TARGETING OF HETEROLOGOUS MEMBRANE-PROTEINS INTO PROLIFERATED INTERNAL MEMBRANES IN SACCHAROMYCES-CEREVISIAE

Overproduction of chimeric proteins containing the HMG2/1 peptide, whi ch comprises the seven transmembrane domains of Saccharomyces cerevisi ae 3-hydroxy-3-methylglutaryl-CoA reductase isozymes 1 and 2, has prev iously been observed to induce the proliferation of internal endoplasm ic reticulum-like membranes. In order to exploit this amplified membra ne surface area for the accommodation of heterologous microsomal prote ins, we fused sequences coding for human cytochrome P4501A1 (CYP1A1) t o sequences encoding the HMG2/1 peptide and expressed the hybrid genes in yeast. The heterologous hybrid proteins were targeted into strongl y proliferated membranes, as shown by electron microscopic and immunof luorescent analysis. Fusion proteins comprising the whole CYP1A1 polyp eptide (HMG2/1-CYP1A1) exhibited 7-ethoxyresorufin-O-deethylase activi ty, whereas fusion proteins lacking the N-terminal 56 amino acids of C YP1A1 (HMG2/1-Delta CYP1A1) were inactive and appeared to be unable to incorporate protoheme. Similar amounts of heterologous protein were d etected in cells expressing HMG2/1-CYP1A1, HMG2/1-Delta CYP1A1 and CYP 1A1, respectively. Replacement of the N-terminal membrane anchor domai n of human NADPH-cytochrome P450 oxidoreductase by the HMG2/1 peptide also resulted in a functional fusion enzyme, which was able to interac t with HMG2/1-CYP1A1 and the yeast endogenous P450 enzyme lanosterol-1 4 alpha-demethylase.