Fig. 4.

The amino acid residues involved in the ligand binding in the crystal are essential for evt-2 localization to REs. (A) The full-length point mutant K20E, in which Lys20 was changed to Glu, was transiently expressed in COS-1 cells. The cells were then fixed and stained for Myc, GM130, TfnR, VPS26, and CD63. Arrowheads indicate puncta where K20E and endosomal markers are juxtaposed or colocalized. (Scale bars, 10 μm.) (B) The PH point mutants PH(K20E), PH(R11E), and PH(R18E), in which Lys20, Arg11, or Arg18 was changed to Glu, were transiently expressed. The cells were then fixed and stained for Myc and GM130. (Scale bars, 10 μm.) (C) Alignment of vertebrate evt-2 PH domains. The secondary structure elements of human evt-2 PH are shown above the sequence. Conserved residues are boxed in white on a black background. Similar residues are boxed in black with a white background. Asterisks indicate the residues directly involved in the interaction of the ligand.