Figure 3.

Identification of critical residues responsible for Rab9 binding in HPS4 by site-directed mutagenesis. A, heatmap analysis of conserved residues of HPS4 from seven vertebrate species: mouse (Mm), human (Hs), rat (Rn), bovine (Bt), chicken (Gg), frog (Xl), and zebrafish (Dr). Conserved residues in the minimal Rab9-binding site of HPS4 (black background) are shown at the bottom of the heatmap. Eight conserved amino acids were selected and subjected to Ala-based site-directed mutagenesis (arrowheads). B, Rab9 binding activity of HPS4 point mutants. Yeast cells containing pGBD-C1-Rab9A/B(CA/CN)ΔCys plasmids and pAct2 plasmids expressing HPS4 point mutants were streaked on SC-LW or SC-AHLW medium (selection medium) and incubated at 30 °C. Note that Ser-541 and Asn-543 of HPS4 are crucial for Rab9(CA) binding.