Fig. 5: RAB33A-mediated recruitment of TBC1D2A inhibits RAB7 activation to inhibit RhoC degradation.

a Localization of RAB33A and endogenous RAB7A in HeLa cells stably overexpressing RAB33A. Arrowheads indicate the colocalization of the two proteins. b Localization of RAB33A and HA-RILP in HeLa cells stably overexpressing RAB33A and transiently transfected with HA-RILP for 48 h. c, d Overexpression of RAB33A in SiHa cells results in reduced RAB7-GTP levels. Active RAB7 in wild-type or RAB33A-overexpressing SiHa cells was detected by a GTP pull-down assay. These results are presented from three independent experiments. The protein levels of RAB7 in the pull-down assay were then quantified (mean ± SD. n = 3 times. two-tailed Student’s t-test). e Localization of Flag-RAB33A, GFP-RAB7 and V5-TBC1D2A in HeLa cells stably overexpressing Flag-RAB33A and transiently transfected with GFP-RAB7 and V5-TBC1D2A for 48 h. The HEK-293T cell line was transiently cotransfected with V5-TBC1D2A (f), distinct TBC1D2A domains (g) and Flag-RAB33A for 48 h, after which the TBC1D2A-RAB33A interaction was assessed by western blotting. h, i The specific colocalization of RAB33A with distinct TBC1D2A domains. Localization of V5-tagged TBC1D2A mutants in HeLa cells stably expressing Flag-RAB33A (h). Cells were transiently transfected with the indicated plasmids for 48 h. The number of puncta indicating Flag-RAB33A and TBC1D2A colocalization in each cell was calculated (i, n = 6 fields). The data are presented as the mean ± SD. P values are shown; two-tailed Student’s t-test. Vector, vector-only control.