A requirement for ubiquinone in ATPase activity and oxidative phosphorylation.

The suggestion that a rapidly sedimenting rough endoplasmic reticulum fraction in close association with mitochondria, is the preferred site of cytochrome P-450 synthesis has been examined. The rate of cytochrome P-450 synthesis in the different subcellular fractions has been evaluated $\text{in}\text{vivo}\text{and}\text{in}\text{vitro}$, using the immunoprecipitation technique. The results indicate that the conventional microsomal fraction (100,000 X g sediment) is the major site of cytochrome P-450 synthesis and that the rapidly sedimenting rough endoplasmic reticulum fraction associated with mitochondria is not a preferred site for the hemoprotein synthesis.     

The role of components of the endoplasmic reticulum in the biosynthesis of cytochrome P-450.

1. Antibodies have been prepared to rat hepatic cytochrome P-450 and their specificity demonstrated. These antibodies have been used to investigate the biosynthesis of cytochrome P-450 in vitro and in situ in various components of the endoplasmic reticulum. 2. A preparation of heavy rough endoplasmic reticulum translocates proteins newly biosynthesized in vitro vectorially into the luminal space and these are released by low concentrations of deoxycholate. A significant proportion of the radioactivity found in this released fraction is incorporated into cytochrome P-450. 3. Following incorporation of [14C]leucine by perfused rat liver, radioactively labelled cytochrome P-450 can be found in the intrascisternal content of heavy rough, light rough and smooth endopalsmic reticulum and also in a solublized Golgi preparation. 4. We suggest that at least part of the newly biosynthesized cytochrome P-450 is translocated into the intracisternal space of the rough endoplasmic and then passes through the other components of the endoplasmic reticulum before insertion at its ultimate membrane locus.     

Isolation of a subfraction of rough endoplasmic reticulum closely associated with mitochondria : Evidence for its role in cytochrome P450 synthesis

A subfraction of rough endoplasmic reticulum (RER) structurally associated with mitochondria (mito-RER complexes) was isolated from crude nuclear fractions of rat liver homogenate. When apocytochrome P450 synthesis (which presumably occurs in RER) and mitochondrial heme synthesis was dissociated by concomitant treatment of rats with phenobarbital and cobaltous chloride, apocytochrome P450 accumulated predominantly in mito-RER complexes. These data suggest that cytochrome P450 synthesis requires structural interaction of mitochondria and RER.     

10-Undecynoic acid, an inhibitor of cytochrome P450 4A1, inhibits ethanolamine-specific phospholipid base exchange reaction in rat liver microsomes.

1,12-Dodecanedioic acid, the end-product of omega-hydroxylation of lauric acid, stimulates in a concentration dependent manner, phosphatidylethanolamine synthesis via ethanolamine-specific phospholipid base exchange reaction in rat liver endoplasmic reticulum. On the other hand, administration to rats of 10-undecynoic acid, a specific inhibitor of omega-hydroxylation reaction catalyzed by cytochrome P450 4A1, inhibits the ethanolamine-specific phospholipid base exchange activity by 30%. This is accompanied by a small but significant decrease in phosphatidylethanolamine content in the endoplasmic reticulum and inhibition of cytochrome P450 4A1. On the basis of these results it can be proposed that a functional relationship between cytochrome P450 4A1 and phosphatidylethanolamine synthesis exists in rat liver. Cytochrome P450 4A1 modulates the cellular level of lauric acid, an inhibitor of phospholipid synthesis. In turn, ethanolamine-specific phospholipid base exchange reaction provides molecular species of phospholipids, containing mainly long-chain polyunsaturated fatty acid moieties, required for the optimal activity of cytochrome P450 4A1.     

Orientation of cytochromes P450 in the endoplasmic reticulum

The orientation of eukaryotic cytochromes P450, with respect to the membrane of the endoplasmic reticulum, has been investigated. There is now good evidence that the tertiary structure of these proteins is essentially the same as that of the soluble bacterial isoenzyme cytochrome P450CI, with the exception of an extension at the N-terminus which is thought to form a membrane-anchoring sequence. The remainder of the molecule protrudes from the cytosolic face of the membrane so that it can interact with substrates and electron-donating proteins. Two models based on this structure have been considered, in which the plane of the heme of cytochrome P450 is oriented either parallel with or perpendicular to the plane of the membrane of the endoplasmic reticulum. The validity of these models has been assessed from the results of studies involving the binding of antipeptide antibodies directed toward known regions of cytochromes P450, modeling of the interaction of cytochrome P450 with cytochrome b5, proposed intramolecular movements of cytochrome P450 during its catalytic cycle, and the partitioning of substrates for cytochrome P450 between the cytosol and membrane. It is concluded that cytochrome P450 is most likely oriented such that the heme is not fixed horizontal to the plane of the membrane of the endoplasmic reticulum and may well lie with the heme perpendicular to the membrane.     

Depression of cytochrome P-450 and alterations of protein metabolism in mice treated with the interferon inducer polyriboinosinic acid X polyribocytidylic acid

Treatment of mice with the interferon inducer polyriboinosinic acid X polyribocytidylic acid [poly(IC)] results in the depression of several hepatic proteins. In this study we examined synthesis and degradation of the proteins of liver cell organelles in mice treated with poly(IC). Effects on synthesis were determined by using [14C]- and L-[3H]leucine incorporation into control and poly(IC)-treated mice, respectively. At selected times after poly(IC) treatment the 3H/14C ratio was established for preparations of nuclei, mitochondria, lysosomes, smooth endoplasmic reticulum, rough endoplasmic reticulum, and 105,000g supernatant (cytosol). Time-dependent alterations in de novo protein synthesis were greatest in lysosomal and rough endoplasmic reticular fractions; both were depressed 9 h after treatment. The effects of poly(IC) on protein degradation were determined with [14C]bicarbonate. Poly(IC) treatment decreased the time required for disappearance of 50% of 14C-labeled protein (t1/2) of smooth and rough endoplasmic reticula. Examination of endoplasmic reticulum marker enzymes showed depression of cytochromes P-450 and b5 from 9 h onward after poly(IC) administration. Tyrosine aminotransferase activity was elevated 6 h after treatment with poly(IC), and then depressed after 9 h. The other organelle marker enzymes were not affected significantly. We conclude that poly(IC) decreases the content of proteins of the hepatic endoplasmic reticulum, including certain cytochrome P-450 isozymes, by decreasing rates of protein synthesis and increasing rates of protein degradation.     

Retention of membrane proteins by the endoplasmic reticulum

We have used a monoclonal antibody specific for a hydrocarbon-induced cytochrome P450 to localize, by electron microscopy, the epitope- specific cytochrome P450. The cytochrome was found in the rough and smooth endoplasmic reticulum (ER) and the nuclear envelope of hepatocytes. Significant quantities of cytochrome P450 were not found in Golgi stacks. We also could not find any evidence of Golgi- associated processing of the Asn-linked oligosaccharide chains of two well-characterized ER membrane glycoprotein enzymes (glucosidase II and hexose-6-phosphate dehydrogenase), or of the oligosaccharides attached to the bulk of the glycoproteins of the ER membrane. We conclude that these ER membrane proteins are efficiently retained during a process of highly selective export from this organelle.     

Biosynthesis of cytochrome P-450 on membrane-bound ribosomes and its subsequent incorporation into rough and smooth microsomes in rat hepatocytes

Intracellular sites of synthesis of cytochrome P-450 and the subsequent incorporation of it into membrane structures of the endoplasmic reticulum (ER) in rat hepatocytes have been studied using an antibody monospecific for phenobarbital-inducible cytochrome P-450. The cytochrome is synthesized mainly on the "tightly bound" type of membrane-bound ribosomes whose release from the membrane requires treatment with puromycin in a high salt buffer (500 mM KCI, 5mM MgCl2, and 50 mM Tris-HCL [pH 7.5]). Subsequently the cytochrome is incorporated directly into the rough ER membranes with its major part exposed to the outer surface to the membrane and accessible to proteolytic enzymes added externally. The newly synthesized molecules, which appeared first in the rough membrane, are translocated to the smooth membrane, and are then distributed evenly between the two types of microsomeal membranes in approximately 1 h. Administration of cycloheximide, an inhibitor of protein biosynthesis, did not significantly inhibit the transfer of the enzyme from the rough to the smooth ER. It is suggested, therefore, that the translocation of the newly synthesized cythochrome P-450 between the rough and smooth microsomes is mainly due to the lateral movement of the molecules in the plane of the membranes rather than to the attachment and detachment of the ribosomes on the microsomal membranes after the ribosomal cycle for protein synthesis.     

Mammalian cytochromes P450—Importance of tissue specificity

Mammals express multiple cytochromes P450 simultaneously in a variety of tissues, including the liver, kidney, lung, adrenal, gonads, brain, and most others. For cytochromes P450 that are expressed in many tissues or cell types, the tissue/cell type-specific expression might be associated with their special physiological roles. Several cytochrome P450 enzymes are found not only in different cell types and tissues, but also in different subcellular compartments. Generally, all mammalian cytochrome P450 enzymes are membrane bound. The two major groups are represented by microsomal cytochromes P450 that reside in the endoplasmic reticulum, and mitochondrial cytochromes P450, that reside in the inner mitochondrial membrane. However, the outer nuclear membrane, different Golgi compartments, peroxisomes and the plasma membrane are also sites where cytochromes P450 were observed. For example, CYP51 is an ER enzyme in majority of tissues but in male germ cells it trafficks through the Golgi to acrosome, where it is stabilized for several weeks. Surprisingly, in brains of heme synthesis deficient mice, a soluble form of CYP1A1 was detected whose activity has been restored by the addition of heme. In the majority of cases each cytochrome P450 enzyme resides in a single subcellular compartment in a certain cell, however, examples of simultaneous localization in different subcellular compartments have also been described, such as endoplasmic reticulum, Golgi and plasma membrane for CYP2E1. This review will focus on the physiological importance of mammalian cytochrome P450 expression and localization in different tissues or cell types and subcellular compartments.     

Mammalian cytochromes P450--importance of tissue specificity

Mammals express multiple cytochromes P450 simultaneously in a variety of tissues, including the liver, kidney, lung, adrenal, gonads, brain, and most others. For cytochromes P450 that are expressed in many tissues or cell types, the tissue/cell type-specific expression might be associated with their special physiological roles. Several cytochrome P450 enzymes are found not only in different cell types and tissues, but also in different subcellular compartments. Generally, all mammalian cytochrome P450 enzymes are membrane bound. The two major groups are represented by microsomal cytochromes P450 that reside in the endoplasmic reticulum, and mitochondrial cytochromes P450, that reside in the inner mitochondrial membrane. However, the outer nuclear membrane, different Golgi compartments, peroxisomes and the plasma membrane are also sites where cytochromes P450 were observed. For example, CYP51 is an ER enzyme in majority of tissues but in male germ cells it trafficks through the Golgi to acrosome, where it is stabilized for several weeks. Surprisingly, in brains of heme synthesis deficient mice, a soluble form of CYP1A1 was detected whose activity has been restored by the addition of heme. In the majority of cases each cytochrome P450 enzyme resides in a single subcellular compartment in a certain cell, however, examples of simultaneous localization in different subcellular compartments have also been described, such as endoplasmic reticulum, Golgi and plasma membrane for CYP2E1. This review will focus on the physiological importance of mammalian cytochrome P450 expression and localization in different tissues or cell types and subcellular compartments.     

Subcellular localization of the enzymes of cholesterol biosynthesis and metabolism in rat liver

We have used isopycnic density gradient centrifugation to study the distribution of several rat liver microsomal enzymes of cholesterol synthesis and metabolism. All of the enzymes assayed in the pathway from lanosterol to cholesterol (lanosterol 14-demethylase, steroid 14-reductase, steroid 8-isomerase, cytochrome P-450, and cytochrome b5) are distributed in both smooth (SER) and rough endoplasmic reticulum (RER). The major regulatory enzyme in the pathway, hydroxymethylglutaryl-CoA reductase, also was found in both smooth and rough fractions, but we did not observe any associated with either plasma membrane or golgi. Since cholesterol can only be synthesized in the presence of these requisite enzymes, we conclude that the intracellular site of cholesterol biosynthesis is the endoplasmic reticulum. This is consistent with the long-held hypothesis. When the overall pathway was assayed by the conversion of mevalonic acid to non-saponifiable lipids (including cholesterol), the pattern of distribution obtained in density gradients verified its general endoplasmic reticulum localization. The enzyme acyl-CoA-cholesterol acyltransferase which removes free cholesterol from the membrane by esterification, was found only in the rough fraction of endoplasmic reticulum. In addition, when the RER was degranulated by the addition of EDTA, the activity of acyl-CoA-cholesterol acyltransferase not only shifted to the density of SER but was stimulated approximately 3-fold. The localization of these enzymes coupled with the stimulatory effect of degranulation on acyl-CoA-cholesterol acyltransferase activity has led us to speculate that the accumulation of free cholesterol in the RER membrane might be a driving factor in the conversion of RER to SER.     

ONTOGENETIC CHANGES OF PROTEINS OF ENDOPLASMIC RETICULUM

The proteins of the smooth and rough endoplasmic reticulum from fetal, immature, and adult male rats were compared after incorporation of two radioactively labeled precursors, ¹⁴C-labeled amino acids and δ-aminolevulinic acid-³H by means of gel electrophoresis. The labeling patterns indicated that protein components present in two major electrophoretic bands underwent significant synthesis in fetal tissue while three actively incorporating protein bands were noted in adult tissue. Although the uptake of the amino acids-¹⁴C decreased for the smooth and rough elements of the endoplasmic reticulum as a whole during liver development, the qualitative patterns were not significantly different in adult and fetal livers. The over-all incorporation (disintegrations per minute per milligram protein) of the heme precursor into the smooth and rough elements also did not change with development. However, a change was noted in the distributional electrophoretic patterns with development. The estimation of molecular weight (by disc electrophoresis) and the incorporation of the heme precursor suggested the similarity of the two major protein bands to cytochrome P-450 and cytochrome b₅, components of the endoplasmic reticulum, thought to be involved in the mixed-function oxidase system. The evidence indicated that in fetal liver, at a time when the oxidase capability was low, the amino acid incorporation into these two protein groups was the same as in the adult. The incorporation of the heme moiety, however, was different, decreasing in the cytochrome b₅ region and increasing in the cytochrome P-450 region during development. These results correlate with the increase in oxidase activity associated with liver development.     

Synthesis and deposition of zein in protein bodies of maize endosperm

The origin of protein bodies in maize (Zea mays L.) endosperm was investigated to determine whether they are formed as highly differentiated organelles or as protein deposits within the rough endoplasmic reticulum. Electron microscopy of developing maize endosperm cells showed that membranes surrounding protein bodies were continuous with rough endoplasmic reticulum membranes. Membranes of protein bodies and rough endoplasmic reticulum both contained cytochrome c reductase activity indicating a similarity between these membranes. Furthermore, the proportion of alcohol-soluble protein synthesized by polyribosomes isolated from protein body or rough endoplasmic reticulum membranes was similar, and the alcohol-soluble or -insoluble proteins showed identical [¹⁴C]leucine labeling. These results demonstrated that protein bodies form simply as deposits within the rough endoplasmic reticulum.

Messenger RNA that directed synthesis of only the smaller molecular weight zein subunit was separated from mRNA that synthesized both subunits by sucrose gradient centrifugation. This result demonstrated that separate but similar sized mRNAs synthesize the major zein components. In vitro translation products of purified mRNAs or polyribosomes were approximately 2,000 daltons larger than native zein proteins, suggesting that the proteins are synthesized as zein precursors. When intact rough endoplasmic reticulum was placed in the in vitro protein synthesis system, proteins corresponding in molecular weight to the native zein proteins were obtained.

     

Effect of phenobarbital administration to rats on the level of the in vitro synthesis of cytochrome P-450 directed by total rat liver RNA.

Total liver RNA has been isolated from male rats at different time points subsequent to a single injection of phenobarbital, and the level of cytochrome P-450 synthesis directed by these RNA preparations in a cell-free translation system has been determined. It is observed that the maximum $\text{in vitro}$ synthesis of cytochrome P-450 occurs at 16 hours (3-fold above uninduced level) which is approximately 30 hours prior to the maximum induction of spectrophotometrically detectable cytochrome P-450 measured in liver homogenates. Thus, while cytochrome P-450 mRNA is involved in the induction process, its synthesis does not appear to be rate limiting. In addition, phenobarbital induced cytochrome P-450 is not synthesized $\text{in vitro}$ in a form larger than that isolated from endoplasmic reticulum, but rather is also found to have a molecular weight of 50,000.     

The roles of cytochrome b5 in cytochrome P450 reactions

Cytochrome b(5), a 17-kDa hemeprotein associated primarily with the endoplasmic reticulum of eukaryotic cells, has long been known to augment some cytochrome P450 monooxygenase reactions, but the mechanism of stimulation has remained controversial. Studies in recent years have clarified this issue by delineating three pathways by which cytochrome b(5) augments P450 reactions: direct electron transfer of both required electrons from NADH-cytochrome b(5) reductase to P450, in a pathway separate and independent of NADPH-cytochrome P450 reductase; transfer of the second electron to oxyferrous P450 from either cytochrome b(5) reductase or cytochrome P450 reductase; and allosteric stimulation of P450 without electron transfer. Evidence now indicates that each of these pathways is likely to operate in vivo.     

Mammalian microsomal cytochrome P450 monooxygenase: structural adaptations for membrane binding and functional diversity

Microsomal cytochrome P450s participate in xenobiotic detoxification, procarcinogen activation, and steroid hormone synthesis. The first structure of a mammalian microsomal P450 suggests that the association of P450s with the endoplasmic reticulum involves a hydrophobic surface of the protein formed by noncontiguous portions of the polypeptide chain. This interaction places the entrance of the putative substrate access channel in or near the membrane and orients the face of the protein proximal to the heme cofactor perpendicular to the plane of the membrane for interaction with the P450 reductase. This structure offers a template for modeling other mammalian P450s and should aid drug discovery and the prediction of drug-drug interactions.     

Membrane topology of the mammalian P450 cytochromes.

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 transmembrane anchor model with an active site buried in the membrane. 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 membrane-bound 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.     

Role of haem in the synthesis and assembly of cytochrome P-450

By using 3-amino-1,2,4-triazole, an inhibitor of haem synthesis, and 2-allyl-2-isopropylacetamide, a drug that degrades the haem moiety of cytochrome P-450, the involvement of haem in cytochrome P-450 synthesis and assembly was investigated. Phenobarbital was used to stimulate apo-(cytochrome P-450) synthesis. Degradation of preformed cytochrome P-450 haem does not result in a concomitant release of the apoprotein from the endoplasmic reticulum. The availability of haem for cytochrome P-450 synthesis in the normal animal is not rate-limiting. Prolonged inhibition of haem synthesis in vivo decreases the rate of apo-(cytochrome P-450) synthesis, although this effect is not discernible under conditions of short-term inhibition of haem synthesis. Under the former conditions exogenous haemin is able to counteract the decrease in the rate of apoprotein synthesis. In animals receiving successive injections of phenobarbital plus 3-amino-1,2,4-triazole, compared with those receiving phenobarbital only, the holo-(cytochrome P-450) content measured spectrally shows a greater decrease than could be accounted for by the decrease in the content of the total apoprotein. In addition to less haem being available under these conditions, the free apoprotein appears to have undergone some modification, such that its haem-binding capacity is considerably decreased. This particular effect could be due to a direct interaction of 3-amino-1,2,4-triazole or its metabolites with cytochrome P-450 rather than a consequence of haem deficiency. Apo-(cytochrome P-450) is capable of binding to the endoplasmic reticulum in a form and at a site, which can be reconstituted with haemin to yield the functional protein.     

The distribution of the components of mixed-function oxidase between the rough and the smooth endoplasmic reticulum of liver cells

Mixed-function oxidase activity, when measured by the N-demethylation of ethylmorphine or the hydroxylation of aniline, is significantly higher in the smooth hepatic endoplasmic reticulum than in the rough. In the rabbit the smooth membrane/rough membrane activity ratios are significantly greater than 1 whether the activities are expressed per g. of liver (ratio 5), per mg. of protein (ratio 3-5), per mug. of phospholipid phosphorus (ratio 2), per unit of cytochrome P-450 (ratio 1.7) or per unit of NADPH-cytochrome c reductase activity (ratio 2). On the other hand, if the activities are normalized to the NADPH-cytochrome P-450 reductase, there is no significant difference between the rough and smooth membranes. These results suggest that, in the rabbit, the rate-limiting step is the reduction of cytochrome P-450. In contrast, in the rat the difference in activities can be explained by differences in the concentration of cytochrome P-450.     

Biogenesis of endoplasmic reticulum membrane in rat liver cells. II. Discharge of the nascent peptides of NADPH-cytochrome c reductase and cytochrome b5 on the cytoplasmic side of the endoplasmic reticulum membrane.

The direction of discharge of the nascent peptides of NADPH-cytochrome c reductase and cytochrome b5 from bound polyribosomes of rough microsomes was investigated in order to elucidate the mechanism of separation of these membrane proteins from secretory proteins, which are also synthesized by the same class of ribosomes of rough endoplasmic reticulum. The nascent peptides of NADPH-cytochrome c reductase and cytochrome b5 in intact rough microsomes were accessible to externally added 125I-Fab's against these proteins, and were susceptible to trypsin digestion, whereas the nascent peptides of serum albumin were not. The nascent peptides of these two microsomal proteins were released into the cytoplasm by puromycin treatment of intact rough microsomes, while the nascent peptides of serum albumin were retained in the microsomal lumen. These observations suggest that the nascent peptides of microsomal proteins, which are present on the cytoplasmic surface of the endoplasmic reticulum membrane, are exposed on the surface of microsomal vesicles, while those of secretory proteins are enclosed inside the vesicles. Therefore, the topographical separation of microsomal membrane proteins from secretory proteins is accomplished at the step of their synthesis by the bound polyribosomes of rough endoplasmic reticulum.