Fig. 5
GORAB, via Scyl1, is sufficient to recruit COPI to membranes. a A schematic diagram depicting the mitochondrial relocation assay where the addition of rapamycin induces mitochondrial relocation of FKBP-tagged GORAB, allowing for recruitment of associated factors to this compartment. b Relocation of GORAB-mycFKBP and co-expressed GFP-Scyl1 to mitochondria. HeLaM cells co-transfected with mito-FRB and GORABK190del-mycFKBP constructs were pretreated with 2.5 µg/mL nocodazole for 2 h and further incubated with 1 µM rapamycin or DMSO for 3 h prior to fixation. Cells were labeled with antibodies to myc and mtHsp70. c Relocation of endogenous Scyl1 to mitochondria by GORAB-mycFKBP. HeLaM cells co-transfected with mito-FRB and GORAB-mycFKBP and treated as described in b and labeled with antibodies to endogenous Scyl1 and the Golgi marker β4GalT1. d Relocation of COPI to mitochondria by GORAB-mycFKBP. HeLaM cells co-transfected with mito-FRB, GORABK190del-mycFKBP and GFP-Scyl1 were treated as in b, and additionally with 5 µg/mL BFA for 15 min (lower panel only). Cells were labeled with antibodies to β’-COP and myc. In b–d, scale bars are 10 µm and white lines indicate the pixels used for the RGB fluorescence intensity profile plots shown on the right, which are representative of data from n = 20 cells
