Figure 3.

SFH proteins are atypical PITPs. The ability of each SFH protein to transfer [³H]PtdIns (A and C) or [¹⁴C]PtdCho (B and D) was determined in either cytosol fractions prepared from salt-stripped yeast membranes (A and B) or cytosol prepared from E. coli strains expressing recombinant SFH gene product (C and D). Activity is represented as the percentage of total input [³H]PtdIns or [¹⁴C]PtdCho transferred from donor membranes to unlabeled acceptor membranes during the course of the experiment (see MATERIALS AND METHODS). Cytosol values are presented as amounts of protein added to the assay cocktail. For the yeast cytosol experiments, the proteins of interest were expressed in strain CTY303 (Table 1), which has no detectable endogenous PtdIns- or PtdCho-transfer activity. CTY303/YEp(URA3) cytosol was prepared and used as a negative control. Assay blanks represent addition of buffer alone to the transfer assay reactions. ♦, Sec14p; ▴, Sfh2p; ▪, Sfh3p; ●, Sfh4p; ×, Sfh5p; ○, vector control. In experiments that used yeast cytosol, 14,155–17,319 cpm of input [³H]PtdIns and 14,963–22,035 cpm of input [¹⁴C]PtdCho was used in each assay. Background values for these PtdIns- and PtdCho-transfer assays were in the range of 521–697 and 76–159 cpm, respectively. All samples contained <170 mM KCl, a salt concentration that itself had no effect on background or signal in these assays. In experiments that used E. coli cytosol, 15,034–15,411 cpm of input [³H]PtdIns and 18,950–22,531 cpm of input [¹⁴C]PtdCho was used in each assay. Background values for assays that used recombinant SFH proteins were in the range of 501–601 for the PtdIns-transfer assays and 547–578 for the PtdCho-transfer assays. No KCl was present in any assays that used E. coli cytosol. These data are representative of at least five independent experiments.