DIP Database of Interacting Proteins 611 Young Drive East; Los Angeles CA 90095; USA http://dip.doe-mbi.ucla.edu/ dip@mbi.ucla.edu 611 Young Drive East; Los Angeles CA 90095; USA http://dip.doe-mbi.ucla.edu/ johnston-2007-8 GTPase acceleration as the rate-limiting step in Arabidopsis G protein-coupled sugar signaling. 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 Proc. Natl. Acad. Sci. U.S.A. (0027-8424) 2007 IMEx Only protein-protein interactions imex curation johnston-2007-6 GTPase acceleration as the rate-limiting step in Arabidopsis G protein-coupled sugar signaling. 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 Proc. Natl. Acad. Sci. U.S.A. (0027-8424) 2007 IMEx Only protein-protein interactions imex curation johnston-2007-1 GTPase acceleration as the rate-limiting step in Arabidopsis G protein-coupled sugar signaling. arath-root Arabidopsis thaliana root tissue root In vascular plants, the root is that organ of a plant body that typically lies below the surface of the soil (compare with stem). However, this is not always the case, since a root can also be aerial (that is, growing above the ground) or aerating (that is, growing up above the ground or especially above water). On the other hand, a stem normally occurring below ground is not exceptional either (see rhizome). So, it is better to define root as a part of a plant body that bears no leaves, and therefore also lacks nodes. There are also important internal structural differences between stems and roots. The two major functions of roots are 1) absorption of water and inorganic nutrients and 2) anchoring the plant body to the ground. Roots also function in cytokinin synthesis, which supplies some of shoot needs. They often function in storage of food. 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 Proc. Natl. Acad. Sci. U.S.A. (0027-8424) 2007 IMEx Only protein-protein interactions imex curation johnston-2007-2 GTPase acceleration as the rate-limiting step in Arabidopsis G protein-coupled sugar signaling. 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 Proc. Natl. Acad. Sci. U.S.A. (0027-8424) 2007 IMEx Only protein-protein interactions imex curation johnston-2007-3 GTPase acceleration as the rate-limiting step in Arabidopsis G protein-coupled sugar signaling. 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 Proc. Natl. Acad. Sci. U.S.A. (0027-8424) 2007 IMEx Only protein-protein interactions imex curation johnston-2007-4 GTPase acceleration as the rate-limiting step in Arabidopsis G protein-coupled sugar signaling. 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 Proc. Natl. Acad. Sci. U.S.A. (0027-8424) 2007 IMEx Only protein-protein interactions imex curation johnston-2007-5 GTPase acceleration as the rate-limiting step in Arabidopsis G protein-coupled sugar signaling. 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 Proc. Natl. Acad. Sci. U.S.A. (0027-8424) 2007 IMEx Only protein-protein interactions imex curation johnston-2007-7 GTPase acceleration as the rate-limiting step in Arabidopsis G protein-coupled sugar signaling. 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 Proc. Natl. Acad. Sci. U.S.A. (0027-8424) 2007 IMEx Only protein-protein interactions imex curation johnston-2007-9 GTPase acceleration as the rate-limiting step in Arabidopsis G protein-coupled sugar signaling. 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 Proc. Natl. Acad. Sci. U.S.A. (0027-8424) 2007 IMEx Only protein-protein interactions imex curation 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 gnao_human Guanine nucleotide-binding protein G(o) subunit alpha GNAO1 protein protein human Homo sapiens Human MGCTLSAEERAALERSKAIEKNLKEDGISAAKDVKLLLLGAGESGKSTIVKQMKIIHEDGFSGEDVKQYKPVVYSNTIQSLAAIVRAMDTLGIEYGDKERKADAKMVCDVVSRMEDTEPFSAELLSAMMRLWGDSGIQECFNRSREYQLNDSAKYYLDSLDRIGAADYQPTEQDILRTRVKTTGIVETHFTFKNLHFRLFDVGGQRSERKKWIHCFEDVTAIIFCVALSGYDQVLHEDETTNRMHESLMLFDSICNNKFFIDTSIILFLNKKDLFGEKIKKSPLTICFPEYTGPNTYEDAAAYIQAQFESKNRSPNKEIYCHMTCATDTNNIQVVFDAVTDIIIANNLRGCGLY 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 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 guanosine 5'-[gamma-thio]triphosphate GTP-gamma-S small molecule small molecule chemical synthesis Chemical synthesis (Chemical synthesis) gdp-tetrafluoroaluminate transition-state analogue GDP.AlF4 small molecule small molecule chemical synthesis Chemical synthesis (Chemical synthesis) GDP.AlF4 is a transition stsate analogue gnao1-gtp GNAO1 binds GTP 721493 N/A 721502 unspecified role unspecified role neutral component neutral component Stoichiometry: 0.9 N/A 721503 unspecified role unspecified role neutral component neutral component direct interaction direct interaction false false false Figure 2A The observed rate of [35S]GTP gamma S binding to AtGPA1K was Kobs = 0.08 per minute. gpa1-gtp-5 GPA1 binds GTP 721493 N/A 721504 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 false ecoli Escherichia coli (strain K12) N/A 721502 unspecified role unspecified role neutral component neutral component Stoichiometry: 0.9 direct interaction direct interaction false false false Figure 2A 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 GPA1 hydrolyses GTP 721494 N/A 721505 stimulator stimulator unspecified role unspecified role ecoli Escherichia coli (strain K12) N/A 721504 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 false ecoli Escherichia coli (strain K12) N/A 721502 enzyme target enzyme target substrate substrate neutral component neutral component enzymatic reaction enzymatic reaction Biochemical reaction Biochemical reaction false false false Figure 2F Supplementary Information Figure 7 Supplementary Information Figure 8 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 GPA1 hydrolyses GTP 721494 N/A 721505 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 false ecoli Escherichia coli (strain K12) N/A 721502 enzyme target enzyme target substrate substrate neutral component neutral component N/A 721504 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 false ecoli Escherichia coli (strain K12) gtpase reaction gtpase reaction false false false Figure 3A 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-at3g26090-3 Direct interaction odf GPA1 and At3g26090 (RGS1) 721495 N/A 721504 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 false N/A 721505 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 false direct interaction direct interaction false false false Figure 1B 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 GPA1 binds GTP 721496 N/A 721506 unspecified role unspecified role prey prey N/A 721502 inhibitor inhibitor prey prey Stoichiometry: 0.9 N/A 721504 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 false ecoli Escherichia coli (strain K12) direct interaction direct interaction false false false Table S1 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 GPA1 binds GTP 721497 N/A 721504 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 false ecoli Escherichia coli (strain K12) N/A 721502 unspecified role unspecified role neutral component neutral component direct interaction direct interaction false false false Figure 2B 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 GPA1 binds GTP 721498 N/A 721504 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 false ecoli Escherichia coli (strain K12) N/A 721502 unspecified role unspecified role neutral component neutral component direct interaction direct interaction false false false Figure 2C Supplementary information Figure 5 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 GPA1 hydrolyses GTP 721499 N/A 721504 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 false ecoli Escherichia coli (strain K12) N/A 721502 enzyme target enzyme target substrate substrate neutral component neutral component enzymatic reaction enzymatic reaction Biochemical reaction Biochemical reaction false false false Figure 2E 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. gpa1-at3g26090-1 GPA1 interacts with At3g26090 721500 N/A 721504 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 false ecoli Escherichia coli (strain K12) N/A 721505 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 false Q8H1F2:p.Glu320Lys mutation disrupting mutation disrupting interaction mutation disrupting certain certain sequence position certain certain certain sequence position certain false ecoli Escherichia coli (strain K12) N/A 721502 unspecified role unspecified role neutral component neutral component direct interaction direct interaction false false false Figure 3B GST-AtRGS1 was immobilized on a biosensor surface. AtRGS1(wild-type) bound AtGPA1 in the transition state for GTP hydrolysis gpa1-at3g26090-2 GPA1 interacts with At3g26090 721501 N/A 721507 unspecified role unspecified role ancillary ancillary N/A 721505 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 false Q8H1F2:p.Glu320Lys mutation disrupting mutation disrupting interaction mutation disrupting certain certain sequence position certain certain certain sequence position certain false c-terminal sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false ecoli Escherichia coli (strain K12) N/A 721504 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 false ecoli Escherichia coli (strain K12) direct interaction direct interaction false false false Supplementary Information Figure 9