Targeting of heterologous membrane proteins into proliferated internal membranes in Saccharomyces cerevisiae

Abstract

Overproduction of chimeric proteins containing the HMG2/1 peptide, which comprises the seven transmembrane domains of Saccharomyces cerevisiae 3-hydroxy-3-methylglutaryl-CoA reductase isozymes 1 and 2, has previously been observed to induce the proliferation of internal endoplasmic reticulum-like membranes. In order to exploit this amplified membrane surface area for the accommodation of heterologous microsomal proteins, we fused sequences coding for human cytochrome P4501A1 (CYP1A1) to sequences encoding the HMG2/1 peptide and expressed the hybrid genes in yeast. The heterologous hybrid proteins were targeted into strongly proliferated membranes, as shown by electron microscopic and immunofluorescent analysis. Fusion proteins comprising the whole CYP1A1 polypeptide (HMG2/1-CYP1A1) exhibited 7-ethoxyresorufin-O-deethylase activity, whereas fusion proteins lacking the N-terminal 56 amino acids of CYP1A1 (HMG2/1-delta CYP1A1) were inactive and appeared to be unable to incorporate protoheme. Similar amounts of heterologous protein were detected in cells expressing HMG2/1-CYP1A1, HMG2/1-delta CYP1A1 and CYP1A1, respectively. Replacement of the N-terminal membrane anchor domain of human NADPH-cytochrome P450 oxidoreductase by the HMG2/1 peptide also resulted in a functional fusion enzyme, which was able to interact with HMG2/1-CYP1A1 and the yeast endogenous P450 enzyme lanosterol-14 alpha-demethylase.

High-yield production of protopanaxadiol from sugarcane molasses by metabolically engineered Saccharomyces cerevisiae

“…Kim et al previously reported that overexpressing INO2 could expand the ER, thus improve the capacity to synthesize ER-associated proteins and cytochrome P450-mediated PPD, and increase available space to accommodate them [ 20 ]. In our study, the cell growth and PPD production of BY-III strains are significantly improved by up-regulation INO2 possibly due to efficient localization of cytochrome P450 in an expanded ER as a possible mechanism, which is consistent with Kim’s studies [ 36 ].

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Functional Expression of Fused Enzymes Between Human Cytochrome P4501A1 and Human NADPH-Cytochrome P450 Oxidoreductase inSaccharomyces cerevisiae

“…Strain YNW64 (MATa,trp5,cprl::ura3A5,ura3A5, derived from strain YES9 by disruption of the URA3 gene present in the cprl::URA3 alíele (Wittekindt et al, 1994).…”

“…In the second fusion protein (HMG2/1-AhOR-CYPlAl), the amino-terminal 47 amino acids that represent the membrane anchor domain of hOR are replaced by a chimera of the seven transmembrane domains of yeast HMG-CoA reducíase isozymes 1 and 2 (HMG2/1). Expression of fusion constructs between the HMG2/1 peptide and human CYP1A1 has previously been shown to induce proliferation of membranes of the endoplasmic reticulum and targeting of the enzymatically active fusion proteins into these membranes (Wittekindt et al, 1994). By analogy, the HMG2/l-AhOR-CYPlAl fusion protein is expected to be targeted into the proliferated membranes and to be stabilized by the yeast HMG2/1 peptide functioning as membrane anchor domain.…”