Specific Mutants of PexRD2 Show Perturbation in Interaction with MAPKKKε
and in Oligomerization.
(A) In Y2H, point
mutations in two Leu residues within PexRD2’s dimerization interface
(Leu109Asp and Leu112Asp), but not the surface presented Lys (Lys104Glu),
abolishes the interaction with StMAPKKKε-KD. Point
mutations at nine other individual surface exposed residues also had no effect
on the interaction (Supplemental Figure 4). Mutation of seven surface presented
residues within a variable region of the WY-domain fold
(PexRD2hepta) weakens the interaction with
StMAPKKKε-KD. The mutation of an additional surface
residue (Ala90Glu) within this region (PexRD2octa) leads to a loss
of interaction with StMAPKKKε-KD. The symbols + and
– denote interaction and absence of interaction, respectively.
(B) In coimmunoprecipitation (IP) from plant tissue, the
GFPPexRD2Leu109Asp and
GFPPexRD2Leu112Asp variants do not interact with
FLAGPexRD2, suggesting they prevent dimerization. All other
GFP-tagged variants tested, that targeted surface-presented residues, retain
interaction with FLAGPexRD2.