Properties of PITPβW202W203 interaction with membranes. (A) Phospholipid-transfer assays. Abilities of each individual PITP to transfer [3H]PtdIns, [14C]PtdCho, or [14C]-SM, (indicated at top) was determined in cytosol fractions prepared from yeast strain CTY303 (sec14Δ cki1Δ) expressing the negative control gene URA3 (black bars), or recombinant versions of the respective PITPs (PITPβ, white bars; PITPβMI74,75AA, hatched bars; PITPβWW202,203AA, stippled bars). Activity is represented as the percentage of total input radiolabeled phospholipid transferred from donor membranes to unlabeled acceptor membranes during the course of the experiment. Values represent the averages of triplicate determinations from a representative experiment, and at least three independent experiments were performed. Assay blanks represented addition of buffer alone to the transfer assay reactions, and corresponding background values were subtracted from the other measurements. In this set of assays, input substrate was 14,792 cpm [3H]PtdIns; 27,940 cpm [14C]PtdCho; 22,216 cpm [14C]SM, respectively. Background values were 295, 485, and 236 cpm for each respective assay. (B) PITPβ WW202,203AA mutants preserve function as assayed in yeast. Serial 10-fold dilutions of isogenic sets of a sec14-1ts strain, derivatives of that strain carrying a high-copy plasmid (YEp) driving expression of either PITPβ, PITPβ-WW202,203AA, PITPβ-MI74,75AA, or a wild-type SEC14 gene (as indicated) were spotted onto YPD agar and incubated at 37°C for 48 h. Strains used were CTY1-1A (sec14-1ts), and CTY1-1A transformed with YEp(SEC14), YEp(PITPβ), YEp(PITPβ-MI74,75AA), and YEp(PITPβWW202,203AA), respectively. The PITPβ genes were driven by the strong and constitutively expressed yeast PGK promoter (PPGK) or the weaker constitutively expressed SEC14 promoter (PSEC14), as indicated.