Targeting of heterologous membrane proteins into proliferated internal membranes in Saccharomyces cerevisiae.
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.
Wittekindt
N E
NE
Institute of Toxicology, Swiss Federal Institute of Technology, Schwerzenbach, Switzerland.
Würgler
F E
FE
Sengstag
C
C
D001483:Base Sequence; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000378:metabolism; D017931:DNA Primers / Q000235:genetics; D004271:DNA, Fungal / Q000235:genetics; D006801:Humans; D006903:Hydroxymethylglutaryl CoA Reductases / Q000235:genetics / Q000378:metabolism; D007425:Intracellular Membranes / Q000378:metabolism; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010957:Plasmids / Q000235:genetics; D015202:Protein Engineering; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D014170:Transformation, Genetic
D017931:DNA Primers; D004271:DNA, Fungal; D007527:Isoenzymes; D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; D003577:Cytochrome P-450 Enzyme System; D006903:Hydroxymethylglutaryl CoA Reductases