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

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© 2010 Pankiv et al.

This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.jcb.org/misc/terms.shtml). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 3.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/3.0/).

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Figure 5. — FYCO1 can dimerize and is recruited to intracellular membranes in an FYVE domain–dependent manner. (A) FYCO1 shows only weak or no colocalization with a PI3P probe 2xFYVE_Hrs. HeLa cells were imaged 24 h after transfection with the indicated constructs. (B) The FYVE domain of FYCO1 is essential but not sufficient for its recruitment to the cytosolic vesicles. HeLa cells were imaged 24 h after transfection with the indicated constructs. (C) FYCO1 can self-interact in a CC-dependent manner. Full-length GFP-FYCO1 was cotranscribed and cotranslated with the indicated deletion mutants of myc-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. (D) Dimers but not a monomer of the FYVE domain from FYCO1 can bind PI3P in protein–lipid overlay assay. PIP Strips were incubated with 1-µg/ml solutions of MBP-FYVE_FYCO1, MBP-2xFYVE_FYCO1, or MBP-FYCO1_863–1,233 for 1 h, and bound proteins were detected by immunostaining with anti-MBP antibody. LPA, lysophosphatic acid; LPC, lysophosphocholine; PI, phosphatidylinositol; PE, phosphatidylethanolamine; PC, phosphatidylcholine; S1P, sphingosine-1-phosphate; PA, phosphatidic acid; PS, phosphatidylserine. (E) The construct containing a tandem fusion of two FYVE domains from FYCO1 (2xFYVE_FYCO1) localizes to the nuclear envelope and ER in HeLa cells. HeLa cells were imaged 24 h after transfection with the indicated constructs. (A and E) Insets show an enlarged field of interest. (F) 2xFYVE_FYCO1 does not colocalize with 2xFYVE_Hrs (left) and PX_p40phox (right). HeLa cells were imaged 24 h after transfection with the indicated constructs. Bars, 10 µm.