(A) In vitro [3H]-PtdIns transfer assays were performed with purified recombinant Sfh5, Sfh5H173A and Sfh5Y175F in a titration series where protein inputs were increased in steps of two-fold (5, 10, 20, 40 µg). The total radiolabeled input for each assay varied between 8377 to 12,070 cpm, and background ranged between 150–575 cpm. The transfer values represent the mean of triplicate assay determinations from at least two independent experiments. (B) Resuscitation of PITP activity in heme-deficient Sfh5 mutants in vivo. Left panel: A sec14-1ts strain was transformed with episomal YEp(URA3) plasmids that drive constitutive ectopic expression of SEC14, SFH5 or sfh5Y175F from the powerful PMA1 promoter. The transformants were spotted in 10-fold dilution series onto YPD plates and incubated at the indicated temperatures for 48 hr before imaging. Growth at 37°C reports rescue of growth defects associated with the sec14-1ts allele at this normally restrictive temperature. Right panel: A sec14Δ ade2 ade3/YEp(SEC14, LEU2, ADE3) strain was transformed with the indicated YEp(URA3) expression plasmids described in (B). Transformants were again spotted in dilution series onto YPD plate as above. Under those conditions, all nutrient selections are relieved and loss of the normally essential YEp(SEC14, LEU2, ADE3) plasmid (which has the dual properties of covering the lethal sec14Δ allele and also the ade3 allele which coverts colony color of ade2 cells from red to white) can be monitored. Appearance of white colonies that are phenotypically Leu auxotrophs signifies loss of the parental YEp(SEC14, LEU2, ADE3) plasmid on the basis of the YEp(URA3) plasmid driving expression of a functional PITP. Uniformly red colonies report the YEp(URA3) expression plasmid does not drive production of a functional PITP with the capacity to provide Sec14-like functions to Sec14-deficient cells. YEp(SEC14) serves as positive control in these experiments. (C) PITP activity is not resuscitated in Sfh5 expressed in yeast cells devoid of heme. Isogenic hem1Δ and hem1Δ sec14-1ts strains deficient in α-aminolevulenic acid (ALA) synthesis were transformed with YEp(URA3) plasmid as mock control or for expression of SEC14, SFH5 or sfh5Y175F as indicated. Transformants were selected on a minimal media plate without uracil and supplemented with 250 µM ALA. After depleting cells for residual heme by growth in ergosterol-containing medium (added to a 20 mg/l final concentration from a 0.2% stock solution in 1:1 ethanol: Tween 80), cells were spotted onto YPD solid medium or onto YPD solid medium supplemented with ALA (250 µM) to rescue the hem1Δ deficiency, or onto YPD solid medium supplemented with 50 µM ergosterol/Tween 80 (Erg/Tween80) to rescue the heme deficiency without permitting heme synthesis. The cells were incubated for 72 hr at the indicated temperatures prior to imaging. The hem1Δ control cells grow under all conditions that either rescue heme synthesis (+ALA) or provide exogenous ergosterol whose synthesis is the sole essential heme-dependent activity in cells cultured under these conditions. Growth of the hem1Δ sec14-1ts mutant at the restrictive temperature of 37C was not rescued by enhanced expression of WT Sfh5 under heme-less conditions (i.e. on YPD supplemented with Erg/Tween80). Expression of SEC14 or the sfh5Y175F heme-binding mutant served as additional positive controls.