Phosphatidylinositol(3)-Phosphate Signaling Mediated by Specific Binding to RING FYVE Domains  Christopher G Burd, Scott D Emr  Molecular Cell  Volume 2, Issue 1, Pages 157-162 (July 1998) DOI: 10.1016/S1097-2765(00)80125-2

Figure 1 FYVE Domain Proteins Yeast and mammalian FYVE domain–containing proteins are schematically illustrated. Amino acid numbers listed below each protein indicate the approximate boundaries of the FYVE motif. Vac1p and EEA1 also contain zinc finger (Zn) and coiled-coil domains (coil), and Vac1p also contains a second RING domain (RING). FGD1 also contains a PH domain (PH) and a Dbl homology domain (DH). Diagrams are not to scale. Molecular Cell 1998 2, 157-162DOI: (10.1016/S1097-2765(00)80125-2)

Figure 2 In Vivo Localization of GFP-FYVE Domain Fusion Proteins (a) Fusion genes encoding GFP fused to the entire coding sequence of PIB1, human EEA1 FYVE domain (amino acids 1257–1411), or an EEA1 C1405S mutant were expressed in wild-type yeast cells and visualized by fluorescence microscopy. (b) GFP-Pib1p and GFP-EEA1(FYVE) were expressed in vps34Δ cells that have a deletion of the gene encoding PI(3)K. For the panels in (a) and (b), the high contrast GFP fluorescence image is on the left and the corresponding Nomarski differential interference image is on the right. Arrowheads point to vacuoles in several fields. (c) Wild-type cells expressing GFP-EEA1(FYVE) were incubated on ice for 30 min in media containing FM4-64, a lipophilic fluorescent dye that labels the plasma membrane under these conditions (Vida and Emr 1995). Fresh media was then added and the culture was warmed to room temperature to induce synchronous endocytosis of the dye to the vacuole. Aliquots were withdrawn at various time points, and fluorescence images of the same field were captured in the rhodamine (FM4-64) or fluoresceine (GFP) channels. Pairs of high contrast images were false-colored and then merged. The 30 min chase point is shown. Molecular Cell 1998 2, 157-162DOI: (10.1016/S1097-2765(00)80125-2)

Figure 3 FYVE Domains of EEA1 and Vps27p Bind Specifically to Liposomes Containing PtdIns(3)P Extracts from E. coli expressing the indicated GST-FYVE domain fusion proteins were incubated with liposomes containing various phosphoinositides at 2.5 mol%, except PtdIns(3)P, which was present at the indicated mol%. (a) GST-EEA1(FYVE) (amino acids 1257–1411 of EEA1); (b) GST-Vps27 FYVE domain (amino acids 40–291). The NaCl concentration of the assay buffer used to assay GST-Vps27 FYVE was 0.5 M. (c) The crude extract containing GST-EEA1(FYVE) domain was preincubated with 10 mM TPEN, a zinc chelating agent, for 10 min prior to testing liposome binding. After the preincubation, the extract was centrifuged and the supernatant was used in the liposome-binding assay (2.5 mol% PtdIns(3)P). A C1405S mutant GST-EEA1(FYVE) fusion protein, and a GST-Vps18p (amino acids 568–918) RING domain fusion protein, which is not a member of the FYVE domain subfamily, were assayed for liposome (2.5 mol% PtdIns(3)P) binding. Molecular Cell 1998 2, 157-162DOI: (10.1016/S1097-2765(00)80125-2)

Figure 4 Summary of PtdIns(3)P Binding Assays A schematic diagram of the RING/FYVE domain with the signature basic patch and the eight zinc-coordinating cysteine residues highlighted is shown at the top. Independent mutations in the basic patch or the C1405S mutant are also indicated. A summary of the binding activities of GST-FYVE domain fusion proteins for liposomes containing no phosphorylated phosphoinositide (gray bars) or 2.5 mol% PtdIns(3)P (black bars), is shown in the bar diagram below. The fraction of protein bound to liposomes is indicated. Assays with GST-Vps27p(FYVE) (amino acids 40–291) and GST-Fab1p(FYVE) (amino acids 2–263) were done in buffer containing 0.5 M NaCl, and assays with GST-EEA1(FYVE) proteins, GST-Pib1p (amino acids 2–286), and GST-Vac1p (amino acids 2–515) were done in standard binding buffer. Molecular Cell 1998 2, 157-162DOI: (10.1016/S1097-2765(00)80125-2)

Figure 5 Regulation of Vacuolar Protein Sorting in Yeast by the Vps34 PI(3) Kinase See text for details. Molecular Cell 1998 2, 157-162DOI: (10.1016/S1097-2765(00)80125-2)