Figure 5.
MON1 Directly Interacts with GTP-Bound ARA7, and the MON1-CCZ1 Complex Is Recruited to Enlarged PVCs and the Tonoplast by Active ARA7 in Protoplasts.
(A) Y2H analysis of the interactions among MON1, CCZ1, and different forms of ARA7 (Rab5). MON1, CCZ1a, and CCZ1b were used as prey. Different forms of ARA7 were used as bait. 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-CCZ1a, Myc-MON1 with wild-type, active, or inactive ARA7 in Arabidopsis protoplasts. Total proteins (input) were subjected to immunoprecipitation with GFP-Trap beads followed by immunoblot analysis.
(C) CFP-ARA7Q69L recruited MON1 and CCZ1 complex to the enlarged PVCs and the tonoplast.
(D) The inactive Rab5 (ARA7S24N) did not change the MON1 or CCZ1 pattern. Confocal images were collected from cells at 12 to 14 h after transfection.
Bars = 10 μm in (C) and (D).