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. 2010 Jan 25;188(2):253–269. doi: 10.1083/jcb.200907015

Figure 7.

Figure 7.

Rab7 forms a complex with FYCO1 and induces the recruitment of FYCO1 to intracellular membranes. (A) Rab7Q67L recruits FYCO11,038–1,233 and FYCO11,091–1,233 but not FYCO11,156–1,233 to the cytosolic vesicles. HeLa cells transfected with GFP-FYCO11,038–1,233 (top), GFP-FYCO11,091–1,233 (bottom), or GFP-FYCO11,156–1,233 (middle right) with or without mCherry-Rab7Q67L were imaged 24 h after transfection. The graph shows the relative number of cells ± SD with GFP-FYCO11,038–1,233 recruited to intracellular membranes without and with cotransfected mCherry-Rab7Q67L. (B) Rab7Q67L recruits the 2xFYVEFYCO1 construct containing a short CC region in front of the first FYVE domain (FYCO11,091–1,233+1,156–1,233) but not a 2xFYVE alone to the perinuclear vesicles. HeLa cells transfected with GFP-FYCO11,091–1,233+1,156–1,233 or GFP-FYCO11,156–1,233+1,156–1,233 with or without mCherry-Rab7Q67L were imaged 24 h after transfection. (C) Rab7 coimmunoprecipitates with FYCO1 from HEK293 cell lysate. HEK293 cells stably transfected with 3xFlag-FYCO1 were cotransfected with GTP-locked mutants of Rab5, -7, -11, and -24 and lysed 24 h after transfection, and protein complexes were immunoprecipitated with anti–Flag M2 agarose affinity gel. Bound proteins were eluted with Flag peptide, resolved by SDS-PAGE, and visualized by immunoblotting with anti-GFP and anti-Flag antibody. IP, immunoprecipitation; WB, Western blot. (D) Leucine-1151 and tryptophan-1152 are important for the recruitment of FYCO1 to Rab7-containing vesicles. HeLa cells transfected with GFP-FYCO1990–1,233L1151A/W1152A with or without mCherry-Rab7Q67L were imaged 24 h after transfection. (B and D) Insets show an enlarged field of interest. (E) RILP but not Rab7 or ORP1L competes with FYCO1 for recruitment to intracellular vesicles. (right) HeLa cells cotransfected with GFP-RILP together with mCherry-FYCO1 and stained with Draq5 were imaged 24 h after transfection. (left) Relative number of HeLa cells cotransfected with mCherry-FYCO1 and the indicated constructs, in which FYCO1 is recruited to the intracellular vesicles. Error bars represent SD. Bars, 10 µm.