Fig. 5

Dopey1 is sufficient to interact with and recruit kinesin-1 to the membrane. a Dopey1 specifically interacts with Kif5b. b Dopey1(1–148) is sufficient to interact with KLC2. c W³⁴ is essential for DEN’s interaction with KLC2. d Dopey1–KLC2 interaction is enhanced by Mon2. Ratios of Dopey1 band intensity of the IP panel to that of the corresponding cell lysate panel were normalized and labeled in the top blot. IPs were similarly performed as in Fig. 1. e The membrane recruitment of KLC2 requires PI4P and Dopey1 and is enhanced by Mon2. Liposomes with PI or PI4P were incubated with cell lysates expressing indicated protein(s) and pull-downs were immunoblotted. Values are normalized ratios of band intensities in pull-down panels to corresponding ones in cell lysate panels. Molecular weights (in kDa) are labeled. f, g The Golgi association of Kif5b decreases upon the knockdown of Dopey1 or Mon2. The experiment and quantification are similar to Fig. 2d, e. h–j Artificially tethering Dopey1 is sufficient to move peroxisomes to the cell periphery. A schematic diagram of fusion proteins is shown in (h). i Cells co-expressing indicated proteins were treated with 50 nM rapamycin followed by live cell imaging. The normalized gyradius is quantified in (j). k–p Particle analysis of peroxisomes with artificially tethered Dopey1. k Instantaneous velocity of a typical peroxisome tethered with indicated fusion protein. Segment speed, time, frequency, distance, and their corresponding centrifugal biases are shown in (l–o). p Statistical analysis of single peroxisome mean velocity. In each case, tracks were identified from time lapses of seven cells in three experiments. n indicates the number of cells (g), segments (l, m, o), or tracks (n, p). Scale bar, 10 µm; error bar, mean ± s.e.m.; P values are from t test (unpaired and two-tailed); N.S. not significant, *P ≤ 0.05, ***P ≤ 0.0005, ****P ≤ 0.00005, *****P ≤ 0.000005. Dotted white lines indicate cell contours (f, i). HEK293T cells were used in a-e while HeLa cells were used in (f, g, i–p). Source data are provided as a Source Data file