Molecular basis and structural determinants for the cellular localization of cytochrome P450 2E1

Cytochrome P450 2E1 (CYP2E1) is the alcohol inducible member of the P450 family and has a role in gluconeogenesis under fasting conditions. However, CYP2E1 is mainly of toxicological significance and metabolizes xenobiotics to their carcinogenic and toxic metabolites and generates active oxygen species that can cause lipid peroxidation and apoptosis, processes observed in alcoholic liver disease (ALD). CYP2E1 like other xenobiotic metabolizing P450s, is mainly located in the membrane of the endoplasmic reticulum (ER), where it is anchored and retained through its hydrophobic NH2-terminus, leaving the large COOH-terminal domain including the catalytic site exposed to the cytosol. CYP2E1 has also been detected in other cellular compartments such as lysosomes and the plasma membrane. CYP2E1 located at the plasma membrane has been suggested to play a role in the immune mediated hepatotoxicity observed in patients suffering from ALD. Other forms of P450 have also been observed at the extracellular side of the plasma membrane and this could be important in the etiology of several forms of drug-induced hepatitis. During the past years much controversy existed regarding the topology of P450s on the plasma membrane and the molecular mechanism behind the appearance of P450 at the cell surface has remained obscure. The involvement of the Golgi apparatus has been implied in the transport of P450s to the plasma membrane in isolated rat hepatocytes, although reports exist where investigators were unable to detect P450 in the Golgi apparatus. Therefore we isolated the Golgi apparatus from rat liver and the distribution of several P450 enzymes in three subfractions of the Golgi apparatus was studied. It was found that there was an enzyme specific appearance of P450 in the isolated Golgi fractions: CYP2E1 CYP4A1 and CYP1A2 together with NADPH cytochrome P450 reductase were present, whereas CYP3A1 was not. This suggested that CYP3A1 was efficiently retained in the ER membrane and others, like CYP2E1, were transported out of the ER to the Golgi apparatus. The molecular mechanism responsible for the exit from the ER and subsequent transport to the plasma membrane of CYP2E1 was studied by constructing several NH2-terminal mutants, since the NH2-terminal domain has been demonstrated to be important for targeting to and retention in the ER membrane. The presence of CYP2E1 and its mutants at the outer surface of the plasma membrane was established by several techniques such as immunofluorescent microscopy and cell surface biotinylation studies. It was demonstrated that N++2E1, which carried two positively charged residues in the NH2-terminus, was present at the plasma membrane at twice the amount as wild-type CYP2E1 Protease protection experiments further demonstrated that CYP2E1 was incorporated in two topologies in the ER membrane, most of the protein was in the expected cytoplasmic orientation while a small fraction (about 2%) was incorporated in a lumenal orientation. N++2E1 appeared to have a higher extent of lumenal orientation than the other variants. Removal or modification of the hydrophobic NH2-terminal transmembrane domain of CYP2E1 resulted in specific targeting to the mitochondria. After mitochondrial import and processing, a soluble and catalytically active protein, called [delta]2E1 (Mr ¡Ö 40 kD), was formed. Low levels of [delta]2E1 were also observed in mitochondria isolated from rat liver, thus showing that [delta]2E1 is present in vivo. Removal or modification of the NH2-terminus of CYP2E1 results in the exposure of a mitochondrial targeting signal that directs the protein to the mitochondria. The mitochondrial targeting signal was identified and demonstrated to be located between amina acid residues 74 and 95, an area rich in positively charged amino acid residues and also containing a hydrophobic region. The role of the NH2-terminus of CYP2E1 in the intracellular targeting was investigated using Saccharomyces cerevisiae as a model system. Wild-type CYP2E1 was shown to be expressed in the ER, while modification of the NH2-terminal transmembrane domain did not result in mitochondrial targeting, as was observed in the mammalian cell line. Furthermore, removal of the NH2-terminal transmembrane domain resulted in mitochondrial targeting but not in processing to the mature form [delta]2E1. The same region that was identified to be responsible for mitochondrial targeting in mammalian cells was also demonstrated to be responsible for directing these NH2-terminally truncated forms of CYP2E1 to the mitochondria in yeast. In addition to the NH2-terminal part, the region between as 64 and 95 was shown to cause membrane interactions of the protein. It is suggested that yeast can be a useful model system as a complement to mammalian cells for studying the intracellular protein targeting of mammalian proteins, but differences such as in the mitochondrial import and processing, do exist. It is concluded that the hydrophobic NH2-terminal transmembrane domain of CYP2E1 plays an important role in the cellular targeting. Removal or modification of this region resulted in the targeting of the protein to the mitochondria where, after import, it was processed to a soluble and catalytically active form. The presence of CYP2E1 at the extracellular side of the plasma membrane was unequivocally demonstrated and the molecular mechanism behind its appearance was resolved by showing the lumenal incorporation of a small fraction of the enzyme during its synthesis, that is not efficiently retained in the ER and transported via the Golgi apparatus to the plasma membrane