Identification and partial characterization of an ER-targeting domain within DFCP1. (A) Sequential truncations of DFCP1 identified an internal domain necessary and sufficient to specify localization in the ER. Residues in red and underlined are required for ER localization. (B) The GFP-[416–543] construct localized to the ER in HEK-293 cells, whereas mutants within this domain (shown here is a W543A construct) were cytosolic. Selected residues important in ER targeting were also mutagenized in the full-length protein as indicated. The full-length DFCP1 (myc-DFCP1) localized to the ER, whereas the DM (myc-dmDFCP1) had ER/Golgi localization (insets in myc-dmDFCP1 panels show colocalization with giantin). Mutagenesis of critical residues resulted in cytoplasmic redistribution (shown here as W/A mutant). All samples were costained with antibodies to calnexin (CLNX), an ER protein. (C) The WT or the W/A mutant of the 416–543 domain were purified as GST fusion proteins (input) and mixed with rat kidney microsomes. Bound material was recovered by centrifugation. Note that the WT-derived protein bound to microsomes, whereas the W/A mutant did not. (D) Microsomes as in C were treated on ice with the indicated units of trypsin before incubation with the GST-[416–543] domain and centrifugation. Note that β-COP, a peripheral protein found on microsomes, was almost completely digested by trypsin; under these conditions, binding of the DFCP1 fragment to microsomes was not changed.