Fig. 4.

VPS41 and VPS33 interact and can bind the SNARE protein SYP22. (A–F) VPS41 and VPS33 interact in vivo and bind the SNARE protein SYP22. BiFC analysis of protein interactions in infiltrated N. benthamiana leaf cells by confocal microscopy. The N-terminal half (nY) or the C-terminal half (cY) of YFP were fused with VPS41, VPS33, SYP22, or VTI11. (A–C) cYFP-VPS41 interacts with nYFP-VPS33 (A), nYFP-SYP22 (B), but not with nYFP-VTI11 (C). (D–F) cYFP-VPS33 interacts with nYFP-VPS41 (D), nYFP-SYP22 (E), but not with nYFP-VTI11 (F). Asterisks indicate background signal that was observed in the negative control. (G) VPS33 binds the SYP22 SNARE in vitro. Recombinant GST-VPS41, GST-VPS33, HIS-SYP22, and HIS-VTI11 protein fusions were expressed in E. coli and purified by affinity chromatography. The purified proteins were mixed as indicated, and immunoprecipitation was carried out with an α-HIS antibody. Proteins from 5% of the input, immunoprecipitate (IP), or 5% of the flowthrough were separated by SDS/PAGE, and GST-VPS41 or GST-VPS33 were detected by Western blot with an α-GST antibody. Arrows indicate the expected sizes of GST-VPS41 and GST-VPS33. The positions of 150-kDa (Top) and 100-kDa (Bottom) molecular markers are indicated with arrowheads. (H–K) Genetic interaction between hypomorphic HOPS mutant alleles and zig-1. Seedlings were stained for 2 h in LysoTracker Red (Invitrogen) and acquired in LSCM. Abnormal vacuole morphology in the maturation zone was observed in zig-1 (H), vps33-1 zig-1 (J), and vps33-2 zig-1 mutants (K). The vacuole phenotype was complemented in the root differentiation zone (DZ) of the double-mutant zig-1 zip2 (I). (L–N) Loss of the VTI11 SNARE induces abnormal VPS41-GFP localization. VPS41-GFP was imaged in the Col-0 WT (L) or the zig-1 mutant background (M and N). The protein was found in bright aggregates at the junction of zig-1 vacuoles or in clusters in the cytosol as shown with the Airyscan mode of the confocal (N). (O–Q). Loss of the VTI11 SNARE induces abnormal VPS33-GFP localization. VPS33-GFP was imaged in the Col-0 WT (O) or zig-1 (P and Q). Details of the aggregates at the junction between unfused vacuoles can be observed in the Airyscan mode (Q). (Scale bars: 10 µm.)