Rab45 and CRACR2a are adaptor proteins for dynein. (A) Domain organizations of Rab45, CRACR2a, Rab11FIP3, and Ninein. EF, EF-hand; CC, coiled-coil. Sizes of the domains are not to scale. (B) Pulldown assay shows that Rab45 and CRACR2a bind to dynein–dynactin. DHC, dynein heavy chain; p150, the p150 subunit of dynactin. Western bands are from the same gel. (C) Sample kymographs of microtubule-based motility of Rab45 or CRACR2a in complex with dynein–dynactin. Fluorescence is from the N-terminal GFP tag on Rab45 or CRACR2a. (D) Quantification of the velocities of dynein–dynactin in complex with Rab45 or CRACR2a. Error bar: SD; n = 40 processive particles from three replicates. (E) Schematic of the inducible peroxisome trafficking assay. U2OS cells were transfected to express GFP-FRB fused with a peroxisome targeting sequence (PEX) and an mCherry-adaptor-FKBP construct. Treatment of the cell with rapamycin induces the localization of the dynein–dynactin adaptor complex to peroxisomes and drives retrograde transport of peroxisomes toward the microtubule minus end. (F) Representative images of the peroxisome transport assay (scale bar: 10 µm). +Rapa, treatment with rapamycin. Quantifications of the percentage of cells with a strong perinuclear cluster of peroxisomes are shown on the right. Data are mean ± SEM of three independent experiments (n = 30–40 cells per experiment). ****, P < 0.0001 between DMSO-treated group and rapamycin-treated group using two-way ANOVA analysis with Sidak’s multiple comparisons test.