CANDIDA-MALTOSA NADPH-CYTOCHROME P450 REDUCTASE - CLONING OF A FULL-LENGTH CDNA, HETEROLOGOUS EXPRESSION IN SACCHAROMYCES-CEREVISIAE AND FUNCTION OF THE N-TERMINAL REGION FOR MEMBRANE ANCHORING AND PROLIFERATION OF THE ENDOPLASMIC-RETICULUM

A full-length cDNA for NADPH-cytochrome P450 reductase from Candida ma ltosa was cloned and sequenced. The derived amino acid sequence showed a high similarity to the reductases from other eukaryotes. Expression in Saccharomyces cerevisiae under control of the GAL10 promoter resul ted in an approximately 70-fold increase in NADPH-cytochrome c reducta se activity in the microsomal fraction. The functional integrity of th e heterologously expressed reductase as an electron transfer component for alkane hydroxylating cytochrome P450 from C. maltosa was shown in a reconstituted system containing both enzymes in a highly purified s tate. The signal-anchor sequence of the reductase was identified withi n the N-terminal region of the protein by means of constructing and ex pressing fusion proteins with the cytosolic form of yeast invertase. T he first 33 amino acids turned out to be sufficient for stable membran e insertion, wild-type membrane orientation and retention in the endop lasmic reticulum. As shown by immunoelectron microscopy, the heterolog ously expressed reductase was integrated into the endoplasmic reticulu m of the host organism. It triggered a strong proliferation of the mem brane system. This membrane-inducing property of the reductase was tra nsferable to the cytosolic reporter protein with the same N-terminal s equences that confer membrane insertion. The nucleotide sequence of th e cDNA of NADPH-cytochrome P450 reductase from C. maltosa is available from the EMBL data library under Accession Number X76226.