GTPase Activity Assay for FgRab1 and FgGyp1

The sequences of FgRab1 cDNA, FgGyp1 cDNA, TBC domain and R357K point mutant of FgGyp1 were cloned into the MBP (Maltose-binding protein) vector pMAL-c2X, using their respective primers listed in Supplementary Table 2, and expressed. The protein products were isolated and purified for GAP activity assay using a GTPase assay kit (Sigma-Aldrich, Catalog Number MAK113) according to the manufacturer’s protocols. GTP hydrolysis was assayed based on previous reports (Xiong et al., 2012 (link); Zheng et al., 2020 (link)). Briefly, the recombinant proteins MBP–FgGyp1, MBP–FgGyp1^TBC (TBC), MBP–FgGyp1^R357K (R357), and MBP–FgRab1 were expressed in BL21 Escherichia coli strain and isolated by Amylose resin (Sangon Biotech, NO.C500096) as per the instructions of the manufacturer. A colorimetry-based kit was used (Sigma-Aldrich, Catalog Number MAK113) to assay for the GTPase activity of Gyp1, and FgRab1 was incubated with FgGyp1, FgGyp1^TBC (TBC), FgGyp1^R357K (R357K), and MBP control, respectively, following the instructions of the manufacturer. The experiment was repeated three times.