Preliminary Studies On Mechanism Of Pdr5P Influencing Ergosterol Metabolism In S.cerevisiae

Antifungal drug resistance has become an increasing problem. Since ergosterol is an unique lipid composition of fungal cell membrane (cholesterol of mammalian cell membrane), it has been the best target in antifungal drug design.Sterols are moved between intracellular compartments by a combination of vesicular and nonvesicular mechanisms and little is known about how nonvesicular sterol transport occurs. The recent research shows ATP-binding Cassette (ABC) Transporters mediate nonvesicular sterol movement besides pleiotropic drug resistance. We use S. cerevisiae as a model system to study the mechanism of Pdr5p,an ABC (ATP-bindingcassette) efflux transporter protein, influencing ergosterol metabolism.Firstly,we construct pdr5、pdr10and yorl single knockout strains respectively. we show that pdr5knockout caused the organism to become hypersensitive to azole(fluconazole), a widely used antifungal drug. Surprisingly, the same knockout conferred growth advantages to the organism on polyenes(Amphotericin B). Amphotericin B is known to interact with ergosterol present in the yeast cell membrane to form ion channels that disrupt membrane function to cause cells death. We found that the content of ergosterol on yeat plasma membrane in pdr5knockout strain reduced by30percent compared to the wild type by HPLC method, while the yeast plama membrane ergosterol content almost have no change in pdr10,yorl single knockout strains.Secondly, this paper also study the changes in transcription level of genes related to ergosterol biosynthesis and some other related genes in pdr5knockout strains by fluorescent quantitative PCR method. The results show only ERG3,a gene related to ergosterol biosynthesis,down-regulated, ARE2, a gene expressing a protein involved in ergosterol esterification,up-regulated and YHE1、YEH2down-regulated in transcription level. This hint ergosterol transportation is affected after pdr5knockout,so Erg3p was down-regulated to reduce ergosterol synthetic amount and ARE2、YEH1、YEH2were regulated to reduce the ergosterol accumulation within yeast cells.As OSH protein families play an important role in the process of lipid transportation, this paper also studied influence on OSH protein family after pdr5knocked out. The result of fluorescent quantitative PCR is that only OSH2was up-regulated in transcription level,which hint OSH protein families may have certain relations with PDR5.