Basic and hydrophobic residues in the Nt are responsible for the distribution in the plasma membrane.

<p>(A) Alignment of the Nt amino acid (aa) sequences of phactr. Human phactr 1–4 (hPh1–4), mouse phactr3 (mouse), chicken phactr3 (bird), zebra fish phactr3 (fish), <i>Drosophila melanogaster</i> phactr (fly), and <i>Caenorhabditis elegans</i> phactr (worm) were aligned. N1–12 indicate substituted with alanine residues. (B) The distribution patterns of GFP-phactr3 Wt, N1, and N2. Cos7 cells expressing each construct were photographed using confocal laser microscopy at 24 h after transfection. The nucleus was stained with Hoechst 33342. Bars, 20 µm. (C) Amino acid residues in the indicated Nt (A) were substituted with alanine residues and expressed in Cos7 cells. The numbers of cells with predominantly plasma membrane (PM) distributions and cytoplasm (CYT) distributions were counted by fluorescence microscopy [(denoted as mean ± standard error (SE)]. The asterisks in the histograms indicate significant differences between Wt and each mutant (Student’s t-test; *<i>P</i><0.05, **<i>P</i><0.005). NS, not significant. (D) The BH plot analysis of the membrane interaction domains <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113289#pone.0113289-Brzeska1" target="_blank">[24]</a> detected a potent membrane interaction domain in Nt. Red bar indicates the experimentally deduced region responsible for the distribution in the plasma membrane.