FIG. 2.

Complementation of S. cerevisiae Δhap1 mutants by KlHAP1. (A) Increase of cytochrome c synthesis by introduction of KlHAP1 into the Δhap1 mutant of S. cerevisiae. KlHAP1 (placed under the control of the S. cerevisiae HAP1 promoter) was inserted into the multicopy vector YEp352, resulting in the plasmid pKl-HAP1. The plasmid pSc-HAP1 containing the S. cerevisiae HAP1 gene was used as a positive control. The Δhap1 mutant W303-ΔHAP1 was transformed with pSc-HAP1 (curve 1), pKl-HAP1 (curve 2), or the empty vector YEp352 (curve 3). All strains were grown at 28°C for 2 days on plates of complete medium containing 2% glucose. Cytochrome spectra were determined at liquid nitrogen temperature in a Cary 400 spectrophotometer according to the protocol of Claisse et al. (13), and three resulting curves were placed at different heights in panel A so that they could be compared with each other. The absorption peak at 550 nm corresponds to cytochrome c. (B) KlHap1p complements the defective phenotype of the S. cerevisiae Δhap1 Δhem1 strain in the presence of ergosterol and Tween 80. Line 1, W303-1A (HEM1 HAP1); line 2, W303-ΔHEM1 (hem1 HAP1); line 3, W303-ΔHEM1-ΔHAP1 (hem1 hap1) transformed with the empty vector YEp352; line 4, W303-ΔHEM1-ΔHAP1 with pKl-HAP1; line 5, W303-ΔHEM1-ΔHAP1 with pSc-HAP1. A serial dilution of cultures was deposited on YP-2% glucose medium supplemented with 30 μg ml⁻¹ ergosterol and 0.2% Tween 80 (right panel, YP-glucose + ET, heme-deficient condition), as well as on the medium supplemented with 30 μg ml⁻¹ δ-ALA as control (left panel, YP-glucose + δ-ALA, heme-sufficient condition). Cells were grown at 28°C for 3 days.