title: Measurement of the heme affinity for yeast dap1p, and its importance in cellular function.
abstract: Current studies on the Saccharomyces cerevisiae protein Dap1p have demonstrated a heme-related function within the ergosterol biosynthetic pathway. Here we present data to further the understanding of the role of heme in the proper biological functioning of Dap1p in cellular processes. First, we examined the role of Dap1p in stabilizing the P450 enzyme, Erg11p, a key regulatory protein in ergosterol biosynthesis. Our data indicate that the absence of Dap1p does not affect Erg11p mRNA, protein expression levels, or the protein degradation rates in S. Cerevisaie. Second, in order to probe the role of heme in the biological functioning of Dap1p, we measured ferric and ferrous heme binding affinities for Dap1p and the mutant Dap1pY138F, as well as equilibrium midpoint reduction potentials of the Fe(III)/Fe(II) couples. Our results show that both wild-type and mutant proteins bind heme in a 1:1 fashion, possessing tight ferric heme affinities, KD values of 400 pM and 200 nM, respectively, but exhibiting weak ferrous affinities, 2 and 10 microM, respectively. Additionally, the measured reduction potential of Dap1p, which was found to be -307 mV, is similar to that of other monotyrosinate hemoproteins. Although previous reports show the weaker affinity of Dap1pY138F for ferric heme lowers the production of ergosterol with respect to wild-type Dap1p in S. pombe, we find that Dap1pY138F expression is still sufficient to rescue the growth sensitivity of dap1Delta to fluconazole and methyl methanesulfonate in S. cerevisiae. Various interpretations of these results are discussed with respect to Dap1p function in the cell.
authors.affiliation: Department of Chemistry and Biochemistry, University of California, Santa Cruz, California 95064, USA.
authors.forename: Alisha M
authors.initials: AM
authors.lastname: Thompson
authors.forename: Amit R
authors.initials: AR
authors.lastname: Reddi
authors.forename: Xiaoli
authors.initials: X
authors.lastname: Shi
authors.forename: Robert A
authors.initials: RA
authors.lastname: Goldbeck
authors.forename: Pierre
authors.initials: P
authors.lastname: Moënne-Loccoz
authors.forename: Brian R
authors.initials: BR
authors.lastname: Gibney
authors.forename: Theodore R
authors.initials: TR
authors.lastname: Holman
mesh_terms: D002942:Circular Dichroism; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000378:metabolism; D015966:Gene Expression Regulation, Fungal; D006418:Heme / Q000378:metabolism*; D009154:Mutation; D016133:Polymerase Chain Reaction; D011485:Protein Binding; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D029701:Saccharomyces cerevisiae Proteins; D006418:Heme; D003577:Cytochrome P-450 Enzyme System; C526348:Erg11 protein, S cerevisiae