Sac2 functions downstream of Rab5 and is part of a complex also comprising OCRL. (A–C) COS7 cells were cotransfected with mCherry (mCh)-Sac2 together with GFP-Rab5^WT, GFP-Rab5^Q79L (constitutively active; B), or GFP-Rab5^S34N (dominant negative; C), as indicated, and imaged by confocal microscopy. mCh-Sac2 colocalizes with WT and constitutively active Rab5. In contrast, it has a predominant cytosolic localization in cells expressing dominant-negative Rab5. Inset panels show enlarged views of the regions boxed in yellow. Bars: (full-size images) 5 µm; (insets) 2 µm for inset image. (D) COS7 cells were cotransfected with 3×Flag-Sac2, HA-OCRL, and GFP-tagged Rab5 constructs (Rab5^WT, GFP-Rab5^Q79L, or GFP-Rab5^S34N). Cell lysates were immunoprecipitated with anti-Flag antibody to enrich for Sac2. Immunoblotting of the starting lysates (left) and of the immunoprecipitates (right) for the Flag, HA, and GFP epitopes confirms enrichment of Sac2 and shows robust coprecipitation of Rab5 and OCRL only from cells expressing GFP-Rab5^WT or GFP-Rab5^Q79L. Recovery of OCRL and Rab5 was dramatically reduced from cells expressing Rab5^S34N.