Measurement of the Heme Affinity for Yeast Dap1p, and Its Importance in Cellular Function

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• 作者：Alisha M. Thompson ; Amit R. Reddi ; Xiaoli Shi ; Robert A. Goldbeck ; Pierre Mo&euml ; nne-Loccoz ; Brian R. Gibney ; Theodore R. Holman
• 刊名：Biochemistry
• 出版年：2007
• 出版时间：December 18, 2007
• 年：2007
• 卷：46
• 期：50
• 页码：14629 - 14637
• 全文大小：330K
• 年卷期：v.46,no.50(December 18, 2007)
• ISSN：1520-4995

文摘

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 theunderstanding 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 inergosterol biosynthesis. Our data indicate that the absence of Dap1p does not affect Erg11p mRNA, proteinexpression levels, or the protein degradation rates in S. Cerevisaie. Second, in order to probe the role ofheme in the biological functioning of Dap1p, we measured ferric and ferrous heme binding affinities forDap1p and the mutant Dap1p^Y138F, 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, K_D values of 400 pM and 200 nM, respectively, but exhibitingweak ferrous affinities, 2 and 10 M, respectively. Additionally, the measured reduction potential ofDap1p, which was found to be -307 mV, is similar to that of other monotyrosinate hemoproteins. Althoughprevious reports show the weaker affinity of Dap1p^Y138F for ferric heme lowers the production of ergosterolwith respect to wild-type Dap1p in S. pombe, we find that Dap1p^Y138F expression is still sufficient torescue the growth sensitivity of dap1 to fluconazole and methyl methanesulfonate in S. cerevisiae. Variousinterpretations of these results are discussed with respect to Dap1p function in the cell.