Protrudin and FYCO1 mediate translocation of mTOR lysosomes to the cell periphery. (A) Confocal micrograph showing colocalization between endogenous LAMP1 and mTOR in HeLa cells grown in complete medium. (B and C) HeLa cells were transfected with GFP Protrudin for 20 h, stained with antibodies against LAMP1 and mTOR (B) or FYCO1 and mTOR (C), and analyzed by confocal microscopy. Insets show magnification of lysosomes in contact with Protrudin-positive ER. (D) HeLa cells were transfected (40 h) with the constructs indicated, labeled for endogenous mTOR, and analyzed by confocal microscopy. Asterisks indicate transfected cells. Arrows point at mTOR in the cell periphery. Insets show magnification of mTOR- and mCherry–FYCO1–positive lysosomes. Note that whereas mCherry–FYCO1WT colocalizes with mTOR in the cell periphery, mCherry–FYCO1ΔKIFBD colocalizes with mTOR in the perinuclear area. (E) Automated quantification of cells treated as in D, using the Olympus ScanR system. Graphs represent the relative total intensities of perinuclear localization of mTOR-positive lysosomes. The transiently transfected cells were compared directly with nontransfected neighboring cells (set to 1) in each sample. Error bars denote ± SEM from independent experiments: GFP–ProtrudinWT (n = 5), GFP–ProtrudinΔFYVE and ΔKIFBD (n = 4), and GFP–ProtrudinWT+mCh-FYCO1WT or ΔKIFBD (n = 3). **, P < 0.01; ***, P < 0.001. Myc–ProtrudinWT and Myc–ProtrudinWT+mChFYCO1WT, unpaired t test. Transfected cells versus control set to 1, one-sample t test. In total 200–800 transfected cells were analyzed per condition.