Chimeric phage-plasmid expression vectors were constructed from pUC18/19 plasmids by cloning a single-stranded DNA (ssDNA) origin of replication from bacteriophage f1 and inserting a bacteriophage T7 promoter within the beta-galactosidase gene. A T7 promoter permits in vivo or in vitro expression of single proteins by the translation of T7 RNA polymerase transcripts. Insertional inactivation of the T7 promoter-containing beta-galactosidase gene permits a simple blue-to-white color cloning assay. Compared with several helper phages that were examined, superinfection with M13K07 resulted in the highest yields of the pTZ plasmids as ssDNA viral particles. These ssDNA promoter plasmids are uniquely suited for protein engineering because they simplify cloning, oligonucleotide directed mutagenesis, verification by enzymatic sequence analysis, and expression of mutant proteins from a single vector. These vectors were utilized to eliminate an efficient transcriptional terminator of T7 RNA polymerase in the cDNA of bovine preproparathyroid hormone by oligonucleotide directed mutagenesis. The mutation changed the codon for phenylalanine-19 in the signal peptide to alanine. In a cell-free system the mutant cDNA transcripts were translated into preproparathyroid hormone, which was converted to proparathyroid hormone in the presence of microsomal membranes.

The NH2-terminal sequences of cytochromes P450 resemble signal peptides, but these sequences are not cleaved during the insertion of these integral membrane proteins into the microsomes. To examine whether these putative signal peptides are functionally equivalent to signal peptides of secretory proteins, cDNA coding for a fusion protein was produced, in which the signal peptide for preproparathyroid hormone was replaced with the putative signal peptide of cytochrome P450IIC2. The translational product of RNA synthesized in vitro from the cDNA was neither processed nor translocated by chicken oviduct microsomal membranes in a reticulocyte cell-free system but was resistant to extraction from the membranes by alkaline solutions. In addition, the translation of the hybrid RNA was arrested by signal recognition particle. Unlike most signal peptides, the cytochrome P450IIC2 NH2-terminal sequence does not contain basic amino acids preceding the hydrophobic core. Introduction by oligonucleotide-directed mutagenesis of lysine and arginine at the NH2 terminus resulted in a fusion protein that was partially processed by the microsomal membranes, with translocation across the membrane of both the processed and unprocessed proteins. The positive charges convert the cytochrome P450IIC2 NH2 terminus from a combination membrane insertion-halt transfer signal to a more classical secretory membrane-insertion signal, possibly by altering the orientation of the signal peptide in the membrane.The integration of proteins into cellular membranes may occur by either a cotranslational signal recognition particle (SRP)-dependent insertion, or by a posttranslational mechanism that does not require SRP (1, 2). The SRP-dependent mechanism is similar to that proposed for the initial steps of protein secretion. In contrast to secreted proteins, the hydrophobic signal sequences of some membrane proteins are not cleaved during the translocation process. The uncleaved signal peptides then serve to anchor the protein to the membrane (2). Because Bacterial Strains and Bacteriophages. Plasmids were propagated in Escherichia coli strain NM522 as described (9) except that strain BW313 (dut-, ung-, thi-1, relA, spoT1/

The molecular organization of microsomal cytochromes P450 (P450s) and formation of complexes with P450 reductase have been studied previously with isolated proteins and in reconstituted systems. Although these studies demonstrated that some P450s oligomerize in vitro, neither oligomerization nor interactions of P450 with P450 reductase have been studied in living cells. Here we have used fluorescence resonance energy transfer (FRET) to study P450 oligomerization and binding to P450 reductase in live transfected cells. Cytochrome P450 2C2, but not P450 2E1, forms homo-oligomeric structures, and this self-association is mediated by the signal-anchor sequence. Because P450 2C2, in contrast to P450 2E1, is directly retained in the endoplasmic reticulum (ER), these results could suggest that oligomerization may prevent transport from the ER. However, P450 2C1 signal-anchor sequence chimera defective in ER retention also formed oligomers, and chimera containing the cytoplasmic domain of P450 2C2, which is directly retained in the ER, did not exhibit self-oligomerization, which indicates that oligomerization is not correlated with direct retention. By using FRET, we have also detected binding of P450 2C2 and P450 2E1 to P450 reductase. In contrast to self-oligomerization, the catalytic domain can mediate an interaction of P450 2C2 with P450 reductase. These results suggest that microsomal P450s may differ in their quaternary structure but that these differences do not detectably affect interaction with the reductase or transport from the ER.The organization of the cytochrome P450 (P450) 1 -containing monooxygenase system in the microsomal membrane is not well understood despite many studies using a wide variety of biophysical methods. This system consists of P450s, NADPHcytochrome P450 reductase (P450 reductase), and for some P450s, cytochrome b 5 . If P450 levels in the endoplasmic reticulum (ER) are induced to maximal concentrations, there are 20 -30 P450s for each P450 reductase molecule. The presence of multiple forms of P450s in the ER membranes combined with limiting amounts of P450 reductase has important implications for the association of the P450s with each other and the reductase and for electron transfer from the reductase to P450. It has been postulated that either a multimeric complex of P450s binds to a molecule of P450 reductase (1, 2) or that the interaction results from random collisions of independently diffusing monomeric proteins that have high rotational and lateral mobility (3-5). Formation of large complexes by P450s has been observed with both isolated proteins and reconstituted systems (6 -8). Studies on rotational mobilities of P450s in microsomal membranes were also consistent with either self-aggregation or association of P450s with other components of the membranes (9 -11). The second model in which monomeric P450 interacts with P450 reductase is supported by observations that high concentrations of some P450s in membranes result in their aggregation and immobilization, but the addition of...

The cytochrome P450 2C1 N-terminal signal anchor sequence mediates direct retention of the protein in the endoplasmic reticulum and consists of a hydrophobic transmembrane domain, residues 3-20, followed by a hydrophilic linker, residues 21-28. Fusions of the N-terminal 21 or 28 amino acids of P450 2C1 to green fluorescent protein resulted in endoplasmic reticulum localization of the chimera in transfected cells. Disruption of microtubules by nocodazole treatment resulted in redistribution into a punctate pattern for the 1-21, but not for the 1-28, chimera indicating that the linker was preventing transport from the endoplasmic reticulum but was not required for retrieval to the endoplasmic reticulum from the pre-Golgi compartment. In the 1-28 chimera, mutations of residues 21-23 (KQS) in the linker resulted in redistribution of the chimera after nocodazole treatment. Mutations in the transmembrane domain affected both direct retention in the endoplasmic reticulum and retrieval from the pre-Golgi compartment, and although structural requirements for each process are distinct, in both cases the arrangement of amino acids and distribution of hydrophobicity are critical. In contrast, the linker region exhibits a sequencespecific requirement for direct retention in the endoplasmic reticulum.

Cytochrome P450 2C2 is a resident endoplasmic reticulum (ER) membrane protein that is excluded from the recycling pathway and contains redundant retention functions in its N-terminal transmembrane signal͞anchor sequence and its large, cytoplasmic domain. Unlike some ER resident proteins, cytochrome P450 2C2 does not contain any known retention͞retrieval signals. One hypothesis to explain exclusion of resident ER proteins from the transport pathway is the formation of networks by interaction with other proteins that immobilize the proteins and are incompatible with packaging into the transport vesicles. To determine the mobility of cytochrome P450 in the ER membrane, chimeric proteins of either cytochrome P450 2C2, its catalytic domain, or the cytochrome P450 2C1 N-terminal signal͞anchor sequence fused to green f luorescent protein (GFP) were expressed in transiently transfected COS1 cells. The laurate hydroxylase activities of cytochrome P450 2C2 or the catalytic domain with GFP fused to the C terminus were similar to the native enzyme. The mobilities of the proteins in the membrane were determined by recovery of f luorescence after photobleaching. Diffusion coefficients for all P450 chimeras were similar, ranging from 2.6 to 6.2 ؋ 10 ؊10 cm 2 ͞s. A coefficient only slightly larger (7.1 ؋ 10 ؊10 cm 2 ͞s) was determined for a GFP chimera that contained a C-terminal dilysine ER retention signal and entered the recycling pathway. These data indicate that exclusion of cytochrome P450 from the recycling pathway is not mediated by immobilization in large protein complexes.