Figure 9.

Phospholipid transfer activity of Ups2–Mdm35. (A) Size-exclusion chromatography of SC Ups2–Mdm35 using a HiLoad 26/60 Superdex 200 PG column. Positions of molecular mass standards (in kilodaltons) are shown. A fraction indicated with a black bar in the elution profile was used in the phospholipid transfer assay. (B) Purified SC Ups2–Mdm35 was analyzed by SDS-PAGE followed by CBB staining. (C) PS transfer activity of SC Ups2–Mdm35 was measured at 25°C by the fluorescent-based phospholipid transfer assay between liposomes (see Materials and methods for details). (D) PS transfer activity of SC Ups2ΔLid–Mdm35 was analyzed as in C. (E) PA, PC, and PE transfer activities of SC Ups2–Mdm35 were analyzed as in C. (F) PS and PA transfer activities of SC Ups2–Mdm35 were measured by the mass spectrometry–based phospholipid transfer assay. SC Ups2–Mdm35 was incubated with heavy donor liposomes containing DOPS or DOPA and light acceptor liposomes. Liposomes were separated by sucrose density-gradient ultracentrifugation. Amounts of DOPS (left) or DOPA (right) in acceptor liposomes relative to those in input donor liposomes were quantified by mass spectrometry. Values are means ± SD (n = 3).