The FYVE domain from FYCO1 containing the dimerizing CC is recruited to late endosomal/lysosomal structures. (A) FYCO1_990–1,233 is the smallest FYVE domain–containing deletion mutant of FYCO1 consistently recruited to cytosolic vesicles. HeLa cells transiently transfected with FYCO1_990–1,233 were imaged 24 h after transfection. (B) FYCO1_990–1,233 is the smallest FYVE domain–containing deletion mutant of FYCO1 that can efficiently interact with the full-length FYCO1. Full-length myc-FYCO1 was cotranscribed and cotranslated with the indicated deletion mutants of GFP-tagged FYCO1 in rabbit reticulocyte lysate. S³⁵-labeled FYCO1 complexes were immunoprecipitated with anti-myc antibody, separated by SDS-PAGE, and visualized by autoradiography. IP, immunoprecipitation. (C) FYCO1_990–1,233 strongly colocalizes with Rab7, LysoTracker red, and Alexa Fluor 647–dextran (10,000 D) but only weakly with PX_p40phox. HeLa cells transfected with the indicated constructs or labeled with LysoTracker red for 60 min and Alexa Fluor 647–dextran (10,000 D) for 4 h were imaged 24 h after transfection. (D) FYCO1 construct containing a CC region followed by a tandem repeat of two FYVE domains from FYCO1 (FYCO1_{990–1,233+1,156–1,233}) localizes to the ER and perinuclear vesicles and only weakly colocalizes with PX_p40phox. HeLa cells were imaged 24 h after transfection with the indicated constructs. (A, C, and D) Insets show an enlarged field of interest. Bars, 10 µm.