Topogenesis of a Microsomal Cytochrome P450 and Induction of Endoplasmic Reticulum Membrane Proliferation inSaccharomyces cerevisiae

Cytochrome P450 52A3 (P450Cm1) is one of the membrane proteins known to trigger by its high-level expression a marked proliferation of the endoplasmic reticulum (ER). To gain insight into the relationship between the expression of a membrane protein and the induction of ER proliferation we have characterized the membrane topology of P450Cm1 and identified the structural determinants required for ER targeting, formation of correct membrane orientation, and ER retention. We show that all these features are interrelated and determined by sequence elements within the NH2-terminal region of P450Cm1. Using several approaches—a protease protection assay followed by probing with peptide-specific antibodies, immunolabeling of the intact membrane-bound P450 protein, and expression of fusion proteins inSaccharomyces 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 membrane, a second hydrophobic segment (48–66) is exposed at the cytoplasmic side, and the remaining part (67–523) forms a large cytosolic domain. Fused to a cytosolic reporter protein, the first 44-amino-acid sequence of P450Cm1 was sufficient to mediate ER targeting, wild-type membrane orientation, and retention in the ER. Similar to wild-type P450Cm1, various fusion proteins were able to induce distinctly organized structures of proliferated ER provided that they were either permanently retained in the ER or accumulated in this compartment due to a delay in further transportation. Thus, we conclude that membrane insertion of the first hydrophobic segment is sufficient to deliver a signal for increased membrane formation.