Figure 5.

The spo20⁺ gene product is a PtdIns/PtdCho-transfer protein. Relative abilities of budding yeast Sec14 and Spo20 to transfer [³H]PtdIns and [¹⁴C]PtdCho were measured in crude cytosolic fractions prepared from S. cerevisiae. Activities are represented as the percentage of total input radiolabeled substrate transferred from donor membranes to unlabeled acceptor membranes. Transfer substrate for each data set is identified at top. In all cases, cytosol was clamped at 0.25 mg per assay. Sec14 and Spo20 were individually expressed in the S. cerevisiae strain CTY303 (see MATERIALS and METHODS), which has no detectable endogenous PtdIns- or PtdCho-transfer activity. Cytosol prepared from the isogenic YEp(URA3) derivative of strain CTY303 (i.e., strain CTY808) served as negative control. Sources of cytosol are identified at the bottom by the expression plasmids introduced into strain CTY303. Corresponding transfer values from mock assays were subtracted from each reaction. Mock samples represented the addition of buffer alone to each transfer reaction. In these experiments, input [³H]PtdIns and [¹⁴C]PtdCho ranged from 14639–19101 to 22428–27656 cpm for each assay, respectively. Background values in mock reactions ranged from 544–657 cpm for PtdIns-transfer assays to 1389–1489 cpm for PtdCho-transfer assays. These data are from a representative experiment.