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–FgGyp1TBC (TBC), MBP–FgGyp1R357K (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, FgGyp1TBC (TBC), FgGyp1R357K (R357K), and MBP control, respectively, following the instructions of the manufacturer. The experiment was repeated three times.
Gtpase assay kit
Manufactured by Merck Group
The GTPase assay kit is a laboratory tool designed to measure the enzymatic activity of GTPases, a class of proteins that play a crucial role in various cellular processes. The kit provides the necessary components and protocols to quantify GTPase activity in a reliable and efficient manner.
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3 protocols using gtpase assay kit
1
GTPase Activity Assay for FgRab1 and FgGyp1
2
GAP Activity Assay for FgBub2 and FgTem1
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pHis-FgBub2 and pGST-FgTem1 vectors were constructed by amplifying FgBUB2 or FgTEM1 coding sequence from the cDNA of PH-1 using the primer pairs listed in S3 Table , then cloned into the pET32a (His) or pGEX-4T (GST) vector, respectively. His-FgBub2, GST-FgTem1 and His proteins were expressed in BL21 Escherichia coli strain and purified, respectively. Next, these proteins were used for GAP activity assay using a GTPase assay kit (Sigma-Aldrich, Catalog Number MAK113) according to the manufacturer’s protocols.
3
Biochemical Analysis of Fungal Rab1 GTPase Regulation
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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.
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