FYVE domain tethered peripherally to the ER translocates to starvation-induced punctate structures in a pathway dependent on PI(3)P. (A) GFP-FYVE domain from FENS-1 bound to PI(3)P-coupled lipid beads, whereas the GFP-ER–targeting domain of DFCP1 showed little binding (first four lanes; in, 5% of input; 3P, the faction bound to PI(3)P-coupled beads). A construct with the FENS-1 FYVE domain fused to the ER domain of DFCP1 (GFP-ERFYVE) maintained PI(3)P binding, whereas a construct with a point mutation in the FYVE domain (GFP-ERFYVE*) showed much-reduced binding. (B) Stable cell lines expressing the four constructs shown in A were left untreated or starved for 45 min before fixation and fluorescence microscopy. Note that the GFP-ERFYVE construct translocated to punctate structures upon starvation. (C) Cells expressing GFP-ERFYVE were starved alone or in the presence of wortmannin or BFA as indicated. (D) Cells expressing GFP-ERFYVE and starved for 45 min were either counterstained for the ER using antibodies to KDEL (top) or cotransfected with mRFP-MAP-LC3 (bottom). Note that the GFP-ERFYVE punctate structures frequently localized on the ER and showed partial colocalization with LC3. The panels on the right show an enlarged view of the boxed regions on the left. Bars, 10 μm.