DIP Database of Interacting Proteins 611 Young Drive East; Los Angeles CA 90095; USA http://dip.doe-mbi.ucla.edu/ dip@mbi.ucla.edu GTPase acceleration as the rate-limiting step in Arabidopsis G protein-coupled sugar signaling. GTPase acceleration as the rate-limiting step in Arabidopsis G protein-coupled sugar signaling. Proc. Natl. Acad. Sci. U.S.A. (0027-8424) 2007 imex curation Johnston CA., Taylor JP., Gao Y., Kimple AJ., Grigston JC., Chen JG., Siderovski DP., Jones AM., Willard FS. Transgenic Arabidopsis plants (ecotype Columbia) expressing epitope tagged proteins were used in this study. Fret analysis was carried out on roots of transgenic plants. fwillard@med.unc.edu 03-Mar-2009: Contacted by JYOTI. 03-Feb-2009: Contacted by JYOTI. jyoti Only protein-protein interactions in vitro In vitro fluorescence fluorescence technology fluorescence predetermined predetermined participant predetermined Johnston CA., Taylor JP., Gao Y., Kimple AJ., Grigston JC., Chen JG., Siderovski DP., Jones AM., Willard FS. Transgenic Arabidopsis plants (ecotype Columbia) expressing epitope tagged proteins were used in this study. Fret analysis was carried out on roots of transgenic plants. fwillard@med.unc.edu Accepted 2008-JUL-6 by JYOTI. 03-Mar-2009: Contacted by JYOTI. Proc. Natl. Acad. Sci. U.S.A. (0027-8424) 2007 jyoti IMEx GTPase acceleration as the rate-limiting step in Arabidopsis G protein-coupled sugar signaling. GTPase acceleration as the rate-limiting step in Arabidopsis G protein-coupled sugar signaling. Proc. Natl. Acad. Sci. U.S.A. (0027-8424) 2007 imex curation Johnston CA., Taylor JP., Gao Y., Kimple AJ., Grigston JC., Chen JG., Siderovski DP., Jones AM., Willard FS. Transgenic Arabidopsis plants (ecotype Columbia) expressing epitope tagged proteins were used in this study. Fret analysis was carried out on roots of transgenic plants. fwillard@med.unc.edu 03-Mar-2009: Contacted by JYOTI. 03-Feb-2009: Contacted by JYOTI. jyoti Only protein-protein interactions arath-root Arabidopsis thaliana root tissue root fret fluorescent resonance energy transfer FRET RET FRET analysis FRET RET FRET analysis fret tag fluorescence tag visualisation by fluorescence tag fluorescence Johnston CA., Taylor JP., Gao Y., Kimple AJ., Grigston JC., Chen JG., Siderovski DP., Jones AM., Willard FS. Transgenic Arabidopsis plants (ecotype Columbia) expressing epitope tagged proteins were used in this study. Fret analysis was carried out on roots of transgenic plants. fwillard@med.unc.edu Accepted 2008-JUL-6 by JYOTI. 03-Mar-2009: Contacted by JYOTI. Proc. Natl. Acad. Sci. U.S.A. (0027-8424) 2007 jyoti IMEx GTPase acceleration as the rate-limiting step in Arabidopsis G protein-coupled sugar signaling. GTPase acceleration as the rate-limiting step in Arabidopsis G protein-coupled sugar signaling. Proc. Natl. Acad. Sci. U.S.A. (0027-8424) 2007 imex curation Johnston CA., Taylor JP., Gao Y., Kimple AJ., Grigston JC., Chen JG., Siderovski DP., Jones AM., Willard FS. Transgenic Arabidopsis plants (ecotype Columbia) expressing epitope tagged proteins were used in this study. Fret analysis was carried out on roots of transgenic plants. fwillard@med.unc.edu 03-Mar-2009: Contacted by JYOTI. 03-Feb-2009: Contacted by JYOTI. jyoti Only protein-protein interactions in vitro In vitro gtpase assay gtpase assay GTPase gtp hydrolisis gtp hydrolisis GTPase predetermined predetermined participant predetermined Johnston CA., Taylor JP., Gao Y., Kimple AJ., Grigston JC., Chen JG., Siderovski DP., Jones AM., Willard FS. Transgenic Arabidopsis plants (ecotype Columbia) expressing epitope tagged proteins were used in this study. Fret analysis was carried out on roots of transgenic plants. fwillard@med.unc.edu Accepted 2008-JUL-6 by JYOTI. 03-Mar-2009: Contacted by JYOTI. Proc. Natl. Acad. Sci. U.S.A. (0027-8424) 2007 jyoti IMEx GTPase acceleration as the rate-limiting step in Arabidopsis G protein-coupled sugar signaling. GTPase acceleration as the rate-limiting step in Arabidopsis G protein-coupled sugar signaling. Proc. Natl. Acad. Sci. U.S.A. (0027-8424) 2007 imex curation Johnston CA., Taylor JP., Gao Y., Kimple AJ., Grigston JC., Chen JG., Siderovski DP., Jones AM., Willard FS. Transgenic Arabidopsis plants (ecotype Columbia) expressing epitope tagged proteins were used in this study. Fret analysis was carried out on roots of transgenic plants. fwillard@med.unc.edu 03-Mar-2009: Contacted by JYOTI. 03-Feb-2009: Contacted by JYOTI. jyoti Only protein-protein interactions in vitro In vitro competition binding competition binding predetermined predetermined participant predetermined Johnston CA., Taylor JP., Gao Y., Kimple AJ., Grigston JC., Chen JG., Siderovski DP., Jones AM., Willard FS. The authors do not provide details of protein purification, it is assumed the His-tagged GPA1 protein was expressed in ecoli and subsequently purified by affinity chromatography. Transgenic Arabidopsis plants (ecotype Columbia) expressing epitope tagged proteins were used in this study. Fret analysis was carried out on roots of transgenic plants. fwillard@med.unc.edu Accepted 2008-JUL-6 by JYOTI. 03-Mar-2009: Contacted by JYOTI. Proc. Natl. Acad. Sci. U.S.A. (0027-8424) 2007 jyoti IMEx GTPase acceleration as the rate-limiting step in Arabidopsis G protein-coupled sugar signaling. GTPase acceleration as the rate-limiting step in Arabidopsis G protein-coupled sugar signaling. Proc. Natl. Acad. Sci. U.S.A. (0027-8424) 2007 imex curation Johnston CA., Taylor JP., Gao Y., Kimple AJ., Grigston JC., Chen JG., Siderovski DP., Jones AM., Willard FS. Transgenic Arabidopsis plants (ecotype Columbia) expressing epitope tagged proteins were used in this study. Fret analysis was carried out on roots of transgenic plants. fwillard@med.unc.edu 03-Mar-2009: Contacted by JYOTI. 03-Feb-2009: Contacted by JYOTI. jyoti Only protein-protein interactions in vitro In vitro gtpase assay gtpase assay GTPase gtp hydrolisis gtp hydrolisis GTPase predetermined predetermined participant predetermined Johnston CA., Taylor JP., Gao Y., Kimple AJ., Grigston JC., Chen JG., Siderovski DP., Jones AM., Willard FS. The authors provide no details of the expression or purification of the proteins used , it is assumed they were expressed and purified from E.coli. Transgenic Arabidopsis plants (ecotype Columbia) expressing epitope tagged proteins were used in this study. Fret analysis was carried out on roots of transgenic plants. fwillard@med.unc.edu Accepted 2008-JUL-6 by JYOTI. 03-Mar-2009: Contacted by JYOTI. Proc. Natl. Acad. Sci. U.S.A. (0027-8424) 2007 jyoti IMEx GTPase acceleration as the rate-limiting step in Arabidopsis G protein-coupled sugar signaling. GTPase acceleration as the rate-limiting step in Arabidopsis G protein-coupled sugar signaling. Proc. Natl. Acad. Sci. U.S.A. (0027-8424) 2007 imex curation Johnston CA., Taylor JP., Gao Y., Kimple AJ., Grigston JC., Chen JG., Siderovski DP., Jones AM., Willard FS. Transgenic Arabidopsis plants (ecotype Columbia) expressing epitope tagged proteins were used in this study. Fret analysis was carried out on roots of transgenic plants. fwillard@med.unc.edu 03-Mar-2009: Contacted by JYOTI. 03-Feb-2009: Contacted by JYOTI. jyoti Only protein-protein interactions in vitro In vitro filter binding filter binding Filter overlay assay Filter overlay assay predetermined predetermined participant predetermined Johnston CA., Taylor JP., Gao Y., Kimple AJ., Grigston JC., Chen JG., Siderovski DP., Jones AM., Willard FS. The authors do not provide details of protein purification, it is assumed the protein was expressed in ecoli and subsequently purified by means unknown. Transgenic Arabidopsis plants (ecotype Columbia) expressing epitope tagged proteins were used in this study. Fret analysis was carried out on roots of transgenic plants. fwillard@med.unc.edu Accepted 2008-JUL-6 by JYOTI. 03-Mar-2009: Contacted by JYOTI. Proc. Natl. Acad. Sci. U.S.A. (0027-8424) 2007 jyoti IMEx GTPase acceleration as the rate-limiting step in Arabidopsis G protein-coupled sugar signaling. GTPase acceleration as the rate-limiting step in Arabidopsis G protein-coupled sugar signaling. Proc. Natl. Acad. Sci. U.S.A. (0027-8424) 2007 imex curation Johnston CA., Taylor JP., Gao Y., Kimple AJ., Grigston JC., Chen JG., Siderovski DP., Jones AM., Willard FS. Transgenic Arabidopsis plants (ecotype Columbia) expressing epitope tagged proteins were used in this study. Fret analysis was carried out on roots of transgenic plants. fwillard@med.unc.edu 03-Mar-2009: Contacted by JYOTI. 03-Feb-2009: Contacted by JYOTI. jyoti Only protein-protein interactions in vitro In vitro fluorescence spectr classical fluorescence spectroscopy fluorescence spectr tag fluorescence tag visualisation by fluorescence tag fluorescence Johnston CA., Taylor JP., Gao Y., Kimple AJ., Grigston JC., Chen JG., Siderovski DP., Jones AM., Willard FS. The authors do not give details of the purification of proteins used in this assay. It is assumed the proteins were expresssed in ecoli and subsequently purified. Transgenic Arabidopsis plants (ecotype Columbia) expressing epitope tagged proteins were used in this study. Fret analysis was carried out on roots of transgenic plants. fwillard@med.unc.edu Accepted 2008-JUL-6 by JYOTI. 03-Mar-2009: Contacted by JYOTI. Proc. Natl. Acad. Sci. U.S.A. (0027-8424) 2007 jyoti IMEx GTPase acceleration as the rate-limiting step in Arabidopsis G protein-coupled sugar signaling. GTPase acceleration as the rate-limiting step in Arabidopsis G protein-coupled sugar signaling. Proc. Natl. Acad. Sci. U.S.A. (0027-8424) 2007 imex curation Johnston CA., Taylor JP., Gao Y., Kimple AJ., Grigston JC., Chen JG., Siderovski DP., Jones AM., Willard FS. Transgenic Arabidopsis plants (ecotype Columbia) expressing epitope tagged proteins were used in this study. Fret analysis was carried out on roots of transgenic plants. fwillard@med.unc.edu 03-Mar-2009: Contacted by JYOTI. 03-Feb-2009: Contacted by JYOTI. jyoti Only protein-protein interactions in vitro In vitro pull down pull down western blot western blot Immuno blot Immuno blot Johnston CA., Taylor JP., Gao Y., Kimple AJ., Grigston JC., Chen JG., Siderovski DP., Jones AM., Willard FS. The authors provide no details of the expression or purification of the proteins used , it is assumed they were expressed and purified from E.coli. Transgenic Arabidopsis plants (ecotype Columbia) expressing epitope tagged proteins were used in this study. Fret analysis was carried out on roots of transgenic plants. fwillard@med.unc.edu Accepted 2008-JUL-6 by JYOTI. 03-Mar-2009: Contacted by JYOTI. Proc. Natl. Acad. Sci. U.S.A. (0027-8424) 2007 jyoti IMEx GTPase acceleration as the rate-limiting step in Arabidopsis G protein-coupled sugar signaling. GTPase acceleration as the rate-limiting step in Arabidopsis G protein-coupled sugar signaling. Proc. Natl. Acad. Sci. U.S.A. (0027-8424) 2007 imex curation Johnston CA., Taylor JP., Gao Y., Kimple AJ., Grigston JC., Chen JG., Siderovski DP., Jones AM., Willard FS. Transgenic Arabidopsis plants (ecotype Columbia) expressing epitope tagged proteins were used in this study. Fret analysis was carried out on roots of transgenic plants. fwillard@med.unc.edu 03-Mar-2009: Contacted by JYOTI. 03-Feb-2009: Contacted by JYOTI. jyoti Only protein-protein interactions in vitro In vitro spr surface plasmon resonance Optical biosensor BIAcore(r) BIAcore(r) Optical biosensor spr predetermined predetermined participant predetermined Johnston CA., Taylor JP., Gao Y., Kimple AJ., Grigston JC., Chen JG., Siderovski DP., Jones AM., Willard FS. The authors provide no details of the expression or purification of the proteins used , it is assumed they were expressed and purified from E.coli. Transgenic Arabidopsis plants (ecotype Columbia) expressing epitope tagged proteins were used in this study. Fret analysis was carried out on roots of transgenic plants. fwillard@med.unc.edu Accepted 2008-JUL-6 by JYOTI. 03-Mar-2009: Contacted by JYOTI. Proc. Natl. Acad. Sci. U.S.A. (0027-8424) 2007 jyoti IMEx guanosine 5'-[gamma-thio]triphosphate GTP-gamma-S small molecule small molecule chemical synthesis Chemical synthesis (Chemical synthesis) rgs1_arath Regulator of G-protein signaling 1 RGS1 MPE11.27 MPE11.28 At3g26090 protein protein arath Arabidopsis thaliana (Mouse-ear cress) Mouse-ear cress MASGCALHGGCPSDYVAVAISVICFFVLLSRSVLPCLIHKAPRTNSSSFWIPVIQVISSFNLLFSIMMSVNLLRFRTKHWWRYCYLWAVWIEGPLGFGLLMSCRITQAFQLYFIFVKKRLPPVKSYIFLPLVLLPWIFGAAIIHATKPLNDKCHMGLQWTFPVAGLHALYVLALIAFTRAVRHVEFRFDELRDLWKGILVSATSIVIWVTAFVLNEIHEEISWLQVASRFVLLVTGGILVVVFFSISSNQPLLSQISLKKRQNFEFQRMGQALGIPDSGLLFRKEEFRPVDPNEPLDKLLLNKRFRHSFMEFADSCYAGETLHFFEEVYEHGKIPEDDSIRRIYMARHIMEKFIVAGAEMELNLSHKTRQEILTTQDLTHTDLFKNALNEVMQLIKMNLVRDYWSSIYFIKFKEEESCHEAMHKEGYSFSSPRLSSVQGSDDPFYQEHMSKSSRCSSPG 43AA65CD0417488E gnao_human Guanine nucleotide-binding protein G(o) subunit alpha GNAO1 protein protein human Homo sapiens Human MGCTLSAEERAALERSKAIEKNLKEDGISAAKDVKLLLLGAGESGKSTIVKQMKIIHEDGFSGEDVKQYKPVVYSNTIQSLAAIVRAMDTLGIEYGDKERKADAKMVCDVVSRMEDTEPFSAELLSAMMRLWGDSGIQECFNRSREYQLNDSAKYYLDSLDRIGAADYQPTEQDILRTRVKTTGIVETHFTFKNLHFRLFDVGGQRSERKKWIHCFEDVTAIIFCVALSGYDQVLHEDETTNRMHESLMLFDSICNNKFFIDTSIILFLNKKDLFGEKIKKSPLTICFPEYTGPNTYEDAAAYIQAQFESKNRSPNKEIYCHMTCATDTNNIQVVFDAVTDIIIANNLRGCGLY 4F182926A8106E3E gtp guanosine triphosphate - GTP 5'-GTP GTP Guanosine 5'-triphosphate guanosine 5'-triphosphoric acid guanosine triphosphate GUANOSINE-5'-TRIPHOSPHATE H4gtp guanosine 5'-(tetrahydrogen triphosphate) small molecule small molecule chemical synthesis Chemical synthesis (Chemical synthesis) InChI=1/C10H16N5O14P3/c11-10-13-7-4(8(18)14-10)12-2-15(7)9-6(17)5(16)3(27-9)1-26-31(22,23)29-32(24,25)28-30(19,20)21/h2-3,5-6,9,16-17H,1H2,(H,22,23)(H,24,25)(H2,19,20,21)(H3,11,13,14,18)/t3-,5-,6-,9-/m1/s1/f/h14,19-20,22,24H,11H2 InChI=1/C10H16N5O14P3/c11-10-13-7-4(8(18)14-10)12-2-15(7)9-6(17)5(16)3(27-9)1-26-31(22,23)29-32(24,25)28-30(19,20)21/h2-3,5-6,9,16-17H,1H2,(H,22,23)(H,24,25)(H2,19,20,21)(H3,11,13,14,18)/t3-,5-,6-,9-/m1/s1/f/h14,19-20,22,24H,11H2 gdp-tetrafluoroaluminate transition-state analogue GDP.AlF4 small molecule small molecule chemical synthesis Chemical synthesis (Chemical synthesis) GDP.AlF4 is a transition stsate analogue GDP.AlF4 is a transition stsate analogue gpa1_arath Guanine nucleotide-binding protein alpha-1 subunit GPA1 At2g26300 T1D16.6 protein protein arath Arabidopsis thaliana (Mouse-ear cress) Mouse-ear cress MGLLCSRSRHHTEDTDENTQAAEIERRIEQEAKAEKHIRKLLLLGAGESGKSTIFKQIKLLFQTGFDEGELKSYVPVIHANVYQTIKLLHDGTKEFAQNETDSAKYMLSSESIAIGEKLSEIGGRLDYPRLTKDIAEGIETLWKDPAIQETCARGNELQVPDCTKYLMENLKRLSDINYIPTKEDVLYARVRTTGVVEIQFSPVGENKKSGEVYRLFDVGGQRNERRKWIHLFEGVTAVIFCAAISEYDQTLFEDEQKNRMMETKELFDWVLKQPCFEKTSFMLFLNKFDIFEKKVLDVPLNVCEWFRDYQPVSSGKQEIEHAYEFVKKKFEELYYQNTAPDRVDRVFKIYRTTALDQKLVKKTFKLVDETLRRRNLLEAGLL DIP protein P18064 original sequence version: 80 46BF8650E067F968 gpa1-at3g26090-3 2 15 unspecified role unspecified role fluorescence accept fluorescence acceptor fluorescence accept region cfp tag cyan fluorescent protein tag CFP cyan fluorescent protein cyan fluorescent protein CFP cfp tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined 11 unspecified role unspecified role fluorescence donor fluorescence donor region yfp tag yellow fluorescent protein tag YFP yellow fluorescent protein yellow fluorescent protein YFP yfp tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined direct interaction direct interaction Figure 1B Here we show, by using FRET, that D-glucose alters the interaction between the AtGPA1 and AtRGS1 in vivo. The authors note that "FRET signals were exclusively observed on or around plastids that were in close proximity to the plasma membrane". D-glucose The GPA1 (P18064) containing construct consisted of the cyan fluorescent protein inserted into the loop between the predicted alpha A and alpha B helices (between alanine 97 and glutamine 98) of the protein. The authors speculated that "AtRGS1is a membrane receptor or coreceptorfor D-glucose that interacts with AtGPA1 upon glucose binding". gpa1-gtp 4 10 unspecified role unspecified role prey prey 13 inhibitor inhibitor prey prey 15 unspecified role unspecified role bait bait region his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined ecoli Escherichia coli (strain K12) direct interaction direct interaction Table S1 indicating that AtGPA1 is selective toward guanine nucleotides and is a bona fide GTPbinding protein. Only guanine nucleotide triphosphates were able to compete with GTP binding to GPA1. Guanosine triposphate was radiolabelled. The authors noted that "recombinant AtGPA1 had a high specific activity, binding [35S]GTP gamma S with a stoichiometry of 0.91 mol of GTP gammaS/mol of protein". gpa1-gtp-2 7 15 unspecified role unspecified role neutral component neutral component region his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined ecoli Escherichia coli (strain K12) 13 unspecified role unspecified role neutral component neutral component direct interaction direct interaction Figure 2B By using this real-time assay, the kobs value for GTP S binding by AtGPA1 was 14.4 min 1, a value 22-fold faster than the most rapidly exchanging G subunits previously described Guanosine triposphate was tagged with the the green-fluorescent BODIPY FL fluorophore. The authors state that "kobs, observed rate of GTPgS binding to G alpha GDP (equivalent to the rate of GDP release from G alpha because it is the rate-limiting step in the nucleotide exchange process)" was 14.4 min per second. gpa1-gtp-3 1 15 unspecified role unspecified role neutral component neutral component region his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined ecoli Escherichia coli (strain K12) 13 unspecified role unspecified role neutral component neutral component direct interaction direct interaction Figure 2C Supplementary information Figure 5 To verify this result by using an independent approach, we measured GTP S binding to AtGPA1 by using intrinsic tryptophan fluorescence, and comparable kinetic data were obtained Guanosine triposphate was radiolabelled. The binding of guanosine triposphate was idenpendent of magnesium ion concentration over a range of 2 micromole to 20 millimole. The authors state that "kobs, observed rate of GTPgS binding to G alpha GDP (equivalent to the rate of GDP release from G alpha because it is the rate-limiting step in the nucleotide exchange process)" was 8.7 min per second. gpa1-gtp-4 3 15 enzyme enzyme neutral component neutral component region his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined ecoli Escherichia coli (strain K12) 13 enzyme target enzyme target substrate substrate neutral component neutral component enzymatic reaction enzymatic reaction Biochemical reaction Biochemical reaction Figure 2E We also directly measured GDP release by using [ -32P]GDP-bound AtGPA1 (Fig. 2D). The rate of GDP dissociation from AtGPA1 (koff 12.6 min 1) was highly concordant with the measured GTP S-binding rate. Guanosine triposphate was radiolabelled with 32P. The authors state that Kcat (rate of hydrolysis of bound GTP by enzyme) was 0.12 minute per second at 20 degrees celsius. gnao1-gtp 6 13 unspecified role unspecified role neutral component neutral component 12 unspecified role unspecified role neutral component neutral component direct interaction direct interaction Figure 2A Human G oA had a kobs value of 0.09 min 1 in this GTP S-binding assay The observed rate of [35S]GTP gamma S binding to AtGPA1K was Kobs = 0.08 per minute. gpa1-gtp-5 6 15 unspecified role unspecified role neutral component neutral component region his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined ecoli Escherichia coli (strain K12) 13 unspecified role unspecified role neutral component neutral component direct interaction direct interaction Figure 2A indicating that AtGPA1 is selective toward guanine nucleotides and is a bona fide GTPbinding protein. The authors noted that "recombinant AtGPA1 had a high specific activity, binding [35S]GTP gamma S with a stoichiometry of 0.91 mol of GTP gammaS/mol of protein". The observed rate of [35S]GTP gamma S binding to AtGPA1K was Kobs = 1.4 per minute. The observed rate of binding of GTP to protein was likely to be an underestimate. The authors state the observed rate is " likely an underestimate because the first time point shows 75% binding in this assay format". GTP was radiolabelled. gpa1-gtp-6 5 11 stimulator stimulator unspecified role unspecified role ecoli Escherichia coli (strain K12) 15 enzyme enzyme neutral component neutral component region his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined ecoli Escherichia coli (strain K12) 13 enzyme target enzyme target substrate substrate neutral component neutral component enzymatic reaction enzymatic reaction Biochemical reaction Biochemical reaction Figure 2F Supplementary Information Figure 7 Supplementary Information Figure 8 We tested these two predictions by performing steady-state GTPase assays by using [ -32P]GTP. The rate of GTP hydrolysis at steady state (ks) at 20�C was 0.063 min 1 ( 0.015 min 1; n 4) (Fig. 2F). Guanosine triposphate was radiolabelled with 32P. The authors state that rate of GTP hydrolysis was 0.063 per minute at steady state at 20 degrees celsius. The authors noted that "a 5-fold molar excess of AtRGS1 gave a 35-fold increase in steady-state GTPase activity". gpa1-gtp-7 5 11 stimulator stimulator unspecified role unspecified role region gst tag glutathione s tranferase tag glutathione S-tranferase tag glutathione S-tranferase tag gst tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined ecoli Escherichia coli (strain K12) 13 enzyme target enzyme target substrate substrate neutral component neutral component 15 enzyme enzyme neutral component neutral component region his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined ecoli Escherichia coli (strain K12) gtpase reaction gtpase reaction Figure 3A AtGPA1 GTPase activity was accelerated by substoichiometric amounts of AtRGS1(wild type), whereas AtRGS1(E320K) was ineffective over a wide range of concentrations. The E320K substitution reduced AtRGS1 GAP activity by at least three orders of magnitude ( Guanosine triposphate was radiolabelled with 32P. The c-terminal region of Q8H1F2 was used in this study (from 249 to 459 amino acid residues). It was demonstrated that mutation of the glutamic acid residue at position 320 in Q8H1F2 to a lysine residue abolished the stimulatory effect of the protein on GTPase activity of GPA1 (P18064). gpa1-at3g26090-1 9 15 enzyme enzyme neutral component neutral component region his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined ecoli Escherichia coli (strain K12) 11 unspecified role unspecified role neutral component neutral component region gst tag glutathione s tranferase tag glutathione S-tranferase tag glutathione S-tranferase tag gst tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined Q8H1F2:p.Glu320Lys mutation disrupting mutation disrupting interaction mutation disrupting certain certain sequence position certain certain certain sequence position certain E K Wed May 02 15:35:30 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'glu320lys' Tue Jun 30 16:06:33 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Glu320Lys' ecoli Escherichia coli (strain K12) 13 unspecified role unspecified role neutral component neutral component direct interaction direct interaction Figure 3B we measured the AtGPA1�AtRGS1 interaction by using surface plasmon resonance spectroscopy. Consistent with published data (12), AtRGS1(wild-type) bound AtGPA1 in the transition state for GTP hydrolysis GST-AtRGS1 was immobilized on a biosensor surface. AtRGS1(wild-type) bound AtGPA1 in the transition state for GTP hydrolysis gpa1-at3g26090-2 8 14 unspecified role unspecified role ancillary ancillary 11 unspecified role unspecified role bait bait region gst tag glutathione s tranferase tag glutathione S-tranferase tag glutathione S-tranferase tag gst tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined Q8H1F2:p.Glu320Lys mutation disrupting mutation disrupting interaction mutation disrupting certain certain sequence position certain certain certain sequence position certain E K Wed May 02 19:17:24 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'glu320lys' Tue Jun 30 18:41:12 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Glu320Lys' c-terminal sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain ecoli Escherichia coli (strain K12) 15 unspecified role unspecified role prey prey region his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined ecoli Escherichia coli (strain K12) direct interaction direct interaction Supplementary Information Figure 9 AtRGS1(E320K) does not interact with AtGPA1. Direct interaction between AtGPA1 and wild-type or mutant forms of the extended RGS domain of AtRGS1 was measured by using a GST (glutatione S-transferase) pull-down (PD) assay as previously described (17).