Fig. 6. The C-terminal region of endofin cannot be replaced by the corresponding region of SARA for interaction with the C-terminal domain of TOM1 and its membrane recruitment. (A) Schematic of wild-type endofin and SARA constructs as well as the chimeric constructs used in the pull-down experiments. (B) The C-terminal region of endofin but not SARA mediates binding to the TOM1 C-terminus. 293T cells were transfected with the Myc-tagged constructs depicted in A and the resultant lysates were incubated with either GST or GST-TOM1(300-492), which were bound to glutathione-Sepharose beads. The inputs (3%) as well as pull-down materials were resolved by SDS-PAGE and analyzed by western blotting with anti-Myc (upper panel) and anti-GST (lower panel) antibodies, the latter to indicate levels of GST or GST fusion proteins used in the experiment. (C) Membrane recruitment of TOM1 by endofin is dependent on the C-terminus of endofin, which cannot be replaced by the corresponding region of SARA. A431 cells were transfected with each of the Myc-tagged constructs depicted in A. Upon fixation and permeabilization, the cells were stained with antibodies against the Myc epitope (left panels) and endogenous TOM1 (right panels). (D) Membrane recruitment of clathrin by endofin is dependent on the C-terminus of endofin, which cannot be replaced by the corresponding region of SARA. A431 cells were transfected with the Myc-tagged constructs depicted in A. Upon fixation and permeabilization, the cells were stained with antibodies against the Myc epitope (left panels) and clathrin heavy chain (HC) (right panels). Bar, 10 µm.