Figure 5.

The UNC-104 PH domain binds selectively to PI(4,5)P₂ and point mutants in conserved basic residues reduce binding. Primary sequence alignment of the PH domains of UNC-104/KIF1A family members: C. elegans (CeUNC-104), D. discoideum (DdUnc104), H. sapiens (HsATSV), M. musculus (MmKIF1A), and D. melanogaster (DmUnc104). Conservation in positively charged amino acids in at least 4 of 5 members are highlighted. (B) A mini-UNC-104 consisting of the first 446 amino acids followed by the PH domain (1446–1584) was loaded at the bottom of a sucrose gradient together with liposomes containing either 100% phosphatidylcholine (PC) or 90% PC and 10% of the indicated phospholipids (PA, phosphatidic acid). After centrifugation in a step sucrose gradient to float liposomes, fractions from the top liposome layer and the bottom unbound layer were collected and analyzed by SDS-PAGE and Coomassie staining (see MATERIAL AND METHODS). The data were expressed as the % of the motor that bound to the 10% PI(4,5)P₂ liposomes. In this experiment, 40% of the added UNC-104 mini-motor (1.5 μM total) was bound to the 10% PI(4,5)P₂ liposomes. The mean and SD are shown for three experiments. (C) Effect of PH domain point mutations on PI(4,5)P₂ binding. Binding efficiency was calculated by comparing the specific binding values to PI(4,5)P₂ vs. PC liposomes, subtracting the corresponding nonspecific PC binding value from the PI(4,5)P₂ binding, and then expressing the ratio between wild-type and mutant PH domain constructs. The nonspecific binding of the point mutants to PC liposomes was similar to wild type. The mean and SD are shown for three experiments.