Fig. 10
SlVPE3 is involved in KTI4 cleavage. a Immunoblot detection of KTI4 cleavage in tomato fruit. Total protein extracts from wild-type (WT) and SlVPE3 RNAi fruit (3-4, 3-12, and 3-15) at 35 and 38 days post-anthesis (dpa) were analyzed by immunoblot analysis using anti-KTI4 or anti-SlVPE3 antibodies. The predicted molecular mass of the full-length KTI4 is ~25 kDa, which is indicated by a blue arrowhead. b Determination of KTI4 cleavage in tobacco (N. benthamiana). Proteins were extracted from tobacco leaves expressing an empty vector (Vec), KTI4 alone (Vec + KTI4), SlVPE3 and KTI4 (SlVPE3 + KTI4), and mutated SlVPE3 and KTI4 (SlVPE3 C69G/C208G  + KTI4) and subjected to immunoblot analysis with an anti-KTI4 antibody. The mutated form of SlVPE3 (SlVPE3C69G/C208G) was generated by site-directed mutagenesis. c Cell-free cleavage of His-tagged KTI4 proteins. His-KTI4 was expressed and purified from Escherichia coli and then incubated for 15 or 30 min at 20 °C with extracts of N. benthamiana expressing an empty vector (Vec), intact SlVPE3 (SlVPE3), or a mutated form of SlVPE3 (SlVPE3 C69G/C208G). An incubation of His-KTI4 with extraction buffer served as the control. The VPE inhibitor (biotin-YVAD-fmk) was applied to determine whether the His-KTI4 was directly cleaved by SlVPE3. In a–c, equal loading was confirmed with an anti-actin antibody. Similar results were obtained from three independent experiments and results from a representative experiment are shown