In-vitro GTPase assay and GEF assay

The GTPase assay and GEF assay were performed using Promega’s GTPase-Glo™ Assay kit. For GTPase assay, 2 × GTP solution was prepared to contain 10 μM GTP and 1 mM DTT with or without 4 μg LepB in GTPase/GAP Buffer. Next, 400 ng purified Rab1 was diluted in GTPase/GAP Buffer. GTP solution (2×) was added and the solution incubated for 2 h to allow the GTPase reaction. The GTPase reaction systems were supplemented with 400 ng Rab, Rab1 R2A, Rab1 R4A, or modified Rab1, 4 μg LepB, and 2 μg DrrA in the GEF Buffer for GEF assay. Reactions were initiated by adding 10 μM GTP in GEF Buffer containing 1 mM DTT and incubated for 2 h at room temperature. All reactions were terminated by the addition of GTPase-Glo Reagent and then incubated for 30 min followed by the addition of Detection Solution. Ten minutes later, the luminescence was measured using a multilabel plate reader (Cytation 5, BioTek).