The first two coiled-coil domains of RUFY1 are required for its function as a dynein cargo adaptor and interaction with dynein subunit LIC1. (A) Schematic representation of the domain architecture of RUFY1 (full-length; FL) and mutants with progressive deletion of its C-terminus and internal deletions lacking either the first (300–400 a.a.) or second (400–500 a.a.) or both coiled-coil regions (300–500 a.a.). The RUFY1 (ΔRUN) mutant lacks the 271 a.a. from the N-terminal. (B–I) Representative confocal images of HeLa cells transiently expressing FRB-Tom70p with 2x-FKBP-GFP-RUFY1 (FL) or mutants (as described in A) and treated without rapamycin, followed by immunostaining with an anti-Tom20 antibody to visualize mitochondria. A white boundary marks the co-transfected cells. Bars: 10 µm. (J) The Tom20 signal intensity profile was quantified with respect to distance from the nucleus of HeLa cells expressing indicated FRB-FKBP fusion proteins upon addition of rapamycin. The values plotted are the mean ± SD from three independent experiments. Experiments are color-coded, and each dot represents the individual data points from each experiment. The total number of cells analyzed is indicated on the bottom of each data set (*P < 0.05, **P < 0.01, ***P < 0.001 ****P < 0.0001; n.s., not significant; unpaired two-tailed t test). (K and L) Recombinant GST and GST-LIC1^WT proteins were immobilized on glutathione-coated-agarose beads and incubated with HEK293T cell lysates expressing 2x-FKBP-GFP-RUFY1 (FL) or mutants (as indicated). The precipitates were immunoblotted (IB) with an anti-GFP antibody and Ponceau S staining was done to visualize the purified proteins. Source data are available for this figure: SourceData F9.