Study On The Biological Function And Mechanism Of Erg28p Protein In Sterol C-4 Demethylated Enzyme System

The plasma membrane of Saccharomyces cerevisiae cell is rich in steroid active substances,which are important intermediates of active vitamin D and hormone drugs.Sterol C-4 demethylation is a very complicated and a key step involved in the sterols biosynthesis.The C-4 demethylation reaction involves three continuous steps of redox reaction,under the catalysis of C-4 oxidase(SMO)encoded by ERG25,C-3 decarboxylase(3?HSD/D)encoded by ERG26 and C-3 ketoreductase(3SR)encoded by ERG27,the double C-4 methyl is oxidized,decarboxylated and ortho ketone is reduced to hydroxyl,after double rounds of demethylation,C-4 dimethyl is finaly removed.In addition to Erg25 p,Erg26p and Erg27 p within the C-4 demethylation complex,Erg28 p,a transmembrane scaffold protein,is also found to play an important role in the ergosterol biosynthesis,while the relevant studies has not been verified till now.Yeast eukaryotic expression system was proposed to study the biological function and molecular mechanism of Erg28 p in assisting other subunits of sterol C-4 demethylation system through the correct endoplasm retinal localization and catalytic interface formation.The major contents in this study includes three parts listed as follows:In the first part of this paper,the Saccharomyces cerevisiae ERG28 gene knockout cell model(BY4741/?ERG28)was established by using lox P-Marker-loxP sequence and Cre-loxP system.The dry weight of wild-type BY4741 and knockout strain BY4741/?ERG28 was compared which indicated that deletion of ERG28 significantly reduced cell growth rate.Erg28 p protein expression vector(pYES2-ERG28)was constructed and it was transfected into BY4741/?ERG28 strain,thus the ERG28 gene was recovered in BY4741/?ERG28-p YES2-ERG28 strain,which offers a reliable cell model for studying the biological function Erg28 p in sterol biosynthetic pathway.In the second part of this paper the biological function of Erg28 p protein was studied,GC-MS method was used to analyse the difference of composition and contents within sterol intermediates in ergosterol metabolism between BY4741/?ERG28 strain and wild strain.It was found that the precursor of ergosterol was accumulated and the content of end-product ergosterol decreased dramatically in BY4741/?ERG28,confirming that the ergosterol synthesis pathway was blocked.However,the precursor was re-removed and end-product ergosterol was re-accumulated in the recovered strain BY4741/?ERG28-pYES2-ERG28,thus clarifying the pivotal role of Erg28 p protein in the sterol synthesis from both positive and negative directions.Besides,the fusion expression vectors of GFP and Erg25 p,Erg26p,Erg27 p and Erg28 p were constructed,it was confirmed that Erg28 p protein assisted the protein folding and endoplasmic reticulum localization of other subunits in C-4 demethyl multiple enzyme system,thus indirectly affecting the C-4 sterol demethylation reaction.The third part of this paper,the key residues involved in sterol C-4 demethylation of Erg28 p protein was predicted by bioinformatics analysis and a set of truncated mutant expression vector of Erg28 p protein were constructed to detect the intermediate metabolites in the ergosterol synthase pathway.Our preliminary data discovered and confirmed that key residues(63-72)within Erg28 p protein significantly affect the sterol C-4 demethylation in S.cerevisiae.