosylation for protein S-nitrosylation the cysteine GSNO the in a of the of Arabidopsis the of Arabidopsis with the of MAT A and is in the thiol group of and the of the and the is we and with an We a of in with the of a of of the and of the the and the with the cysteine in and the for the of resulted in the of the NO group of and of the of of S-nitrosylation of recombinant with GSNO for and with of the and MAT1, to S-nitrosylation of for the different to the and the to the in a of S-nitrosylation of recombinant with GSNO for and with of and to S-nitrosylation of of a of Arabidopsis the to that is for the inhibition of the recombinant with and the activity in GSNO the to and activity with incubation of the with the activity to for the of GSNO and on recombinant with GSNO and for and activity activity of the to for the different to in the GSNO and of In a we S-nitrosylation which is for for NO in We Arabidopsis with different of the NO GSNO and GSNO we a inhibition of the of a incubation the inhibition of to and with GSNO to of and the is with incubation A mechanism for of the S-nitrosylation of the data we different in of the different and and cysteine and the activity of the Arabidopsis MAT is NO we first to the of the Arabidopsis proteins. the of the of and we on the isoforms and of the of three MAT isoforms and the in on the three showed the molecular of in effect of NO on the activity of the different MAT isoforms tested the with GSNO and activity of to the activity of of activity of the of with different of GSNO showed that a of the activity to the mechanism of MAT we tested the of the cysteine and on the activity of the different MAT is a that and cysteine thiol is an that of a and cysteine thiol in a with proteins. in to to of MAT1, and activity to and We a with and showed a activity of and MAT in a with the and this with the Arabidopsis MAT to the of MAT a to the of the Arabidopsis MAT MAT is a of which the of the and an the of the cysteine of the MAT isoforms to cysteine for of the cysteine and for the cysteine of is to the of the this cysteine is a for S-nitrosylation, we the this cysteine to the for protein S-nitrosylation and to the for the of cysteine of the cysteine and of the of the cysteine a is of an in the the is GSNO first for in the for data and to in for protein S-nitrosylation, and In the GSNO for is the of Arabidopsis that the and of the S-nitrosylation to the group of a of the the of the thiol group of to the group of is and to the group of and is and that for GSNO and in a of and to the thiol group of the is we and with an We a of in with the of a of of the and of the the and the with the cysteine in and the for the of resulted in the of the NO group of and of the of of a of Arabidopsis the to that is for the inhibition of the recombinant with and the activity in GSNO the to and activity with incubation of the with the activity to for the plays a role in and in the the of of NO mechanism NO protein is the of protein Here we the inhibition of Arabidopsis MAT isoforms NO and the of inhibition of in we Arabidopsis MAT for protein S-nitrosylation the In Arabidopsis MAT isoforms with of for and MAT protein and for of activity suggests the of cysteine in the of MAT Arabidopsis MAT isoforms to in to the effect of NO on Arabidopsis MAT Our data showed that incubation of the purified, recombinant with GSNO resulted in inhibition of MAT1, and inhibition the to the reversible effect of NO is for protein S-nitrosylation and for the activity of and the cysteine of the three MAT isoforms we the of the of inhibition of MAT1, this is the of this and is in and and evidence for the of this for inhibition the effect of GSNO of that this cysteine is the for inhibition of of that of is GSNO and GSNO to activity for the of to NO the of we the for protein S-nitrosylation to the GSNO and residues, which S-nitrosylation of the the of the thiol group of the of the NO In the is to the thiol group of on the of the group of on the the of S-nitrosylation of GSNO in a that the NO group of GSNO is to the group of the molecular and data and to in which to to S-nitrosylation of GSNO is the of GSNO and the of the of the In the of for of and and the thiol group of in of GSNO for an NO a of the of NO to the a in a of the NO of MAT1, the NO of and the of that is the for regulation of of the with the and resulted in of activity for of cysteine of and the to in protein which a effect on the for the activity of and with the the effect is is for and inhibition of to and the of to GSNO is a of S-nitrosylation of of the that of cysteine that the for is that is the and of is for and for the activity of is the of for the regulation of S-nitrosylation of of a effect on the activity of that cysteine to redox-based in the of which protein of of animals, protein S-nitrosylation regulates many different and protein In of the of protein S-nitrosylation in is the a of S-nitrosylation is for Arabidopsis protein is to NO of and NO a role in NO is evidence that of S-nitrosylation of cysteine of in that GSNO of Arabidopsis to and in and GSNO the of in of GSNO to on the of in a of plants, NO and to for and and NO of and that of and in with a of NO and of that NO to in and A of NO and in and in and and the effect of NO in the of with that NO of Arabidopsis that NO inhibition of activity S-nitrosylation in of the and we that this protein a for NO and MAT is the NO showed that in of different NO in the of the that NO the S-nitrosylation of and methionine of the and of the In we the first detailed molecular characterization of S-nitrosylation in plants. of of of the a on plays a role in and in the the of of NO mechanism NO protein is the of protein Here we the inhibition of Arabidopsis MAT isoforms NO and the of inhibition of in we Arabidopsis MAT for protein S-nitrosylation the In Arabidopsis MAT isoforms with of for and MAT protein and for of activity suggests the of cysteine in the of MAT Arabidopsis MAT isoforms to in to the effect of NO on Arabidopsis MAT Our data showed that incubation of the purified, recombinant with GSNO resulted in inhibition of MAT1, and inhibition the to the reversible effect of NO is for protein S-nitrosylation and for the activity of and the cysteine of the three MAT isoforms we the of the of inhibition of MAT1, this is the of this and is in and and evidence for the of this for inhibition the effect of GSNO of that this cysteine is the for inhibition of of that of is GSNO and GSNO to activity for the of to NO the of we the for protein S-nitrosylation to the GSNO and residues, which S-nitrosylation of the the of the thiol group of the of the NO In the is to the thiol group of on the of the group of on the the of S-nitrosylation of GSNO in a that the NO group of GSNO is to the group of the molecular and data and to in which to to S-nitrosylation of GSNO is the of GSNO and the of the of the In the of for of and and the thiol group of in of GSNO for an NO a of the of NO to the a in a of the NO of MAT1, the NO of and the of that is the for regulation of of the with the and resulted in of activity for of cysteine of and the to in protein which a effect on the for the activity of and with the the effect is is for and inhibition of to and the of to GSNO is a of S-nitrosylation of of the that of cysteine that the for is that is the and of is for and for the activity of is the of for the regulation of S-nitrosylation of of a effect on the activity of that cysteine to redox-based in the of which protein of of animals, protein S-nitrosylation regulates many different and protein In of the of protein S-nitrosylation in is the a of S-nitrosylation is for Arabidopsis protein is to NO of and NO a role in NO is evidence that of S-nitrosylation of cysteine of in that GSNO of Arabidopsis to and in and GSNO the of in of GSNO to on the of in a of plants, NO and to for and and NO of and that of and in with a of NO and of that NO to in and A of NO and in and in and and the effect of NO in the of with that NO of Arabidopsis that NO inhibition of activity S-nitrosylation in of the and we that this protein a for NO and MAT is the NO showed that in of different NO in the of the that NO the S-nitrosylation of and methionine of the and of the In we the first detailed molecular characterization of S-nitrosylation in plants. of of of the a on We for