Membrane

of the:HI,

topology

SHAUN

D. BLACK

of Medicinal Chemistry and Phann The Comprehensive Cancer Center, The OLio Columbus Ohio 43210-1291, USA Division

The membrane topology of the mammalian P450 cytochromes has been studied intensively by computational approaches, proteolysis, chemical modification, genetic engineering, and immunochemistry. Initial results for the cytochromes of the endoplasmic reticulum appeared to indicate a polytopic, four to eight transABSTRACT

membrane

membrane.

anchor

model

with

an active

site buried

in the

However,

recent findings show that the microsomal P450s are bound to the endoplasmic reticulum by only one or two transmembrane peptides located at the NH2-terminal end, and that the active site is part of a large cytoplasmic domain that may have one or two additional peripheral membrane contacts. The membranebound state is viewed as rather rigid, and the plane of the heme lies between perpendicular and parallel to the plane of the endoplasmic reticulum. The mitochondrial P450 cytochromes lack a hydrophobic NH2 terminus in the mature form, and thus differ from the microsomal isozymes in this significant way. However, although the exact topology of cytochrome P450 in the inner mitochondrial membrane remains to be elucidated, certain features are clearly comparable to those of microsomal P450. Therefore, the membrane topology of the P450 gene superfamily may follow a similar pattern. Black, S. D. Membrane topology of the mammalian P450 cytochromes. FASEBJ. 6: 680-685; 1992. -

Key Words: cytochrome P450 structure-function

membrane topology

topography

P450 MONOOXYGENASE SYSTEM IS distributed widely in nature, and in mammals is found associated with all membranous fractions of the cell, notably in mitochondria and the endoplasmic reticulum (1-3). Metabolic properties of cytochrome P450’ are characterized generally by broad substrate specificity and by catalysis of more than 10 reaction types. NADPH serves as the proximate electron donor in eukaryotes. In the endoplasmic reticulum, NADPH-cytochrome P450 reductase (which contains FAD and FMN) functions as the intermediate electron acceptor, whereas in mitochondria this role is served by two proteins, a ferredoxin-type reductase (which contains FAD) and a ferredoxin (which contains an iron-sulfur center). One of the many isoforms of cytochrome P450 serves as the terminal oxidase in these electron transfer chains. Cytochrome P450 is a gene superfamily, with at least 25 isozymes per mammalian species being the norm. The enzymes are discrete gene products of about 57,000 daltons (-500 residues), and contain one equivalent of b-type heme per polypeptide chain. Purified preparations exhibit a highly amphipathic character and exist as micellar aggregates of approximately six monomers. To date, more than 160 primary structures have been characterized, and structural homology has been observed among all of the sequences. The hemeTHE

680

#{149}vsivenau

binding cysteinyl residue near the COOH terminus is invariant. Sequences of the microsomal cytochromes begin with a highly hydrophobic segment of about 20 amino acid residues that is followed by a short polycationic segment; the remainder of the sequence exhibits alternating hydrophiic and hydrophobic character, but typically not of greater nonpolarity than that of the NH2-terminal region. Mitochondrial P450 is first synthesized in the cytosol with a transient targeting sequence of 24-39 residues at the NH2 terminus; the amphipathic target sequences are rich in Leu, Ala, and Arg and are proteolytically removed after movement of the precursor to the inner membrane of the mitochondrion. Some data are available concerning the secondary structure of P450 cytochromes via circular dichroism and from computation, but the three-dimensional structure is not known for any eukaryotic P450. Thus far, these membrane proteins have resisted crystallization for x-ray diffraction studies. The tertiary structure is known only for soluble P450 101 (P45Ocam) from the bacterium, Pseudornonas pulida. Current knowledge suggests that P4SOcam may serve as an archetypal model for the mammalian P450 cytochromes. Nonetheless, many advances have been made to elucidate aspects of the three-dimensional structure of the mammalian P450s, especially regarding the membrane topology. Approaches for characterizing the membrane-bound state have included predictive computation, protease susceptibility, chemical modification, genetic engineering, and immunochemical assays. It is the purpose of this research review to summarize recent data on membrane topology for mammalian microsomal (endoplasmic reticulum) and mitochondrial isoforms of cytochrome P450. The reader is also guided to related reviews on this topic (4-8).

MEMBRANE FROM THE

TOPOLOGY ENDOPLASMIC

Mechanisms retention

of membrane

OF

CYTOCHROME RETICULUM

insertion

P450

and organelle

Signal peptides direct the insertion of secreted and membranous proteins into the endoplasmic reticulum (9). Some gene products are completely translocated through the membrane into the lumen. Those destined for secretion are carried to the Golgi apparatus after removal of the signal peptide; before secretion, the polypeptide may be glycosylated at Asn-X-Thr or Asn-X-Ser sequences. Those soluble proteins that remain resident in the lumen of the endoplasmic reticu-

‘Abbreviations: for which

endoplasmic fluorescein

both

Cytochrome a trivial

and

P450 refers to a gene superfamily

systematic

nomenclature

exist

reticulum; SRP, signal recognition particle; isothiocyanate; MNT, 2-methoxy-5-nitropone;

3-(trifluoromethyl)-3-(m-[

(1). ER,

Flit, TID,

‘251]iodophenyl)diazirine.

0892-6638/9210006-0680/$01

.50. © FASEB

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lum are processed similarly, but usually contain a COOHterminal sequence of Lys-Asp-Glu-Leu (KDEL), which serves as an ER retention signal (10). Integral membrane proteins of the endoplasmic reticulum may undergo insertion, translocation, cleavage, and glycosylation as described above, but remain fixed in the ER by an anchor peptide located at the COOH-terminal end; retention signals exist in this region but are not well characterized at present. Still other membranous proteins may be inserted into the ER by an NH2-terminal signal peptide, but one that is not cleaved and also serves an an anchor; the signal-anchor sequence is followed by a halt-transfer signal which is a short Segment of high cationic amino acid content. It is into this lattermost class that the P450 cytochromes fall (2, 3, 11). Of note also is the absence of N-linked glycosylation or any KDEL-like sequence at the COOH-terminus. The ER translocation machinery is complex and consists of the signal recognition particle (SRP), SRP-receptor/docking protein, chaperone proteins, and a ribosome receptor (9, 12, 13). This mechanism appears to be used in the case of the P450 cytochromes (11, 14). The signal may be inserted into the membrane in a vectorial fashion or as a hairpin (9, 12, 15), but the molecular determinants for this selection are not well understood and mechanistic details for the microsomal P450 cytochromes have not been worked out. Possible topologies for microsomal P450s in light of currently accepted mechanisms of membrane insertion are given in Fig. 1. Alternative 1 is a so-called monotopic orientation (i.e., touching only one leaflet of the membrane), somewhat similar to the conformation known for cytochrome b5, but appears to be extremely unlikely as the correct topology for cytochrome P450. Alternatives 2 and 3 are bitopic in character (i.e., touching both leaflets of the membrane), and alternatives 4 and 5 are polytopic (i.e., multiple membrane crossing), twoanchor models; these represent theoretically possible topologies for the monooxygenases. Data from a variety of sources (see below) suggest that only alternatives 3 and 4 are tenable.

1

1.4

LUtN

I..,..)

Figure 1. Theoretically feasible integral membrane topologies for the mammalian P450 cytochromes of the endoplasmic reticulum. Models 1-3 depict cases where only one membrane anchor exists, whereas models 4-5 represent scenarios with two membrane anchors, potentially inserted as a “hairpin.” The figure is rendered schematically for illustrative purposes and is not drawn to exact scale.

Hartmann et al. (20) developed an algorithm to calculate the charges that flanked the NH2-terminal membrane anchor in a large variety of membranous proteins, including a selection of P450 cytochromes. The longitudinal charge moment of the transmembrane peptide was aligned with the membrane potential to determine the orientation. For the P450s these authors calculated the general balance of charge on the amino side of the membrane anchor as negative relative to the carboxyl side, and therefore asserted that the topology must be “NexoCcyt”, shown as model 3 in Fig. 1. Tretiakov et al. (21) used secondary structure, hydrophobicity, and hydrophobic moment calculations on 19 microsomal P450

Prediction

of membrane

topology

Principal approaches taken to predict the membrane topology of the eukaryotic P450 cytochromes from the ER have involved computational techniques in which hydrophobicity, hydrophobic moments, secondary structure, or sequence alignments were determined. The early studies by Tarr et al. (16) suggested that the membrane-bound P450 2B4 (IIB4, isozyme 2, LM2) might contain eight transmembrane helical segments. Later, hydrophobicity computations showed that no more than four membranous segments were tenable, and that “a simple topology such as was proposed for NADPH-cytochrome P450 reductase still remains a possibility” (2) (analogous to model 3 of Fig. 1). Calculations by Hudecek and Anzenbacher (17) yielded a similar conclusion. The heme and active site were envisaged to be in a globular, cytoplasmic domain, rich in alpha helix content. Nelson and Strobel used databases of 34 (18) and 52 (19) aligned P450 sequences to examine, respectively, collective hydrophobicity and secondary structure calculations. In both studiues they concluded that the most consistent topology for the superfamily was a helical hairpin at the NH2 terminus, and a large globular heme domain with the plane of the prosthetic group parallel to the membrane. Both the NH2 terminus and the heme domain would be located on the cytosolic side of the ER, and the general structure of the catalytic portion would bear homology to P45Ocam (3, 19). This topology is represented by model 4 in Fig. 1.

CYTOCHROME

P450 MEMBRANE

TOPOLOGY

HN

model tion

cytochromes

to

develop

for the enzymes. Their that the microsomal

a

quasi

three-dimensional

conclusions cytochromes

affirmed were

the assersimilar to

P45Ocam, and that only a single NH2-terminal transmembrane anchor was present (as in model 3 of Fig. 1). In addition, they predicted that two /3a/3 motifs (Rossmann folds) were present, and that peripheral membrane interactions might occur between the membrane and, for example, regions 312-332 and 466-486 of P450 2B4. The hydrophobic moment profile can be a powerful tool to locate secondary structural features of the P450s; Black and Mould (22) recently

showed

that

all

14 alpha

helices

of P4SOcam

can

be

correctly predicted by this method. Another possible approach to predict the position of membrane signal-anchors is to examine intron-exon boundaries located in genomic sequences of P450 cytochromes. Others have suggested that such boundaries may fall between membranous domains. However, this assertion does not yet seem practical due to the small database of previously analyzed sequences for comparison and the existence of known violations where intron-exon boundaries occur within transmembrane anchor segments (7).

Membrane

topology

studied

by protease

susceptibility

Proteases have been shown in a large variety of investigations to be impermeable to membranes. Thus, proteolysis of peptide bonds in a membrane-bound protein implies the availability of the hydrolyzed region on the exterior surface of the

681

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membrane. For membrane fractions such as microsomes, the “outside” is faithfully equivalent to the cytosolic side of the ER (4, 5), and thus topological information can potentially be extracted from proteolysis data. In the early studies by Vlasuk et al, (23), phenobarbitaltreated microsomes were exposed to trypsin, chymotrypsin, or subtilisin, and proteolysis was monitored by twodimensional electrophoresis. P450 cytochromes were hydrolyzed to varying extents, suggesting that various isoforms were at least partially exposed on the exterior (cytosolic) side of the microsome. Brown and Black (24) used trypsin to treat barbiturateinduced microsomes for extended periods. Peptides were purified by high-performance liquid chromatography after stringent washing with high ionic-strength buffers. Peptides were sequenced, and the sequences were matched to a database of P450 primary structures. Fragments were obtained from seven different isozymes. Matches were normalized to the structure of P450 2B4 and a topological model was developed, as shown in Fig. 2. Two alternative models (“a” and “b”) were derived as explanations consistent with the data; these correspond roughly to models 3 and 4 of Fig. 1. Alternative “a” is a bitopic NexoCcyt topology, and alternative “b” is a polytopic, two-anchor hairpin model. In both cases strong peripheral contacts between the catalytic heme domain and the membrane are indicated, perhaps mediated by peptide segment 316-330. Residues 20-30 are rich in Lys and Arg, but no tryptic peptides were recovered from this region. Vergeres et al. (25) incorporated microsomal isozyme 2B1 into phospholipid vesicles, and showed that trypsin proteolysis of the membrane-bound cytochrome yielded a single peptide, 1-21, as spanning the bilayer. Chemical modification failed to label the amino terminus of the membranous cytochrome. Their conclusion was analogous to model 3 of Fig. 1. Thus, proteolysis studies show that one or two transmembrane signal-anchor sequences at the NH2 terminus bind the P450s to the ER. Strong peripheral interactions involving another region (or regions) of the polypeptide have also been detected.

Membrane topology studied and biophysical methods

by chemical

modification

Bernhardt et al. (26) used the fluorescent reagent fluorescein isothiocyanate (FITC) to label the NH2-terminal amino group of rabbit microsomal cytochrome P450 2B4. The labeled protein was isolated from electrophoresis gels and was sequenced to reveal the identity of the polypeptide. These data indicate that the NH2 terminus is located at the cytosolic side of the ER membrane, and would be consistent with

model 4 in Fig. 1. These authors also studied general amine labeling of P450 2B4 with the reagent 2-methoxy-5-nitropone (MNT) in the presence and absence of P450 reductase (27). Lysine residues at positions 139, 251, and 384 were selectively protected by the reductase, thereby suggesting that these may form part of the reductase-binding site on the P450 2B4. It is expected that this site would not be membrane bound, and that these lysyl residues are extramembranous. Recent results of Richter and colleagues (28) are in sharp contrast to those of Bernhardt et al. (26) discussed above. Rat microsomes were treated with FI’TC, but the NH2 terminus of P450 2B1 could be labeled only if the membrane had been solubiized with detergent. Such data clearly support model 3 of Fig. 1. Vergeres et al. (8) have studied the spectroscopic properties of the sole tryptophan residue (Trp121) in liposomeincorporated P450 2B1, with and without trypsin digestion. Both fluorescence spectra and fluorescence polarization changed dramatically when the cytochrome was proteolyzed. The results suggested that upon polypeptide cleavage, Trpis, became displaced to a more polar environment and that motion of the indole side chain was considerably relaxed. Thus, the region of Trp,at is partially buried but readily accessible to protease, implying that this region is located in the large cytoplasmic domain of P450 2B1. Many investigators have explored the cAMP-dependent protein kinase-catalyzed phosphorylation of P450 cytochromes. The site of phosphorylation has been localized to Ser,28 in P450 2B4 (29) and to the equivalent site in other isoforms. Clearly, this residue must be a surface feature of the cytosolic heme domain. The compound, 3-(trifluoromethyl)-3-(m-[ 1251]iodophenyl) diazirine (TID), has been used to label a substrate-binding site of P450 2B1 in hepatic microsomes (30). Hydrocarbonand barbiturate-induced microsomes showed enhanced binding relative to control microsomes, and substrate inhibited binding in a concentration-dependent manner. Trypsin proteolysis released a single - 4,000-dalton radiolabeled peptide from the microsomes. The results show that the substratebinding region of the P450s is located on the cytosolic side of the ER, that this region is not membrane enmeshed, and that TID may be useful in further experiments aimed at localization of residues involved in substrate binding. Krainev et al. (31) have used a homologous series of thioalkylpyridine diphosphonates to attempt measurement of the distance from the P450 active site to the surface of the microsomal membrane. Competition experiments with substrates and proton NMR spectroscopy led to the conclusion that the heme iron atom was some -1.8 nm from the membrane surface. Alternatively, the results may be interpreted to show that the hydrophilic surface of the globular heme domain at the entrance to the substrate-binding channel is -1.8 nm from the heme iron. Saturation transfer EPR spectroscopy of liposomal cytochrome P450 2B4 has been used in conjunction with freezefracture electron microscopy experiments (32) to suggest limited rotational diffusion of an oligomeric P450 membrane aggregate. A model was developed in which a possibly hexameric membrane complex of cytochrome molecules rotates slowly about an axis perpendicular to the membrane surface.

Membrane LUMEN

Figure

2.

microsomal

682

Vol. 6

a

b

Experimentally cytochrome

January

derived P-450

1992

2B4

membrane

(taken

from

topologies ref 24).

for

topology

studied

by genetic

engineering

Sakaguchi et al. (33) constructed six hybrid cDNAs that contained varying amounts of the NH2-terminal coding sequence for a rabbit P450 cytochrome and a passenger protein (either yeast porin or interleukin 2). Expression of these in a cell-free system with rough microsomes showed that the

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BLACK www.fasebj.org by UNIVERSITY OF TOLEDO LIBRARIES (18.218.56.169) on August 14, 2018. The FASEB Journal Vol. ${article.issue.getVolume()}, No. ${article.issue.getIssueNumbe

constructions with 29 or greater residues of the signal-anchor sequence were inserted into the ER membrane cotranslationally

but

were

neither

translocated

nor

proteolytically

processed. Tryptic hydrolysis showed that a majority of the polypeptide was available on the exterior (cytosolic) surface of the microsomes. These results suggested that the first 29 residues were sufficient not only as an insertion signal but also as a halt-transfer signal. In addition, it was proposed that the cytochrome was bound to the membrane by only a single NH2-terminal anchor, equivalent to alternative 3 in Fig. 1. Szczesna-Skorupa and Kemper (34) altered the structure of cytochrome P450 2C2 by replacing Asps-’Lys2 and Leus-Args. These mutations changed the net charge of the NH2 terminus from 0 to +3. When the mutant cytochrome was synthesized in an in vitro translation system in the presence of rough microsomes, the protein was completely translocated across the membrane, was not cleaved by signal peptidase, became glycosylated, and was membrane bound. The results suggest that the membrane topology had been converted essentially to that of model 2 in Fig. 1 via alteration of the charge at the NH2 terminus. In addition, because the protein was completely translocated, no other portion of the primary structure has the capacity to serve as a halttransfer signal but that located on the carboxyl side of the signal-anchor sequence. Monier et al. (11) constructed 14 P450 2B1 deletion mutants or chimeras containing various segments of pregrowth hormone or opsin. Cell-free translation of the derived mRNAs in the presence or absence of microsomal membranes, protease digestion, or endoglycosidaseH treatment enabled demonstration that the first 43 residues were required for cotranslational membrane insertion and that only this segment and the region of residues 167-185 could serve as halt-transfer signals. Furthermore, evidence for an initial loop-insertion mechanism was obtained, but it was determined that the NH2 terminus could reorient to face the lumen of the membrane. These data support alternative 3 in Fig. 1. Yabusaki et al. (35) modified the structure of cytochrome P450 1A1 by removing the first 30 residues, which include the entire hydrophobic membrane anchor and part of the halt-transfer signal. The altered protein was expressed in yeast but, surprisingly, remained associated with the microsomal fraction. However, the activity toward 7-ethoxycoumarin was diminished by a factor of four compared with the intact P450 1A1 expressed in the same organism. These results argue strongly for additional modes of membrane binding, perhaps through peripheral contacts such as those indicated in Fig. 2. Larson et al. (36) engineered rabbit cytochrome P450 2E1 by deletion of residues 3-29. This mutant is missing the entire NH2-terminal hydrophobic membrane anchor and halt-transfer signal. Yet, when the protein was expressed in Escherichia coil, it was found tightly associated with the bacterial inner membrane. Unlike P450 1AI described above, the shortened P450 2E1 was fully active catalytically. Their data argue convincingly that the signal! anchor/halt-transfer region is not solely required for membrane binding, and is not necessary for correct protein folding and full catalytic activity. Again, the existence of alternate determinants of membrane binding is clearly indicated. Membrane

topology

studied

by immunochemistry

Early studies by Thomas et al. (37) focused on antibody inhibition of microsomal metabolism. Specific antibodies raised against highly purified isoforms of P450 were used in inhibi-

tion experiments in which the activities of five substrates were examined in various microsomes. Results showed that blocking of catalysis was dependent on the particular substrate and reaction as well as the type of microsome preparation and antibody. They concluded that the membranebound form was at least partially exposed on the exterior (cytosolic side) of the membrane. De Lemos-Chiarandini et al. (38) prepared synthetic peptides to a wide variety of sites in cytochrome P450 2B1 and used these to raise specific antipeptide antibodies. The antibody specific for residues 1-31 bound to the purified cytochrome but bound only very poorly to the microsomal enzyme. This would indicate that the NH2-terminal hydrophobic region is membrane bound in the microsome. Antibodies directed against regions 61-72, 108-116, 122-131, and 398-408 were strongly recognized in both the purified and membrane-bound forms, implying that such peptides are exposed on the cytosolic side of the membrane in the large heme-containing domain, These investigators concluded that the most likely membrane topology would have only a single transmembrane anchor, analogous to model 3 in Fig. 1. A similar approach was taken by Edwards et al. (39) wherein antipeptide antibodies were raised to 10 regions of cytochrome P450 2E1 from rabbit. All of these readily recognized the respective epitopes when tested in microsomes. In addition, these authors built a molecular model of P4SOcam with a hydrophobic NH2-terminal anchor peptide and attempted to address the tilt of the heme plane relative to the plane of the membrane. They concluded that a model in which the heme was nearly perpendicular to the plane of the membrane was the most likely. Their data were interpreted in favor of a topological model akin to alternative 3 of Fig. 1, but with a rather rigid character similar to model “a” of Fig. 2.

MEMBRANE FROM THE

TOPOLOGY OF MITOCHONDRION

Mechanisms

of membrane

CYTOCHROME

P450

insertion

Mitochondrial membrane proteins use amphipathic NH2terminal targeting sequences to gain access to the inner membrane of the mitochondrion. The precursor membrane protein binds to the mitochondrial surface at a point where the inner and outer membranes are in close apposition, and translocation to the inner membrane occurs in an ATPdependent process (9, 40, 41). The import machinery is quite complex and is not yet fully understood. The transit peptide is removed by a specific peptidase in the mitochondrial matrix and the resulting mature membrane protein can then assume the native conformation. P450 hAl (P4SOscc) and P450 11B1 (P450 11/3) are steroidogenic mitochondrial membrane proteins essential in the production of pregnenolone and cortisol, respectively. Whereas the precursor forms of these P450s are known to contain targeting sequences and are cleaved to mature forms upon insertion into the mitochondrion, the specific mechanisms of targeting are yet unexplored. However, the mature forms of these P450 cytochromes lack any highly hydrophobic sequence that might be identified as a membrane anchor, such as was seen at the NH2 terminus of the microsomal enzymes. Such an observation suggests the possibility that peripheral membrane associations may be the primary determinants of membrane binding for these steroid-metabolizing monooxygenases.

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Membrane

topology

studies

Topology investigations of steroidogenic mitochondrial P45Oscc have taken the form of proteolysis studies and immunochemical experiments with domain-specific antibodies (42). Tryptic cleavage of P45Oscc yields two prominent fragments, F, (Mr 29,800, containing the NH2 terminus) and F2 (Mr 26,600, containing the COOH terminus). These fragments were generated by hydrolysis of peptide bonds in the region of residues 250-257 in the primary structure; proteolysis of this region can occur with the purified cytochrome or in the membrane-bound state, but cleavage does not release F1 or F2 from the membrane. Furthermore, antibodies raised to each of these “domains” cause extensive inhibition of catalysis when bound, and binding occurs only in submitochondrial particles but not in mitoplasts. These results indicate that P45Oscc is bound to the inner mitochondrial membrane, with a significant fraction of the polypeptide facing the matrix side. The membrane topology of cytochrorne P450 11/3 has been studied by means of trypsin proteolysis (43). Incorporation of the cytochrome into phospholipid vesicles followed by trypsinolysis yields a major 34,000-dalton peptide that remains bound to the membrane and contains the heme prosthetic group. Similar results were obtained with submitochondrial particles. The data show that a majority of the polypeptide chain is available on the matrix side of the inner mitochondrial membrane, that the cAMP-dependent protein kinase site of phosphorylation can be removed from the membrane by proteolysis, and that the general conformation of P450 11/3 or the 34-kDa fragment was similar, as shown by identical circular dichroism spectra.

CONCLUSION Significant progress has been made in our understanding of membrane topological features of the mammalian P450 cytochromes. The enzymes from the endoplasmic reticulum are integral membrane proteins anchored by one or two transmembrane segments located at the NH2 terminus. This region serves as an insertion signal, anchor, and halt-transfer signal; apparently no other portion of the primary structure can serve to arrest polypeptide translocation. Membrane insertion is accomplished via the signal recognition particle and associated cellular machinery. The microsomal P450s are also bound to the ER membrane by one or more peripheral contacts. The large heme-containing domain probably makes use of amphipathic helices to bind to the cytosolic surface of the membrane and thereby assumes a somewhat rigid character. However, the exact nature of these peripheral interactions has not been characterized in detail. The plane of the active-site heme is probably positioned at an angle somewhat greater than parallel to the membrane surface. Our current concepts of membrane topology for the microsomal P450 monooxygenases are summarized in Fig. 2. Considerable data have been obtained in support of both models “a” and “b?’ However, it is not clear at present whether one or both alternatives are correct, although it is important to indicate that more data are presently in support of model “a.” Further experimentation will be required to resolve this controversy. The membrane topology of mitochondrial P450 is less well characterized. However, certain features, such as the lack of an NH2-terminal hydrophobic anchor, are clearly different from the microsomal cytochromes. The mitochondrial P450s appear to be tightly bound to the inner mitochondrial mem-

brane, with a majority of the polypeptide Whether membrane binding is peripheral, remains

to be

facing the matrix. integral, or both

determined.

Nonetheless, present data suggest the possibility that all mammalian P450 monooxygenases may share similar topological features. Further work is needed to clarify this hypothesis. Certainly, a detailed knowledge of membrane topology will be quite important in terms of our understanding of conformational dynamics, interactions with accessory redox proteins, substrate accessibility, and mechanisms of membrane insertion. This work was supported in part by Public Health Service grants GM-38261 from the National Institutes of General Medical Sciences and 2 P30 CA-16058-16A1 from the National Cancer Institute.

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10. Vaux, D., Tooze, J., and Fuller, 5. (1990) Identification by antiidiotype antibodies of an intracellular membrane protein that recognizes a mammalian endoplasmic reticulum retention signal. Nature (London) 345, 495-502 11. Monier, S., van Luc, P., Kreibich, G., Sabatini, D. D., and Adesnik, M. (1988) Signals for the incorporation and orientation of cytochrome P-450 in the endoplasmic reticulum membrane. J. Cell Biol. 107, 457-470 12. Verner, K., and Schatz, G. (1988) Protein translocation across membranes. Science 241, 1307-1313 13. Savitz, A. J., and Meyer, D. I. (1990) Identification of a ribosome receptor in the rough endoplasmic reticulum. Nature (London) 346, 540-544 14. Sakaguchi, M., Mihara, K., and Sato, R. (1984) Signal recognition particle is required for co-translational insertion of cytochrome P-450 into microsomal membranes. Proc. Nail. Acad. Sd. USA 81, 3361-3364 15. Shaw, A. W., Rottier, P. J. M., and Rose, J. K. (1988) Evidence for the loop model of signal-sequence insertion into the endoplasmic reticulum. Proc. Nati. Atad. Sci USA 85, 7592-7596

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