Fig. 2. SYT5 stimulates SYP132-VAMP721/722 interaction.

(A) Ca²⁺-stimulated direct interaction between SYT5 and SYP132 in vitro. Equimolar recombinant HA-tagged SYP132 (HA-SYP132) and the TM-lacking GST-fused SYT5 (GST-SYT5ΔTM) were incubated in the presence or absence of 1 mM CaCl2. Their interaction was analyzed by immunoblot with anti-HA antibody to detect HA-SYP132 in the precipitates with GST-SYT5ΔTM. To test a Ca²⁺ effect on their interaction, 1 mM EDTA was added during incubation. Equal loading was visualized by immunoblot with anti-GST antibody to detect GST-SYT5ΔTM. GST was used as a negative control. (B) SYT5-induced interaction between SYP132 and VAMP722 in the presence of Ca²⁺ in vitro. Equimolar recombinant HA-SYP132 and GST-VAMP722 were incubated with increasing amounts of SYT5ΔTM (+, equimolar; ++, 5-fold more; +++, 10-fold more amounts) in the presence or absence of 1 mM CaCl2. The SYP132-VAMP722 interaction was analyzed by immunoblot with anti-HA antibody to detect HA-SYP132 in the precipitates with GST-VAMP722. To show the size of HA-SYP132, 1% of used HA-SYP132 was subject to immunoblot with anti-HA antibody. To test a Ca²⁺ effect, 1 mM EDTA was added during incubation. Equal loading was visualized by immunoblot with anti-GST antibody to detect GST-VAMP722. (C) Reduced SYP132-VAMP721/722 interaction in syt5 plants. Protein extracts from the indicated genotype plants leaves were precipitated (IP) with anti-VAMP721/722 antibody and the precipitates (Co-IP) were analyzed by immunoblot (IB) with anti-SYP132 antibody. To show expression levels of SYT5, SYP132, SNAP33, and VAMP721/722 in the indicated genotype plants, 3% of protein extracts used for Co-IP were subject to immunoblot with anti-SYT5, anti-SYP132, anti-VAMP721/722 antibody or anti-SNAP33 antiserum. Equal loading was visualized by staining Rubisco with Ponceau S.