Figure 5. SdhA interrupts Rab5 binding.
(A) High-affinity binding of SdhA variants to OCRL. ASH-RhoGAP was immobilized and challenged with indicated GST-tagged proteins. EC50 were calculated and expressed as nM (STAR Methods).
(B) Competitive binding of Rab5 and SdhA to ASH domain. Immobilized ASH domain was challenged with 22 nM Rab5a (Q79L) in combination with increasing amounts of GST-fused SdhA constructs.
(C) Same as in (B), except ASH/RhoGAP domain was immobilized.
(D) SdhA binding to ASH domain of OCRL was not affected by challenge with Rab5a. Same as in (B), but constant amounts of SdhA (CBM2 = 22 nM; CBM2A = 7.4 nM) and increasing amounts of Rab5a (Q79L) are shown.
(E) Same as in (D), but ASH/RhoGAP domain was immobilized.
(A–E) Data represent mean values ± SD of three wells (technical replicates) for each dilution. Shown is representative of two experiments.
(F) SdhA-CBM2 does not affect the Ses1- and Pacsin2-stimulated 5-phosphatase activity of OCRL. Phosphatase activity was measured as described (STAR Methods). His-OCRL (50 nM) was incubated with indicated proteins and with 200 μM of PI(4,5)P2-containing liposomes. Ses1:SdhA refers to concentration of SdhA-CBM2 relative to Ses1. Data expressed as percentage of 5-phosphatase activity compared to incubation of OCRL with liposomes are shown. Data represent mean values ± SE (standard error) of three independent experiments (statistical significance was tested using two-tailed unpaired Student’s t test; *p < 0.05; **p < 0.01; ***p < 0.001).
(G) SdhA-CBM2 binding to OCRL during 5-phosphatase assay in (F) demonstrated by pull-down assay. His-OCRL and SdhA-CBM2 were collected with Ni2+ resin and analyzed by Coomassie staining. The amount input (10%), unbound FT (flowthrough) (20%), and bound PD (50%) are displayed.