Figure 3.

The MON1-CCZ1 Complex Serves as the Rab7 GEF in Arabidopsis.

(A) Y2H analysis of the interaction between MON1 and CCZ1. MON1, CCZ1a, and CCZ1b were used as either bait or pray. Interactions between two proteins were tested using the HIS3 reporter gene. AD, activation domain; BD, binding domain.

(B) Co-IP assays were performed after transient coexpression of YFP or YFP-CCZ1a with Myc-MON1 in Arabidopsis protoplasts. Total proteins (input) were subjected to immunoprecipitation with GFP-Trap beads followed by immunoblot analysis.

(C) and (D) In vitro GEF assay of MON1-CCZ1 complex. Nucleotide exchange on RABG3f (Rab7) (C) and RHA1 (Rab5) (D) was measured by monitoring tryptophan autofluorescence in the absence (yellow) or presence of 0.25 μM (green), 0.5 μM (blue), or 1 μM (red) GST-CCZ1a-12×His-MON1. The jagged lines represent the raw data, and the bold lines represent the trend lines.

(E) Co-IP assays were performed after transient coexpression of YFP-CCZ1a, Myc-MON1 with wild-type, active, or inactive RABG3f in Arabidopsis protoplasts. Total proteins (input) were subjected to immunoprecipitation with GFP-Trap beads followed by immunoblot analysis.