©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The Carboxyl Terminus of the Membrane-binding Domain of Cytochrome b Spans the Bilayer of the Endoplasmic Reticulum (*)

(Received for publication, September 19, 1994; and in revised form, December 6, 1994)

Guy Vergères (1) (2) Jeremy Ramsden (2) Lucy Waskell (1)(§)

From the  (1)Department of Anesthesia, University of California, Veterans Administration Medical Center, San Francisco, California 94121 and the Department of Biophysical Chemistry, (2)Biocenter of the University of Basel, Basel, Switzerland

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Preliminary studies (Vergères, G., and Waskell, L.(1992) J. Biol. Chem. 267, 12583-12591) have suggested that the carboxyl-terminal membrane-binding domain of cytochrome b(5) traverses the membrane and that the carboxyl terminus is in the lumen of the endoplasmic reticulum. In order to confirm and extend these studies, additional experiments were conducted. The gene coding for rat cytochrome b(5) was transcribed and the resulting mRNA was translated in vitro in a rabbit reticulocyte lysate in the presence of microsomes. The binding and topology of cytochrome b(5) were investigated by treating microsomes containing the newly incorporated cytochrome b(5) with carboxypeptidase Y and trypsin. Our studies indicate that cytochrome b(5) is inserted both co- and post-translationally into microsomes in a topology in which the membrane-binding domain spans the bilayer with its COOH terminus in the lumen. Cytochrome b(5) is also incorporated into microsomes pretreated with trypsin in a topology indistinguishable from the one resulting from the insertion of the protein into untreated microsomes, reconfirming that cytochrome b(5) does not use the signal recognition particle-dependent translocation machinery. Our results do not allow a distinction to be made between a spontaneous insertion mode or some other trypsin-resistant receptor-mediated mechanism.

A role for Pro in the middle of the membrane-binding domain of cytochrome b(5) was also examined by mutating it to an alanine and subsequently characterizing the ability of the mutant protein to be incorporated into membranes. The mutant protein inserted more slowly in vitro into microsomes as well as into pure lipid bilayers by a factor of 2 to 3.


INTRODUCTION

Microsomal cytochrome b(5) is a heme protein involved in the desaturation of fatty acids (Strittmatter et al., 1974; Lee et al., 1977; Oshino, 1982), biosynthesis of cholesterol (Grinstead and Gaylor, 1982), as well as in various hydroxylation reactions of the monooxygenase system (Hildebrandt and Estabrook, 1971; Canova-Davis et al., 1985). The liver protein is an amphipathic molecule which contains two domains, namely a heme-binding domain and a hydrophobic membrane-anchoring domain at the COOH terminus (Ozols, 1989).

Cytochrome b(5) is synthesized on cytosolic ribosomes and post-translationally inserted into the microsomal membrane independently of signal recognition particle (SRP) (^1)(Rachubinski et al., 1980; Bendzko et al., 1982; Okada et al., 1982; Anderson et al., 1983). The conclusion that cytochrome b(5) is inserted post-translationally into microsomes was based on the finding that cytochrome b(5) which had been added to microsomes subsequent to synthesis could not be removed by treatment with 0.5 M NaCl or sodium carbonate at pH 11.5 (Bendzko et al., 1982; Okada et al., 1982; Anderson et al., 1983). The exact topology of the COOH terminus was, however, not investigated in these studies. Two models have been proposed for the membrane topology of the COOH terminus which contains a hydrophobic stretch of 22 uncharged amino acids (Ser to Tyr) (Fig. 1). In the transmembrane model, the membrane-binding domain spans the bilayer with the COOH terminus in the lumen of the microsomes. In the hairpin loop model, the hydrophobic peptide only penetrates the external leaflet of the membrane and the COOH terminus loops back into the cytosol.


Figure 1: Models for the topology of cytochrome b(5). The COOH-terminal membrane-binding domain (Ser-Asp) of cytochrome b(5) is shown in the transmembrane (solid circles) and hairpin loop (broken circles) conformations. The hydrophobic peptide (Ser to Tyr) is in bold. The arrows denote the putative sites of cleavage by trypsin. The two methionines labeled with S are at position 125 and 130. The heme-binding domain consists of residues 1-93, with residues 94-103 forming a linker peptide.



It has been shown in vitro that cytochrome b(5) binds to liposomes in two different ways: when added to preformed large unilamellar vesicles, the protein binds in the so-called ``loose-binding form,'' i.e. it can be transferred to other vesicles (Roseman et al., 1977; Enoch et al., 1979). When incorporated during formation of unilamellar vesicles by the detergent-dialysis technique, the protein binds in the so-called ``tight-binding form'' and cannot be transferred to other vesicles (Enoch et al., 1979). These two forms of binding can be further distinguished by digestion with carboxypeptidase Y (CPY), which releases the heme domain of the loose-binding form of cytochrome b(5) from the membrane whereas the tight-binding form cannot be removed in this manner (Enoch et al., 1979). A correlation between the two forms of binding and the topology of the COOH terminus was proposed by Khorana and co-workers (Takagaki et al., 1983a, 1983b). Cross-linking studies suggest that the COOH terminus of cytochrome b(5) is in a hairpin loop conformation when bound to vesicles in the loose-binding form (Takagaki et al., 1983a) and in a transmembrane conformation when bound to vesicles in the tight-binding form (Takagaki et al., 1983b). Other cross-linking studies, however, suggested that the tight-binding form of cytochrome b(5) was in the loop conformation (Arinc et al., 1987). In microsomes, cytochrome b(5) binds in a tight-binding form as judged by its inability to transfer to vesicles (Enoch et al., 1979). Two proteolytic studies, with contradictory results, have addressed the topology of the microsomal protein. In one study treatment of microsomes with trypsin was reported to result in the generation of the membrane-binding peptide of cytochrome b(5) lacking the last 6 amino acids 128-133 (see Fig. 1), suggesting that the COOH terminus is exposed to the cytosol (Ozols, 1989). In the second study CPY could not solubilize the heme-binding domain of microsomal cytochrome b(5), suggesting that the COOH terminus is luminal (Enoch et al., 1979). Preliminary site-directed mutagenesis experiments from this laboratory suggested that the carboxyl terminus of cytochrome b(5) is located in the lumen of the endoplasmic reticulum (Vergères and Waskell, 1992). In order to provide more definitive information about the topology of the membrane-binding domain of cytochrome b(5), we have expressed cytochrome b(5) in a cell-free system and used proteolytic enzymes to probe the location of the carboxyl terminus of cytochrome b(5) inserted either co- or post-translationally into membranes. Herein it is reported that the transmembrane domain of cytochrome b(5) spans the membrane with its carboxyl terminus located in the lumen of the endoplasmic reticulum.

Prolines induce unique structural and electron configurations in proteins, namely, disruption of secondary structures (e.g. helix-breaking) due to the lack of hydrogen bonding of the backbone carbonyl group and its exposure to the solvent (Barlow and Thornton, 1988; Richardson and Richardson, 1989). Although prolines occur with relatively high frequency in the putative alpha-helical transmembrane segments of many integral membrane proteins, a function unique to this amino acid in membrane proteins has not been elucidated (Williams and Deber, 1991). The peptide bond of proline residues can exist in either the cis or trans configuration due largely to destabilization of the transXaa-Pro amide bond (Richardson and Richardson, 1989). Isomerization at proline residues occurs spontaneously and is also catalyzed by the enzyme prolyl isomerase (Schmid, 1993). The second issue which was explored was whether the ability of the transmembrane anchor to isomerize at the Ile-Pro bond and assume either a hairpin-like (cis) or merely a kinked structure (trans) (Fig. 1) would influence the ability of cytochrome b(5) to be incorporated co- and or post-translationally into membranes. Pro was therefore mutated to an alanine which presumably generated a rigid alpha-helical transmembrane segment. The co- and post-translational insertion of the mutant protein into microsomes was decreased suggesting that Pro modulates the insertion of cytochrome b(5) into the membranes.


EXPERIMENTAL PROCEDURES

Materials

The gene coding for rat liver cytochrome b(5) was a gift of Dr. Steven Sligar, University of Illinois, Urbana, IL (Beck von Bodman et al., 1986). The transcription plasmid pGM 3Zf(-), canine pancreas microsomes, DNase-free RNase, RNase-free DNase, ribonuclease inhibitor (RNasin), SP6 DNA polymerase, T7 DNA polymerase, as well as an in vitro translation kit containing the micrococcal nuclease-treated rabbit reticulocyte lysate, amino acids minus methionine, and the control mRNA coding for alpha factor were obtained from Promega (Madison, WI). [S]Methionine was purchased from ICN Biomedical Inc. (Costa Mesa, CA). HPLC-purified trypsin and carboxypeptidase Y (both sequencing grade) were from Sigma. Restriction enzymes were obtained from New England Biolabs (Beverly, MA). Yeast tRNA was from Boehringer (Mannheim, Germany). Palmitoyl oleoyl phosphatidylcholine (POPC) was from Avanti Polar Lipids (Alabaster, AL). Planar optical waveguides made from SiTiO(2) incorporating a diffraction grating were obtained from Artificial Sensing Instruments AG (Zürich, Switzerland).

In Vitro Transcription of Cytochrome b(5) Genes

The genes coding for the wild type cytochrome b(5) as well as for the P115A mutant (Vergères and Waskell, 1992) were inserted in the EcoRI site of the plasmid pGM 3Zf(-). The plasmids were linearized with the restriction endonuclease HindIII, for the inserts in the T7 orientation, or with NdeI, for the inserts in the SP6 orientation. The efficiency of transcription of the cytochrome b(5) genes was dramatically increased by RNase pretreatment of the linearized plasmids followed by phenol/chloroform extraction and ethanol precipitation of the DNAs. A typical transcription reaction contained 10 µl of a 5-fold concentrated SP6 or T7 transcription buffer (Promega), 1.25 µl of a 10 mM solution of ATP, GTP, CTP, and UTP, 1 µl of RNasin (40 units), 1.0 µl of SP6 or T7 DNA polymerase (80 units), 5 µl of dithiothreitol (100 mM), 10 µl of DNA (1-2 µg), and nuclease-free water to 50 µl. The solution was incubated at 40 °C for 1 h.

Cell-free Translation of Cytochrome b(5) mRNAs

In order to improve the efficiency of translation, DNA present in the transcription reaction mixture was removed by digestion with RNase-free DNase followed by two rounds of ethanol precipitation. A standard translation solution consisted of 17.5 µl of rabbit reticulocyte lysate, 0.5 µl of amino acids minus methionine, 2 µl of [S]methionine (20 µCi, 1200 Ci/mmol), 0.5 µl of RNasin (20 units), 1 µl of RNA coding for cytochrome b(5) (about 0.1 µg), 1 µl of microsomes, if needed, and water to a final volume of 25 µl. To allow for complete translation the solution was incubated for 90 min at room temperature. Longer incubation times did not increase the level of expression of cytochrome b(5).

Preparation of Microsomes

Rabbit liver microsomes were prepared as described by Haugen and Coon(1976). Endogenous mRNA was removed by treatment of the microsomes with EDTA (Walter and Blobel, 1983). Microsomes containing alpha factor were prepared by expressing 0.1 µg of the mRNA coding for alpha factor co-translationally in the presence of canine pancreas microsomes as described above. Microsomes were treated with trypsin as described (Meyer and Dobberstein, 1980). After centrifugation in 1.5-ml microcentrifuge tubes in a TL-100.3 rotor (table top ultracentrifuge, Beckman, Palo Alto, CA) for 30 min at 4 °C and 50,000 rpm (100,000 times g), microsomes were resuspended in the original volume of ``membrane buffer'' (50 mM Tris, pH 7.5, at room temperature, 0.25 M sucrose, 1 mM dithiothreitol) using a hand homogenizer (Bolab Products) fitting the 1.5-ml microcentrifuge tubes. A volume of at least 10 µl of microsomes was necessary to ensure quantitative recovery.

Assays for Binding of Cytochrome b(5) to the Various Microsomal Membranes

For post-translational insertion of cytochrome b(5) into microsomes, 50 µl of a solution in which cytochrome b(5) has been translated to completion for 90 min in the absence of microsomes was centrifuged in 1.5-ml microcentrifuge tubes in a TL-100.3 rotor for 30 min at 4 °C and 50,000 rpm to remove the insoluble population of cytochrome b(5). 24 µl of the supernatant were incubated with 1 µl of microsomes at room temperature for 90 min. For co-translational insertion of cytochrome b(5) into microsomes, insoluble cytochrome b(5) could not been removed by centrifugation at 100,000 times g since it coprecipitates with microsomes.

5 µl of a translation reaction mixture, in which cytochrome b(5) has been incubated either co- or post-translationally with microsomes for 90 min at room temperature, was added to a microcentrifuge tube containing 250 µl of an ice-cold solution (either 50 mM Tris, pH 7.5, at room temperature, 0.5 M NaCl, or 100 mM sodium carbonate, pH 11.5). The solution was then incubated on ice for 30 min and the microsomes precipitated by centrifugation in a TL100.3 rotor as described above. The fraction of cytochrome b(5) present in the pellet was compared to the total amount present in the translation reaction mixture prior to centrifugation by analysis of the equivalent of 0.25 µl of the translation reaction mixture on SDS gels and volume scanning densitometry of the autoradiograms.

To quantitate the recovery of microsomes after centrifugation, cytochrome b(5) was added to canine pancreas microsomes in which alpha factor had previously been inserted co-translationally as an internal standard. Alternatively the mRNAs coding for alpha factor and cytochrome b(5) were co-translated in the presence of microsomes.

Treatment of Microsomes with Proteases

In a standard proteolysis assay, 1 µl of the translation reaction mixture was digested with various proteases in a volume of 10 µl (see below). The reactions were stopped by addition of 10 µl of sample buffer (0.25 M Tris, pH 6.8, at room temperature, 20% glycerol, 2% SDS (w/v), 0.025% bromphenol blue, 5% mercaptoethanol) containing 2 mM phenylmethylsulfonyl fluoride (100 mM stock in isopropyl alcohol) followed by heating at 95 °C for 5 min (Morimoto et al., 1983). Tergitol NP-10 (Sigma) was used to eliminate the protective effect of the microsomal membrane. Processed alpha factor could not be digested unless the microsomes were treated with 0.2% Tergitol, indicating that microsomes are impermeable to proteases and that Tergitol can solubilize or, at least, permeabilize the microsomal membrane. Tergitol has no inhibitory effect on any of the proteases used in this study since cytochrome b(5) translated in the absence of microsomes could be digested in the absence as well as in the presence of 0.2% Tergitol.

CPY Treatment

A standard reaction mixture contained 1 µl of 1 M Tris (pH 6.7) at room temperature, 1 µl of translation reaction mixture described in the section under ``Cell-free Translation of Cytochrome b(5) mRNAs,'' 1 µl of HPLC-purified CPY (sequencing grade, 2 mg/ml), 1 µl of 2% Tergitol NP-10 (final concentration 0.2%), if needed, and RNase-free water to 10 µl. The solution was incubated for 15 min at 37 °C.

Trypsin Treatment

A standard reaction mixture contained 1 µl of 1 M Tris, pH 8.1, at room temperature, 1 µl of 10 mM EDTA, 1 µl of translation reaction mixture described in the section under ``Cell-free Translation of Cytochrome b(5) mRNAs,'' 1 µl of HPLC-purified trypsin (sequencing grade, 2 mg/ml), 1 µl of 2% Tergitol NP-10 (final concentration 0.2%), if needed, and RNase-free water to 10 µl. The solution was incubated on ice for 1 h.

The presence of an insoluble population of cytochrome b(5) or of the protein which has not been incorporated into microsomes obscures the proteolytic analysis of the protein topology. Removal of these populations is therefore critical in order to obtain unambiguous results. For digestion of cytochrome b(5) in the absence of microsomes, the translation reaction mixture was first centrifuged at 50,000 rpm in a TL 100.3 rotor, as described above. The supernatant was treated with proteases. For digestion of cytochrome b(5) inserted either co- or post-translationally into microsomes, the translation reaction mixture was first centrifuged at 50,000 rpm and the microsomal pellet resuspended in membrane buffer to the original volume of translation reaction mixture.

Gel Analysis

0.25-0.5 µl of the translation reaction mixture was typically analyzed on sodium dodecyl sulfate (SDS)-polyacrylamide gels. For translation and binding assays in which the undigested form of cytochrome b(5) was analyzed, 12.5% glycine gels were used (Laemmli, 1970). The molecular weight of the expressed cytochrome b(5) was estimated with a low molecular mass standards kit (97.4 to 14.5 kDa) from Bio-Rad. For analysis of the proteolytic degradation product of cytochrome b(5), 15% glycine or 16.5% Tricine gels (Schägger and von Jagow, 1987) were used and the molecular masses were estimated using a kit of low molecular mass peptides derived from myoglobin (17 to 2.5 kDa) (Sigma). Detection of the radioactive bands was performed by fluorography with Amplify (15 min incubation) (Amersham, Buckinghamshire, United Kingdom) followed by exposure (3-16 h) on x-ray film at -80 °C.

Scanning and Quantification of Radioactive Bands

Bands labeled with [S]methionine were analyzed following detection on x-ray film by volume integration with local background substraction at 100-µm resolution on a Personal Densitometer from Molecular Dynamics. Care was taken not to overexpose and saturate the x-ray film in order to assure an accurate and reliable quantitation of the radioactive bands (Molecular Dynamics pamphlet: Image Quant v3.0). The fraction of cytochrome b(5) binding to microsomes was corrected for the amount of microsomes lost during the centrifugation. Recovery of microsomes after centrifugation was determined by using processed alpha factor as an internal control. Quantitation of the data by volume scanning densitometry was done on a single set of experiments which faithfully reflects what had been observed in previous experiments.

Expression and Purification of Cytochrome b(5) in Yeast

Expression of wild type cytochrome b(5) and of the P115A mutant in the yeast Saccharomyces cerevisiae as well as their purification to homogeneity have been described previously (Waskell et al., 1991; Vergères and Waskell, 1992).

Determination of the Amount of Cytochrome b(5) Bound to a Planar Optical Waveguide-supported Lipid Bilayer Using a Novel Integrated Optics Technique

The lipid bilayer was formed on the waveguide as described previously (Ramsden, 1993a). A waveguide is a thin film of high refractive index material along which light propagates laterally by total internal reflection (Tien, 1971; Adams, 1981). The waveguide incorporates a diffractive grating which allows an external light beam incident on it to be converted into a guided light mode propagating along the waveguide (Tien, 1971). A 1 mM solution of POPC in hexane:ethanol (9:1) was spread on the surface of buffer (50 mM Tris-HCl, pH 7.5, at room temperature, 1 mM EDTA, 100 mM NaCl) in a Langmuir trough and compressed to a monolayer of POPC with a surface pressure of 32 millinewton/m. The first monolayer was deposited on the waveguide (which had been previously rapidly lowered vertically through the monolayer without deposition of lipids) with the polar headgroups oriented next to its hydrophilic surface by slowly (0.14 mm/s) withdrawing the waveguide vertically out through the monolayer. The waveguide was dried in air for 3 min. The second monolayer was deposited by rapidly lowering the waveguide horizontally onto the floating lipid monolayer. The waveguide was removed from the trough without letting it cross another air-water interface, and mounted on the goniometer table of an IOS-1 scanner (Artificial Sensing Instruments AG). A small circular cuvette (diameter 1 cm) which could be filled with 300 µl of buffer or protein solution was fixed over the grating region of the waveguide, such that the waveguide formed one wall of the cuvette as described by Ramsden and Schneider (1993; Tien, 1971; Ramsden, 1993b). The IOS-1 instrument allows the phase velocities of guided light modes to be measured to a precision of about 1 ppm. In the present experiment, changes in the composition of the membrane-solution interfacial region, and hence the reflectivity of the interface and hence the phase velocity of the guided modes, result from the binding of cytochrome b(5) to the membrane. From the velocities of the zero order modes perpendicular and parallel to the interface, the mass of protein deposited on the membrane can be calculated to a precision of ± 1 ng/cm^2 (Ramsden, 1992, 1993b; Ramsden and Schneider, 1993). Phase velocity measurements were repeated every 30 s. The cuvette was filled with pure buffer to establish the baseline, following which all but 100 µl was then removed and 200 µl of cytochrome b(5), typically at 1 µM, was added and rapidly mixed with the buffer. Temperature was measured with a platinium resistance thermometer embedded in the goniometer table.


RESULTS

Cell-free Expression of Cytochrome b(5)

DNA coding for the membrane-bound form of rat liver cytochrome b(5) was transcribed and the corresponding mRNA translated in vitro in a rabbit reticulocyte lysate in the presence of [S]methionine (Walter and Blobel, 1983). A single radioactive product whose molecular weight corresponds to the membrane-bound form of cytochrome b(5) (17 kDa) is expressed (Fig. 2, lane 2). In the absence of cytochrome b(5) mRNA, the protein is not synthesized (lane 1).


Figure 2: Expression of cytochrome b(5) in a cell-free system. mRNA coding for cytochrome b(5) was translated in a rabbit reticulocyte lysate in the presence of [S]methionine and analyzed on a 12.5% SDS gel by fluorography as described under ``Experimental Procedures.'' Lane 1, translation in the absence of cytochrome b(5) mRNA. Lane 2, translation in the presence of mRNA coding for cytochrome b(5).



Cytochrome b(5) Is Inserted into Microsomal Membranes both Co- and Post-translationally

In order to determine whether cytochrome b(5) was inserted into microsomal membranes co- and/or post-translationally, cytochrome b(5) was either translated in the presence of microsomes or added to microsomes following completion of translation. The amount of cytochrome b(5) which had been incorporated into the microsomes was then quantitated by volume scanning densitometry of autoradiograms of SDS-polyacrylamide gels following separation of the microsomes from the cytosolic fraction by centrifugation at 100,000 times g. Microsomes previously loaded with the yeast mating pheromone, alpha factor, served as an internal standard for microsomal recovery. Fig. 3(lane 1) demonstrates that cytochrome b(5) is readily translated in the absence of microsomes. When the translation mixture is centrifuged at 100,000 times g in the presence of 0.5 M NaCl (lane 2), 5-10% of the cytochrome b(5) is found in the pellet. This fraction is referred to as the insoluble cytochrome b(5) and presumably represents cytochrome b(5) aggregated with itself and/or other macromolecules in the translation mixture. Since the insoluble cytochrome b(5) population increased with time and coprecipitates with microsomes, the binding of cytochrome b(5) to microsomes was assayed within hours of completion. In addition, post-translational insertion of cytochrome b(5) into microsomes was studied with a translation reaction in which the fraction of insoluble cytochrome b(5) was removed by centrifugation prior to incubation with microsomes.


Figure 3: Binding of cytochrome b(5) to microsomal membranes in the presence of 0.5 M NaCl and sodium carbonate, pH 11.5. Cytochrome b(5) was translated in the absence of microsomes (-mic: lanes 1-3) or in the presence of canine pancreas microsomes (co mic: lanes 7-9). Alternatively cytochrome b(5) was translated and microsomes were subsequently added (post mic: lanes 4-6). The mixture was incubated for 90 min as described under ``Experimental Procedures.'' Microsomes in which alpha factor had previously been inserted were used in order to have an internal control to follow the recovery of microsomes. Binding was assessed by comparing the total amount of cytochrome b(5) present in the translation reaction (lanes 1, 4, and 7) with the fraction associated with the pellet after centrifugation at 100,000 times g in 0.5 M NaCl (lanes 2, 5, and 8) or in sodium carbonate at pH 11.5 (lanes 3, 6, and 9). Processed glycosylated alpha factor (p-alpha fac: 30 kDa) was used as an internal control to quantitate the recovery of microsomes. The equivalent of 0.25 µl of translation reaction was loaded on a 12.5% gel. Quantification was performed by volume scanning densitometry of the bands. u-alpha fac, unprocessed alpha factor (18.6 kDa).



When cytochrome b(5) was translated in the presence of canine pancreas microsomes (Fig. 3, lanes 4 and 5) or added to microsomes following completion of translation (Fig. 3, lanes 7 and 8), 65-70% of the S-labeled cytochrome b(5) could be retrieved in the microsomes in the 100,000 times g pellet in 0.5 M NaCl. This is slightly less than the 85% recovery of the internal standard, which contained glycosylated alpha factor in the microsomal lumen. Luminal glycosylated alpha factor also served as a control for the integrity of the vesicles. The yeast pheromone, alpha factor, is translated in a SRP-dependent manner in canine pancreas microsomes and is translocated into the lumen of the microsomes where it undergoes core glycosylation by various glycosyltransferases (Schachter and Roseman, 1980). The unprocessed unglycosylated protein has a molecular mass of 18.6 kDa, whereas the fully glycosylated protein has a molecular mass of 30 kDa (Promega pamphlet TM231). In an effort to elucidate whether cytochrome b(5) was inserted into the microsomal membrane as a peripheral or integral membrane protein, microsomes containing S-labeled cytochrome b(5) and alpha factor were pelleted in the presence of 0.5 M NaCl, to remove peripheral membrane proteins. In a second experiment the same microsomes were pelleted in the presence of 100 mM sodium carbonate, pH 11.5, which dissociates peripheral membrane proteins and releases luminal proteins such as alpha factor (Morimoto et al., 1983). High salt (0.5 M NaCl) did not release either cytochrome b(5) or alpha factor, indicating that cytochrome b(5) is not a peripheral membrane protein and that microsomes are intact (Fig. 3, lanes 5 and 8). In the presence of sodium carbonate, pH 11.5, cytochrome b(5) remained associated with the microsomal membrane indicating it had become an integral membrane protein while alpha factor was completely released from the microsomal lumen (Fig. 3, lanes 6 and 9).

Interestingly, cytochrome b(5) was also incorporated co- and post-translationally into phenobarbital-induced rabbit liver microsomes, even though the rabbit liver microsomes were incompetent in the translocation of alpha factor due to their known lower intrinsic activity in the translocation machinery and in protein secretion (Walter and Blobel, 1983). These results confirm previous reports that cytochrome b(5) is incorporated into microsomes by a process that does not require SRP (Anderson et al., 1983).

Topological Analysis of Microsomal Cytochrome b(5)

The binding assays described above indicate that cytochrome b(5) is incorporated into microsomal membranes as an integral membrane protein both co- and post-translationally. However, they do not provide information about whether the membrane anchor spans the membrane or forms a hairpin loop with the carboxyl terminus outside of the microsomal lumen (see Fig. 1). In order to explore the topology of the membrane anchor of cytochrome b(5), microsomes containing the newly translated S-labeled cytochrome b(5) were subjected to treatment with water-soluble proteases which were assumed to be unable to enter the microsomal lumen. Cytochrome b(5) contains a total of 3 methionines; one at position 1 and two near the carboxyl terminus at residues 125 and 130 (see Fig. 1). In vitro translation of a mRNA coding for a truncated form of cytochrome b(5) (amino acids 1-97) lacking the membrane-binding domain and methionines 125 and 130, did not result in the labeling of cytochrome b(5) with [S]methionine, indicating that Met^1 is removed following translation in the reticulocyte assay. Therefore the susceptibility of the Met and Met containing the COOH-terminal membrane-binding domain of cytochrome b(5) to proteolysis and consequently its topology relative to the membrane can be directly and selectively analyzed. Inability of exogenously added proteases to remove S-labeled cytochrome b(5) peptides from microsomes would provide evidence for a transmembrane spanning membrane anchor. If proteases completely or partially remove S-labeled cytochrome b(5) peptides from the microsomes, it would indicate that the carboxyl terminus is extraluminal. Proteases could release 50 or 100% of the S radioactive labeling from the microsomes depending on the accessibility of Met. This residue is close to the membrane boundary and might be protected from proteolysis by the lipid bilayer.

Digestion of Free and Membrane-bound Cytochrome b(5) with Carboxypeptidase Y

For most cases of topological analysis of proteins, nonspecific proteases are sufficient to determine their membrane topologies since the size of the domain(s) translocated into the luminal space is large enough to analyze the proteolytic pattern on SDS gels. In the present experiments the object was to determine whether the carboxyl terminus (Arg to Asp) containing Met and Met was in the microsomal lumen or on the outside of the microsomal vesicles (see Fig. 1). CPY is a serine exoprotease which catalyzes the removal of amino acids, including prolines, from the COOH terminus of proteins (Morimoto et al., 1983). CPY can digest cytochrome b(5) in the absence of microsomes as seen in Fig. 4(lanes 1-2). The protease removes >95% of the radioactivity indicating that the COOH terminus can be digested at least as far as Met (see Fig. 1). When cytochrome b(5) is inserted post-translationally into microsomes, 90-95% of the protein is resistant to digestion by CPY demonstrating that the COOH terminus of membrane-bound cytochrome b(5) is not accessible to CPY (lanes 3 and 4) and indicating that the hydrophobic peptide of cytochrome b(5) is in the transmembrane conformation. In order to obtain this protective effect, sequencing-grade CPY, which has been purified by reverse phase HPLC, must be used. If microsomal cytochrome b(5) is digested with non-purified CPY, presumably containing endoproteases, this protection is substantially decreased and results in the degradation of cytochrome b(5). The observation that sequencing-grade CPY digests cytochrome b(5) in the absence of microsomes but not when incorporated in membranes, clearly indicates that the enzyme preparation is not contaminated with endoproteases. Treatment of microsomes with Tergitol abolishes the protective effect since >95% of the protein is digested by CPY as seen by the complete disappearance of the radioactively labeled cytochrome b(5) (lane 5).


Figure 4: Digestion of free and membrane-bound cytochrome b(5) with carboxypeptidase Y. Cytochrome b(5) translated in the absence of microsomes (-mic: lanes 1 and 2), cytochrome b(5) to which canine microsomes were added after translation (post mic: lanes 3-5) and cytochrome b(5) translated in the presence of microsomes (co mic: lanes 6-8) was digested with CPY (lanes 2, 4, 5, 7, and 8) in the absence (lanes 2, 4, and 7) or in the presence (lanes 5 and 8) of 0.2% Tergitol and analyzed as described under ``Experimental Procedures.''



CPY treatment of cytochrome b(5) inserted co-translationally follows qualitatively the same pattern described for the post-translational insertion. In the presence of microsomes, 80% of the protein is protected from digestion (Fig. 4, lane 7). Upon treatment of microsomes with Tergitol, this fraction is decreased to 25% (lane 8). The analysis is complicated by the presence of the population of the insoluble cytochrome b(5) (see Fig. 3, lanes 2 and 3), which is presumably not incorporated into microsomes and which cannot be removed in the co-translational assay since it coprecipitates with microsomes at 100,000 times g. This insoluble cytochrome b(5) is not digested by CPY even in the presence of Tergitol (not shown), and is presumably the origin of the protein visible in lane 8 of Fig. 4. This population, however, only represents a minor fraction of cytochrome b(5) and does not alter the interpretation of the results.

Treatment of unprocessed, extramicrosomal alpha factor with CPY reduces the radioactive labeling associated with the protein as analyzed by autoradiography of SDS-polyacrylamide gels, whereas processed alpha factor, which is translocated into the lumen of microsomes, is protected from degradation by the protease (not shown) indicating that alpha factor is a substrate for CPY and that the microsomal membrane is impermeable to the protease.

Digestion of Free and Membrane-bound Cytochrome b(5) with Trypsin

In view of our observation that the COOH terminus of cytochrome b(5) is protected from proteolysis by CPY when bound to microsomes, the results obtained by Ozols(1989) in which the membrane-binding peptide of cytochrome b(5) (residues 91-127), lacking the COOH-terminal amino acids 128-133, was purified from a rabbit liver microsomal fraction treated with trypsin are rather contradictory since they indicate that the membrane-binding domain of cytochrome b(5) is in the hairpin loop conformation. We have therefore analyzed the relative susceptibilities of the COOH terminus of cytochrome b(5) toward proteolysis by trypsin, in the absence of microsomes as well as for membrane-bound cytochrome b(5). The pattern of the radioactive 4-5 kDa hydrophobic tryptic peptide should be the same for cytochrome b(5) digested in the absence of microsomes and for cytochrome b(5) inserted in a hairpin loop conformation into microsomes since trypsin would cleave at Lys and Arg in both case (see Fig. 1). On the other hand, trypsin would cleave cytochrome b(5) inserted into microsomes in the transmembrane conformation at Lys, only resulting in the appearance of a larger degradation peptide containing the two labeled methionines 125 and 130 and hence more radioactivity than for the protein digested in solution which would only contain S-labeled Met. Treatment of cytochrome b(5) either in the absence of microsomes or incorporated post-translationally into microsomes with trypsin results in complete disappearance of the intact S-labeled protein (Fig. 5, lanes 2 and 4) and the appearance of two radioactive peptides, with estimated molecular masses of 4-5 and 3-4 kDa (lane 2). These low molecular weight peptides are consistent with the peptides Pro-Asp and Pro-Arg since the hexapeptide Leu-Asp, containing S-labeled Met, would be too small to be detected on a SDS-polyacrylamide gel. The 4-5 and 3-4 kDa peptides contain 16 and 24%, respectively, of the total radioactivity of the undigested protein. The remainder of the radioactivity is presumably associated with the undetectable Leu-Asp hexapeptide. The broad band with weak radioactivity (arrow) migrates between intact cytochrome b(5) and its degradation products and is expressed even in the absence of mRNA coding for cytochrome b(5), showing that it is due to a contamination in the reticulocyte lysate and does not interfere with the studies since radioactive peptides derived from this band cannot be detected on SDS gels. When cytochrome b(5) inserted post-translationally into microsomes was treated with trypsin, a 4-5 kDa protected peptide was formed (lanes 3 and 4). In contrast to cytochrome b(5) translated in the absence of microsomes this peptide contains all of the radioactivity originally present in the intact cytochrome b(5), indicating that both Met and Met of cytochrome b(5) inserted in microsomes are protected from digestion by trypsin and that the membrane-anchor of cytochrome b(5) is in the transmembrane conformation. Digestion of non-microsomal bound cytochrome b(5) in the presence of Tergitol results in the formation of a single 4-5-kDa protected peptide (not shown). The most likely explanation is that detergent binds to cytochrome b(5) near Arg and consequently inhibits hydrolysis of this bond by trypsin. Tergitol could, therefore, not be used to test the accessibility of the COOH terminus of cytochrome b(5) to trypsin upon treatment of the microsomal membrane with this detergent.


Figure 5: Digestion of free and membrane-bound cytochrome b(5) with trypsin. Cytochrome b(5) translated in the absence of microsomes (-mic: lanes 1 and 2) or incubated post-translationally (post mic: lanes 3 and 4) for 90 min with canine pancreas microsomes was digested with trypsin (lanes 2 and 4) and analyzed as described under ``Experimental Procedures'' on a 16.5% Tricine gel. The arrow indicates the position of the contaminating band expressed even in the absence of mRNA coding for cytochrome b(5). PROT. p., protected peptide.



It should be pointed out that, when cytochrome b(5) which was translated in the absence of microsomes is digested, the soluble proteins of the reticulocyte lysate, such as hemoglobin, are present in huge excess relative to cytochrome b(5). In contrast, the reticulocyte lysate proteins, including hemoglobin, are not present during the digestion of membrane-bound cytochrome b(5) since the microsomes have been precipitated by centrifugation and then resuspended in buffer. This difference does not alter the analysis or conclusions about the topology of cytochrome b(5) since the proteases cannot digest the COOH terminus of membrane-bound cytochrome b(5) even at a protease/protein substrate ratio which dramatically increases upon removal of the proteins of the reticulocyte lysate. Furthermore, re-addition of the reticulocyte extract to the microsomal pellet had no influence on the proteolytic pattern of membrane-bound cytochrome b(5) (not shown).

Insertion of Cytochrome b(5)into Microsomes Pretreated with Trypsin

In order to determine whether treatment of microsomes with trypsin would destroy their ability to incorporate the amphipathic cytochrome b(5) molecule, canine pancreas microsomes were pretreated with trypsin and then tested for their ability to post-translationally incorporate cytochrome b(5). Treatment of microsomes with trypsin inactivates the SRP-dependent translocation machinery (Meyer and Dobberstein, 1980) as seen in Fig. 6. Control microsomes process alpha factor to its mature form (Panel A, lane 2) whereas trypsin-pretreated microsomes are incompetent in the translocation of alpha factor (lane 3). The trypsin-pretreated microsomes can, however, still effectively incorporate cytochrome b(5) in a topology which is indistinguishable from the topology resulting from the incorporation of cytochrome b(5) in control microsomes. Cytochrome b(5) can be recovered in the trypsin-treated microsomes after centrifugation in 0.5 M NaCl or at pH 11.5 in sodium carbonate to the same extent as cytochrome b(5) incorporated into control microsomes (Fig. 6B, lanes 1-6). Also the susceptibility of cytochrome b(5) to digestion by CPY (Fig. 6C, lanes 1-6) is unchanged compared to that for cytochrome b(5) inserted in control microsomes, indicating that inactivation of the SRP-dependent translocation machinery by trypsin has no effect on the insertion and topology of cytochrome b(5).


Figure 6: Post-translational insertion of cytochrome b(5) into microsomes pretreated with trypsin. Microsomes were treated with trypsin and assayed for their ability to incorporate alpha factor into the microsomal lumen and to bind cytochrome b(5) as described under ``Experimental Procedures.'' A, alpha factor was synthesized either in the absence of microsomes (lane 1) or co-translationally in the presence of control canine pancreas microsomes (lane 2) or microsomes pretreated with trypsin (lane 3). The translation reactions were analyzed on a 12.5% SDS gel. p-alpha fac, processed alpha factor; u-alpha fac, unprocessed alpha factor. B, cytochrome b(5) was incubated post-translationally with either control (mic: lanes 1-3) or trypsin-pretreated microsomes (t-mic: lanes 4-6). Following precipitation by centrifugation in 0.5 M NaCl (lanes 2 and 5) or in sodium carbonate at pH 11.5 (lanes 3 and 6), the microsomal pellets were electrophoresed and analyzed as described under ``Experimental Procedures.'' C, cytochrome b(5) was incubated post-translationally with control (mic: lanes 1-3) or trypsin-pretreated microsomes (t-mic: lanes 4-6). The cytochrome b(5) containing microsomes were exposed to carboxypeptidase Y (lanes 2, 3, 5, and 6) in the absence (lanes 2 and 5) or presence (lanes 3 and 6) of 0.2% Tergitol as described under ``Experimental Procedures.''



Insertion of the P115A Mutant Cytochrome b(5) into Microsomal Membranes

Pro which is in the middle of the putative transmembrane domain of cytochrome b(5) is capable of isomerizing at the Ile-Pro peptide bond. In the cis conformation a hairpin loop will result while in the trans conformation the membrane-binding domain should exist as a transmembrane alpha-helix with a kink of about 29° at Pro (Richardson and Richardson, 1989; Schmid, 1993; Vergères and Waskell, 1992). In order to investigate whether the ability of Pro to isomerize and hence cause a marked conformational change in the transmembrane domain might influence the ability of cytochrome b(5) to become incorporated into microsomes, Pro was mutated to an alanine. The P115A mutant should have a rigid uninterrupted hydrophobic alpha-helical membrane anchor. The wild type and mutant cytochrome b(5) were synthesized in the reticulocyte cell-free system for 90 min in the presence of canine pancreas microsomes and the cytochrome b(5) containing microsomes were precipitated in the presence of 0.5 M NaCl and subjected to electrophoresis (Fig. 7). In order to have an internal control for the recovery of microsomes, alpha factor was translated in the same reaction mixture with wild type or P115A mutant cytochrome b(5). Quantitation of the autoradiogram shown in Fig. 7by volume scanning densitometry shows that the co-translational insertion of the P115A mutant into microsomes is diminished by a factor of 2 to 3 compared to the wild type. In this assay, 80% of the wild type cytochrome b(5) is inserted in microsomes (Fig. 7, lanes 1 and 2) whereas only 35% of the P115A mutant is recovered in the microsomal fraction precipitated in the presence of a high salt concentration (lanes 3 and 4). The remainder of the P115A mutant protein can be recovered in the supernatant (not shown). Post-translational insertion of the P115A mutant is diminished to a comparable extent (not shown).


Figure 7: Comparison of the co-translational insertion of wild type and P115A mutant cytochrome b(5) into microsomes. The mRNAs coding for alpha factor (lanes 1-4) and either wild type (WT, lanes 1 and 2) or P115A mutant (P115A, lanes 3 and 4) cytochrome b(5) were simultaneously translated in the presence of canine pancreas microsomes (lanes 1 and 3). The solutions were then centrifuged in 0.5 M NaCl and the proteins in the microsomal pellet (lanes 2 and 4) were analyzed by SDS-gel electrophoresis as described under ``Experimental Procedures.''



Apart from this decreased efficiency in binding, the mutation has no observable effect on the topology of cytochrome b(5) in the microsomal membrane as judged by the resistance of the membrane-bound fraction to extraction with 0.5 M NaCl or at pH 11.5 and by the radioactive degradation products formed following digestion with CPY (not shown).

Insertion of the Purified Wild Type Cytochrome b(5) and the P115A Mutant Protein into a Waveguide-supported Planar Lipid Bilayer

The spontaneous binding of the wild type and P115A mutant cytochrome b(5) to a single lipid bilayer consisting exclusively of POPC was investigated (Tretyachenko-Ladokhina et al., 1993). These studies provide information about how the mutant and wild type proteins bind to a bilayer in the absence of proteins which might be involved in the insertion of cytochrome b(5) into the microsomal membrane. Fig. 8clearly shows that the wild type protein binds much more quickly than the mutant protein to the POPC bilayer, indicating that the mutation alters an intrinsic property of cytochrome b(5). The soluble form of cytochrome b(5) lacking the membrane anchor does not significantly bind to the bilayer. (^2)The apparent plateau of the binding of cytochrome b(5) does not reflect saturation of the POPC bilayer but merely the deceleration of the diffusion-limited flux of the protein due to its progressive depletion in the solution near the surface of the bilayer (von Smoluchowski, 1916; Ramsden, 1992). Log-log plots of the data were linear up to 2 h (data not shown), indicating that the bilayer was not saturated. Assuming a molecular mass of 15 kDa and an area of 800-Å^2 per cytochrome b(5) molecule, a deposited mass of 0.01 µg/cm^2 corresponds to an occupancy of 1.4% of the surface of the lipid bilayer (Mathews et al., 1972).


Figure 8: Time course of the binding of purified wild type cytochrome b(5) (WT) and of the P115A mutant (P115A) to a waveguide-supported planar POPC bilayer. Temperature of the measuring chamber was 26.4 ± 0.2 °C. The cytochrome b(5) concentration in the solution above the bilayer was 0.67 µM.




DISCUSSION

Membrane Topology

What are these proteolysis assays telling us about the membrane topology of cytochrome b(5)? Cleavage of membrane-bound cytochrome b(5), radioactively labeled with [S]methionine at positions 125 and 130 with trypsin, generates a 4-5-kDa radioactive COOH-terminal peptide in agreement with a topology in which the heme-binding domain is cytosolic and the COOH-terminal domain is membrane-bound and hence protected from water-soluble proteases. A closer look at the radioactive patterns of the degradation peptides provides information on the topology of the COOH terminus. With cytochrome b(5) in a transmembrane topology, exogenous trypsin would cleave at Lys but not at Arg (see Fig. 1). The COOH-terminal protected peptide would contain 40 amino acids (Pro-Asp) and have a calculated molecular mass of 4.6 kDa. This peptide would also be labeled to the same extent as undigested cytochrome b(5) since it would include both Met and Met. With cytochrome b(5) in a loop configuration, trypsin would cleave at Lys and Arg. The protected peptide would contain 34 amino acids (Pro-Arg) and have a calculated molecular mass of 3.8 kDa. This partially degraded peptide lacking Met would contain 50% of the radioactivity compared to undigested cytochrome b(5). Due to its small size (0.8 kDa) the radioactive COOH-terminal peptide Leu-Asp, containing Met, would not be detected on SDS gel. Finally, digestion of cytochrome b(5) with trypsin in the absence of microsomes would give the same pattern as for the protein bound to microsomes in the loop conformation. Since SDS gels cannot provide accurate molecular weights, especially for low molecular weight peptides, which do not have a statistical distribution of amino acids, it is impossible to differentiate between a transmembrane or a loop conformation solely on the basis of the apparent size of the degraded peptides. However, the size and intensities of the radioactive peptides resulting from digestion of cytochrome b(5) in the absence of microsomes can be compared. The size of the radioactive peptide resulting from digestion of membrane-bound cytochrome b(5) with trypsin (estimated to 4-5 kDa) is larger than for the protein digested in the absence of microsomes (estimated to 3-4 kDa) (see Fig. 5). Also, and in contrast to cytochrome b(5) digested in the absence of microsomes, the proteolytic radioactive peptide of membrane-bound cytochrome b(5) contains all of the radioactivity originally present on undigested cytochrome b(5). These observations are consistent with the pattern expected for the digestion of cytochrome b(5) binding to microsomes in a transmembrane topology.

The transmembrane topology is further supported by the finding that CPY has access to the COOH terminus of cytochrome b(5) in solution but not when incorporated into microsomes unless these microsomes have been treated with detergent. Ozols(1989) found that the COOH-terminal hexapeptide Leu-Asp of cytochrome b(5) incorporated into microsomes in vivo was cleaved by trypsin. The discrepancy between our results and those of Ozols (Ozols, 1989) may be due to the fact that sequencing-grade trypsin was used in the studies reported herein but was not used in the experiments of Ozols. In addition, the relative quantities of the recovered peptide were not reported. Therefore it is not known whether the recovered hexapeptide represented a major or minor fraction of the total cytochrome b(5) population. In summary the proteolysis studies strongly favor a transmembrane topology of cytochrome b(5) but the possibility cannot be totally excluded that the carboxyl terminus of cytochrome b(5) is extraluminal and is sterically hindered from interacting with the proteases unless detergent is present (Jennings, 1989).

Mode of Insertion of Cytochrome b(5)

In keeping with the two modes of binding of cytochrome b(5) to vesicles and to microsomes (see Introduction), we have asked whether the COOH terminus of cytochrome b(5) assumes different topologies when it is inserted during translation or when the protein is added to microsomes following translation. Previous studies have clearly demonstrated that exogenous cytochrome b(5) can be incorporated into microsomes and that it functions in electron transfer (Rachubinski et al., 1980; Okada et al., 1982; Anderson et al., 1983; Vergères et al., 1993) but have not addressed the exact topology of the protein under these conditions. The studies described here demonstrate that, in vitro, both co- and post-translational insertions of cytochrome b(5) result in the same membrane topology as judged by the susceptibility of the protein to CPY. The COOH terminus of cytochrome b(5) can therefore be translocated post-translationally into the lumen of microsomes.

Intracellularly cytochrome b(5) is found almost exclusively in the microsomal membrane (Mitoma and Ito, 1992; D'Arrigo et al., 1993) even though it can apparently spontaneously insert into microsomal membranes in vitro. The ability of cytochrome b(5) to insert into trypsin-pretreated microsomes establishes that cytochrome b(5) does not use a trypsin-sensitive receptor to mediate its microsomal incorporation but does not rule out a non-trypsin-sensitive receptor-mediated mechanism (Borgese et al., 1993).

The population of insoluble cytochrome b(5) might not only be present in our cell-free system but also in vivo. In the light of the function of chaperones in protein folding as well as in their targeting to subcellular locations and insertion into membranes (Craig, 1993; High and Stirling, 1993; Lithgow et al., 1993), it would be interesting to characterize this population in order to understand whether chaperones are involved in the biosynthesis and intracellular targeting of cytochrome b(5).

Function of Pro

Pro in the middle of the transmembrane peptide of cytochrome b(5) does not determine the topology of the protein in the membrane since the P115A mutant cannot be distinguished from wild type cytochrome b(5) with protease and sedimentation assays. Also overexpression of the P115A mutant in the yeast S. cerevisiae induces proliferation of perinuclear membranes, called karmellae, as observed with the wild type protein (Vergères et al., 1993). This mutant also functions in electron transport in the endoplasmic reticulum in a manner similar to that of the wild type protein (Vergères and Waskell, 1992). Since this particular response of the cell, namely karmellae formation, seems to be modulated by the nature of the interaction of cytochrome b(5) with the membrane, it was previously proposed, in agreement with the findings reported here, that the topology of the P115A mutant would be similar to the wild type topology (Vergères et al., 1993). On the other hand replacement of Pro with an alanine decreases the binding of the protein in vitro by a factor 2 to 3 compared to the wild type protein to both microsomal membranes and a POPC bilayer suggesting that Pro may facilitate the insertion of cytochrome b(5) into membranes. The decreased ability of the mutant protein to bind to a membrane can be explained either by a change in an intrinsic property of the protein or an alteration of its interaction with the lipid bilayer. For instance, cytochrome b(5) in solution is in equilibrium between the membrane-insertion competent monomeric and the membrane-insertion incompetent octameric form and replacement of the proline might shift the equilibrium toward the octameric form as indicated by our observation that the P115A mutation renders the COOH terminus of cytochrome b(5) more resistant to proteolysis by trypsin (Calabro et al., 1976; Leto and Holloway, 1979; Greenhut et al., 1993). This interpretation is supported by a recent study demonstrating that mutations in the tryptophan residues 108, 109, and 112 in the membrane-binding domain of cytochrome b(5) (see Fig. 1) have profound effects on the strengths of self- and membrane-association of the protein (Tretyachenko-Ladokhina et al., 1993). Alternatively, Pro might directly facilitate the binding of cytochrome b(5) to bilayers via cis-trans isomerization of the Pro peptide bond. In this respect an attractive two-step model for the insertion of cytochrome b(5) into the microsomal membrane would be that cytochrome b(5) first spontaneously binds to the membrane in a loop conformation with the Ile-Pro bond in the cis configuration. Subsequent isomerization of this bond to the trans configuration would then promote translocation of the carboxyl terminus into the lumen of the endoplasmic reticulum.


FOOTNOTES

*
This research was supported by National Institutes of Health Grant GM 35333 (to L. W.) and by the Commission pour l'encouragement de la recherche scientifique, Switzerland. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Anesthesiology (129), VA Medical Center, 4150 Clement St., San Francisco, CA 94121. Tel.: 415-750-2069; Fax: 415-750-6946.

(^1)
The abbreviations used are: SRP, signal recognition particle; CPY, carboxypeptidase Y; POPC, palmitoyl oleoyl phosphatidylcholine; RNasin, ribonuclease inhibitor; Tricine, N-[1-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; HPLC, high performance liquid chromatography.

(^2)
J. J. Ramsden, G. I. Bachmanova, and A. I. Archakov, unpublished results.


ACKNOWLEDGEMENTS

We thank Drs. Ma and Lingappa for many fruitful discussions.


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