Source was yeast cell line; bait produced as transgenic fusion protein; prey produced as transgenic fusion protein (prey from ovary cDNA expression library).
83 of 202 positives represented Su(var)205 clones. Su(var)205 interacts only with piwi, and not aub, AGO1, AGO2, AGO3 or ftz. piwi interacts with Su(var)205 (HP1a), but not with HP1b or HP1c. Interaction abolished by mutation of the first PxVxL motif (V30A) but not the second motif (V130A).
Two-hybrid system: yeast LexA-BD/B42-AD
Source was embryonic nuclear extract of wild-type fly line; bait produced from endogenous gene; prey produced from endogenous gene.
Source was embryonic nuclear extract of wild-type fly line; bait produced from endogenous gene; prey produced from endogenous gene.
Interaction in vitro; bait produced as a recombinant fusion protein in bacterial system; prey synthesized in vitro.
S199E, S202E
Interaction in vitro; bait produced as a recombinant fusion protein in bacterial system; prey peptide chemically synthesized and labeled in vitro.
E205Q, 2-fold decrease
D206N, 3-fold decrease
L147K, 3-fold decrease
L153K, 10-fold decrease
V177K, over 25-fold decrease
V181K
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.
Source was adult ovaries; bait produced from endogenous gene; prey produced from endogenous gene.
