Figure 1.

PI4Kα1 interacts with proteins from the NPG, HYC, and EFOP families. A, Schematic representation of PI4Kα1 (At1g49340) gene. Boxes and lines represent exons and introns, respectively. The kinase domain is shown in red. The T-DNA positions of the pi4kα1-1 and pi4kα1-2 alleles are indicated. Parts of the protein used for the yeast-two-hybrid screen and the antibody production are also shown. The regions targeted by microRNA#2 and #3 are indicated in green. B, Co-IP of PI4Kα1 with NPGR2. Arabidopsis transgenic plants overexpressing NPGR2–mCITRINE (NPGR2–mCt) or Lti6b–mCITRINE (Lti6b–mCt) were used for IP using anti-GFP beads. Immunoblots used anti-PI4Kα1 (upper) and anti-GFP (lower). C, Venn diagram of proteins identified by mass spectrometry from IP of mCITRINE–PI4Kα1, NPGR2–mCITRINE, mCITRINE–NES–mCITRINE, and myristoylation–2x-mCITRINE. D, Co-IP of NPGR2 with HYC2. Arabidopsis transgenic plants overexpressing HYC2–mCITRINE (HYC2-mCt) or Lti6b–mCITRINE were used for IP using anti-GFP beads. Immunoblots used anti-GFP (lower) and anti-NPGR2 (upper). E, Co-IP of PI4Kα1 with EFOP2 and HYC2. Arabidopsis transgenic plants overexpressing EFOP2–mCITRINE (EFOP2–mCt), HYC2–mCITRINE, or Lti6b–mCITRINE were used for IP using anti-GFP beads. Immunoblots used anti-PI4Kα1 (upper) or anti-GFP (lower). F, Yeast-two hybrid assay of HYC1 with NPG1, HYC2 with NPG1 or NPGR1 and NPGR2. Indicated combinations of interactions between HYCCIN-CONTAINING and NPG proteins were assessed by growth on plates with yeast growth media lacking Leu, Trp, and His (-LTH). Yeast growth on plates lacking Leu and Trp (-LT) shows the presence of the bait and prey vectors. The absence of growth when cycloheximide was added (+CHX) shows the absence of auto-activation of the DB vectors. The addition of 3-amino-1,2,4-triazol (+3AT) shows the strength of the interaction. G, Summary of experiments showing interactions among PI4Kα1, NPG, HYC, and EFOP2 proteins.