Figure 7.

The LjPLP-IV Protein Complements the Temperature-Sensitive Phenotype of the Yeast sec14 Mutant and Exhibits PtdIns and PtdCho Transfer Activity.

(A) The growth phenotype of yeast strain CTY1079 (sec14-1^ts Δspo14) harboring the indicated constructs (see Methods). Individual transformants were streaked on selective (−Ura) media and grown at permissive (28°C) and nonpermissive (37°C) temperatures.

(B) and (C) Phospholipid transfer activities. The ability of the recombinant LjPLP-IV Sec14p domain (LjPLP-IV–Sec14p) to transfer ³H-PtdIns (B) or ¹⁴C-PtdCho (C) was measured in cytosol prepared from E. coli. Activities are represented as the percentage of total input ³H-PtdIns or ¹⁴C-PtdCho transferred from donor membranes to unlabeled acceptor membranes during a 30-min incubation at 37°C. Cytosol values are presented as amounts of protein added to the assay. Closed circles, Sec14p; open squares, LjPLP-IV–Sec14p; open circles, E. coli cytosol.

Assay blanks represented the addition of buffer alone to the transfer assay reactions. These reactions defined assay background, and background values were subtracted from each assay to yield transfer values. Ranges of 12,695 to 15,121 cpm and 22,697 to 28,560 cpm of input ³H-PtdIns and ¹⁴C-PtdCho, respectively, were used in each assay. Background values for these assays ranged from 237 to 374 for the PtdIns transfer assays and from 238 to 391 for the PtdCho transfer assays. The data shown are from a representative experiment of at least three independent assays.