TOPOGENESIS OF A MICROSOMAL CYTOCHROME-P450 AND INDUCTION OF ENDOPLASMIC-RETICULUM MEMBRANE PROLIFERATION IN SACCHAROMYCES-CEREVISIAE

Cytochrome P450 52A3 (P450Cm1) is one of the membrane proteins known t o trigger by its high-level expression a marked proliferation of the e ndoplasmic reticulum (ER). To gain insight into the relationship betwe en the expression of a membrane protein and the induction of ER prolif eration we have characterized the membrane topology of P450Cm1 and ide ntified the structural determinants required for ER targeting, formati on of correct membrane orientation, and ER retention. We show that all these features are interrelated and determined by sequence elements w ithin the NH2-terminal region of P450Cm1 Using several approaches-a pr otease protection assay followed by probing with peptide-specific anti bodies, immunolabeling of the intact membrane-bound P450 protein, and expression of fusion proteins in Saccharomyces cerevisiae-the membrane topology was defined as follows: residues 2-16 are located in the ER lumen, only the first hydrophobic segment (residues 17-34) spans the m embrane, a second hydrophobic segment (48-66) is exposed at the cytopl asmic side, and the remaining part (67-523) forms a large cytosolic do main. Fused to a cytosolic reporter protein, the first 44-amino-acid s equence of P450Cm1 was sufficient to mediate ER targeting, wild-type m embrane orientation, and retention in the ER. Similar to wild-type P45 0Cm1, various fusion proteins were able to induce distinctly organized structures of proliferated ER provided that they were either permanen tly retained in the ER or accumulated in this compartment due to a del ay in further transportation. Thus, we conclude that membrane insertio n of the first hydrophobic segment is sufficient to deliver a signal f or increased membrane formation. (C) 1996 Academic Press, Inc.