Source was yeast cell line; bait produced as transgenic fusion protein; prey produced as transgenic fusion protein (prey from embryo cDNA expression library).
Two-hybrid system: yeast LexA-BD/B42-AD
Interaction in vitro; bait derived from wild-type embryonic extract; prey synthesized by in vitro translation.
Two-hybrid system: yeast LexA-BD/B42-AD
coordinates relative to (Block H, Block G and the 19aa linker between the two) Su(var)2-HP2-PA
V2468E,R2472E,W2478E, coordinates relative to Su(var)2-HP2-PA
Mutation of the H block abrogates binding, however the H block is not sufficient for binding. G and H blocks plus the 19aa linker between the 2 are sufficient for binding.
Interaction in vitro; bait produced and labeled by in vitro translation; prey produced and labeled by in vitro translation.
Source was embryos of wild-type fly line; proteins produced from endogenous genes.
Source was adults of wild-type fly line; bait produced from endogenous gene; prey produced from endogenous gene.
Positive control.
coordinates relative to Su(var)2-HP2-PA
Interaction in vitro; proteins produced as recombinant fusion proteins in bacterial system.
NMR suggests Trp 2478 of Su(var)2-HP2 involved in binding Su(var)205 CSD.
coordinates relative to Su(var)2-HP2-PA
Interaction in vitro; protein produced as a recombinant fusion protein in bacterial system.
Su(var)2-HP2 monomer binds to Su(var)205 dimer.
coordinates relative to Su(var)2-HP2-PA
V2468E, coordinates relative to Su(var)2-HP2-PA
Interaction in vitro; bait produced and labeled by in vitro translation; prey produced and labeled by in vitro translation.
coordinates relative to Su(var)2-HP2-PA
Interaction in vitro; proteins produced as recombinant fusion proteins in bacterial system.
Stoichiometry is 2 Su(var)205 : 1 Su(var)2-HP2.
S199E, S202E
coordinates relative to Su(var)2-HP2-PA
Interaction in vitro; bait produced as a recombinant fusion protein in bacterial system; prey peptide chemically synthesized and labeled in vitro.
E205Q, 4-fold decrease
D206N, 4-fold decrease
L147K, 3-fold decrease
L153K, 30-fold decrease
V177K, over 20-fold decrease
V181K
L165K, 20-fold decrease
Interaction in vitro; bait produced as a recombinant fusion protein in bacterial system; prey peptide chemically synthesized and labeled in vitro.
Su(var)205 binds various proteins containing the PXVXL with different affinities. Binding is strongest to Su(var)2-HP2 (Kd = 0.27 uM). Su(var)205 also binds woc (Kd = 2.47 uM) and piwi (12.8 uM) much more weakly.
The Su(var)205 R188Q mutation, which affects Su(var)205 homodimerization, has little effect on the binding constant of this interaction.
Residue Leu 165 of Su(var)205 is critical for selective high-affinity binding of Su(var)205 to Su(var)2-HP2 over piwi. Mutation L165K drastically reduces the Su(var)205-Su(var)2-HP2 interaction, with little effect on binding to piwi.
Source was cross-linked nuclear extract of S2 cell line; bait produced from transfected construct; prey produced from endogenous gene.
Crude nuclear extracts were cross-linked using formaldehyde, sonicated, and subjected to ProteinA affinity purification with rabbit IgG agarose beads eluted under denaturing conditions, and subsequently using streptavidin agarose beads for Bio affinity purification. Purified peptides were eluted by on-bead trypsin digestion and mass spectrometric analysis.
Enrichment of prey protein was compared to input, mock and unrelated BioTAP affinity purifications using a statistical method (Bamse) developed by the authors to control for multiple sources of bias.
Source was cross-linked nuclear embryonic extract of transgenic fly line; bait produced from tagged transgenic construct; prey produced from endogenous gene.
Crude nuclear extracts were cross-linked using formaldehyde, sonicated, and subjected to ProteinA affinity purification with rabbit IgG agarose beads eluted under denaturing conditions, and subsequently using streptavidin agarose beads for Bio affinity purification. Purified peptides were eluted by on-bead trypsin digestion and mass spectrometric analysis.
Enrichment of prey protein was compared to input, mock and unrelated BioTAP affinity purifications using a statistical method (Bamse) developed by the authors to control for multiple sources of bias.
Source was cross-linked nuclear larval extract of transgenic fly line; bait produced from tagged transgenic construct; prey produced from endogenous gene.
Crude nuclear extracts were cross-linked using formaldehyde, sonicated, and subjected to ProteinA affinity purification with rabbit IgG agarose beads eluted under denaturing conditions, and subsequently using streptavidin agarose beads for Bio affinity purification. Purified peptides were eluted by on-bead trypsin digestion and mass spectrometric analysis.
Enrichment of prey protein was compared to input, mock and unrelated BioTAP affinity purifications using a statistical method (Bamse) developed by the authors to control for multiple sources of bias.
Source was cross-linked nuclear extract of transgenic fly line; bait produced from tagged transgenic construct; prey produced from endogenous gene.
Crude nuclear extracts were cross-linked using formaldehyde, sonicated, and subjected to ProteinA affinity purification with rabbit IgG agarose beads eluted under denaturing conditions, and subsequently using streptavidin agarose beads for Bio affinity purification. Purified peptides were eluted by on-bead trypsin digestion and mass spectrometric analysis.
Enrichment of prey protein was compared to input, mock and unrelated BioTAP affinity purifications using a statistical method (Bamse) developed by the authors to control for multiple sources of bias.
