Sac2/INPP5F is an inositol 4-phosphatase that functions in the endocytic pathway

Colocalization of Sac2 with OCRL on early endosomes and at the last stage of clathrin-mediated endocytosis. Live spinning-disc confocal microscopy. (A) Colocalization of GFP-Sac2 and mCherry-OCRL. The majority of the fluorescent puncta positive for both proteins are expected to be early endosomes, given the close colocalization of both proteins with Rab5 (see Fig. 1 A and Hyvola et al., 2006; Erdmann et al., 2007). Insets show enlarged views of the regions boxed in yellow. (B and C) Time course of CLC-mRFP and either GFP-Sac2 (B) or GFP-OCRL (C) fluorescence intensities at very late stage clathrin-coated pits demonstrating that Sac2 and OCRL appear when clathrin is disappearing, and that Sac2 and OCRL display similar recruitment patterns. The disappearance of the Sac2 and OCRL fluorescence reflects the newly formed vesicle moving out of the focal plane. Bars: (A) 5 µm; (B and C) 1 µm.

Recruitment of Sac2 to macropinosomes. Mouse fibroblasts expressing GFP-Sac2 and either RFP-PH^AKT (A) or RFP-EEA1 (B) were also cotransfected with H-Ras^V12G to induce the formation of macropinosomes. The gallery of confocal images shows that Sac2 is recruited to these vesicles when the AKT signal, which primarily reflects PI(3,4,5)P₃, is disappearing, and remains associated with the vesicles as they mature to EEA1-positive endosomes. Bars, 1 µm.

Defect in the internalization of transferrin in Sac2 KO MEFs. Internalization of biotinylated transferrin over time was analyzed biochemically in WT and Sac2 KO MEFs using an ELISA-based assay (see Materials and methods for details). Data were expressed as mean ± SD (error bars) of three independent experiments (Student’s t test, **, P < 0.01; ***, P < 0.001).

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.