Characterization of VPS13 mutant constructs. (A) Immunoprecipitation of GFP-tagged VPS13 protein from yeast cells expressing the protein from an integrated genomic copy under control of a GAL1 promoter. From left to right: WT, mut1, and mut2. Proteins in each lane are purified from equal numbers of cells, and the indicated yield is normalized to WT VPS13 protein. (B) Negative staining of 3xFLAG-tagged S. cerevisiae VPS13_1-1350 purified from Expi293 cells, with representative 2D-class averages shown below. Numbers of particles included in averages are indicated. From left to right: WT, mut1, and mut2. (C) Lipid binding gel shift assay with S. cerevisiae VPS13_1-1350. Equal amounts of VPS13_1-1350 constructs are coincubated with the indicated fluorescent lipids or no lipids and separated on a native gel. Gel imaging was performed both by fluorescence imaging and Coomassie staining. From left to right: WT, mut1, and mut2. PA, phosphatidic acid; PC, phosphatidylcholine.