Microsomal cytochrome b
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
is
synthesized on cytosolic ribosomes and post-translationally inserted
into the microsomal membrane independently of signal recognition
particle (SRP) (
)(Rachubinski et al., 1980; Bendzko et al., 1982; Okada et al., 1982; Anderson et
al., 1983). The conclusion that cytochrome b
is inserted post-translationally into microsomes was based on the
finding that cytochrome b
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
. The COOH-terminal membrane-binding domain
(Ser
-Asp
) of cytochrome b
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
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
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
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
was in the loop conformation
(Arinc et al., 1987). In microsomes, cytochrome b
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
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
, 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
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
, we have expressed
cytochrome b
in a cell-free system and used
proteolytic enzymes to probe the location of the carboxyl terminus of
cytochrome b
inserted either co- or
post-translationally into membranes. Herein it is reported that the
transmembrane domain of cytochrome b
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
-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
to be incorporated co- and or
post-translationally into membranes. Pro
was therefore
mutated to an alanine which presumably generated a rigid
-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
into the membranes.
EXPERIMENTAL PROCEDURES
Materials
The gene coding for rat liver cytochrome b
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
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 Si
Ti
O
incorporating a diffraction grating were obtained from Artificial
Sensing Instruments AG (Zürich, Switzerland).
In Vitro Transcription of Cytochrome b
Genes
The genes coding for the wild type cytochrome b
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
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
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
(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
.
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
factor were prepared by expressing 0.1 µg of the mRNA coding
for
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
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
to the
Various Microsomal Membranes
For post-translational insertion of cytochrome b
into microsomes, 50 µl of a solution in
which cytochrome b
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
. 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
into
microsomes, insoluble cytochrome b
could not been
removed by centrifugation at 100,000
g since it
coprecipitates with microsomes.5 µl of a translation reaction
mixture, in which cytochrome b
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
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
was added to canine pancreas microsomes in which
factor had previously been inserted co-translationally as an
internal standard. Alternatively the mRNAs coding for
factor and
cytochrome b
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
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
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
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
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
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
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
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
was analyzed, 12.5% glycine gels were
used (Laemmli, 1970). The molecular weight of the expressed cytochrome b
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
,
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
binding to microsomes was corrected for the amount of microsomes
lost during the centrifugation. Recovery of microsomes after
centrifugation was determined by using processed
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
in Yeast
Expression of wild type cytochrome b
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
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
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
(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
, 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
DNA coding for the membrane-bound form of rat
liver cytochrome b
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
(17 kDa) is
expressed (Fig. 2, lane 2). In the absence of
cytochrome b
mRNA, the protein is not synthesized (lane 1).
Figure 2:
Expression of cytochrome b
in a cell-free system. mRNA coding for cytochrome b
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
mRNA. Lane 2, translation in
the presence of mRNA coding for cytochrome b
.
Cytochrome b
Is Inserted into Microsomal
Membranes both Co- and Post-translationally
In order to
determine whether cytochrome b
was inserted into
microsomal membranes co- and/or post-translationally, cytochrome b
was either translated in the presence of
microsomes or added to microsomes following completion of translation.
The amount of cytochrome b
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
g. Microsomes previously
loaded with the yeast mating pheromone,
factor, served as an
internal standard for microsomal recovery. Fig. 3(lane
1) demonstrates that cytochrome b
is readily
translated in the absence of microsomes. When the translation mixture
is centrifuged at 100,000
g in the presence of 0.5 M NaCl (lane 2), 5-10% of the cytochrome b
is found in the pellet. This fraction is
referred to as the insoluble cytochrome b
and
presumably represents cytochrome b
aggregated with
itself and/or other macromolecules in the translation mixture. Since
the insoluble cytochrome b
population increased
with time and coprecipitates with microsomes, the binding of cytochrome b
to microsomes was assayed within hours of
completion. In addition, post-translational insertion of cytochrome b
into microsomes was studied with a translation
reaction in which the fraction of insoluble cytochrome b
was removed by centrifugation prior to incubation with
microsomes.
Figure 3:
Binding of cytochrome b
to microsomal membranes in the presence of 0.5 M NaCl
and sodium carbonate, pH 11.5. Cytochrome b
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
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
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
present in the translation reaction (lanes 1, 4, and 7) with the fraction associated with
the pellet after centrifugation at 100,000
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
factor (p-
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-
fac, unprocessed
factor (18.6
kDa).
When cytochrome b
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
could be retrieved in the microsomes in the 100,000
g pellet in 0.5 M NaCl. This is slightly less than the 85%
recovery of the internal standard, which contained glycosylated
factor in the microsomal lumen. Luminal glycosylated
factor also
served as a control for the integrity of the vesicles. The yeast
pheromone,
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
was inserted into the microsomal membrane as a
peripheral or integral membrane protein, microsomes containing
S-labeled cytochrome b
and
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
factor (Morimoto et
al., 1983). High salt (0.5 M NaCl) did not release either
cytochrome b
or
factor, indicating that
cytochrome b
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
remained associated with the microsomal membrane
indicating it had become an integral membrane protein while
factor was completely released from the microsomal lumen (Fig. 3, lanes 6 and 9).
Interestingly,
cytochrome b
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
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
is incorporated into microsomes by a
process that does not require SRP (Anderson et al., 1983).
Topological Analysis of Microsomal Cytochrome
b
The binding assays described above indicate that
cytochrome b
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
, microsomes containing the newly
translated
S-labeled cytochrome b
were subjected to treatment with water-soluble proteases which
were assumed to be unable to enter the microsomal lumen. Cytochrome b
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
(amino acids
1-97) lacking the membrane-binding domain and methionines 125 and
130, did not result in the labeling of cytochrome b
with [
S]methionine, indicating that
Met
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
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
peptides from
microsomes would provide evidence for a transmembrane spanning membrane
anchor. If proteases completely or partially remove
S-labeled cytochrome b
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
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
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
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
is not accessible to CPY (lanes 3 and 4) and indicating that the hydrophobic peptide of cytochrome b
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
is digested with non-purified CPY, presumably
containing endoproteases, this protection is substantially decreased
and results in the degradation of cytochrome b
.
The observation that sequencing-grade CPY digests cytochrome b
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
(lane 5).
Figure 4:
Digestion of free and membrane-bound
cytochrome b
with carboxypeptidase Y. Cytochrome b
translated in the absence of microsomes
(-mic: lanes 1 and 2), cytochrome b
to which canine microsomes were added after
translation (post mic: lanes 3-5) and cytochrome b
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
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
(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
g. This insoluble cytochrome b
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
and does not alter
the interpretation of the results.
Treatment of unprocessed,
extramicrosomal
factor with CPY reduces the radioactive labeling
associated with the protein as analyzed by autoradiography of
SDS-polyacrylamide gels, whereas processed
factor, which is
translocated into the lumen of microsomes, is protected from
degradation by the protease (not shown) indicating that
factor is
a substrate for CPY and that the microsomal membrane is impermeable to
the protease.
Digestion of Free and Membrane-bound Cytochrome b
with Trypsin
In view of our observation that the COOH
terminus of cytochrome b
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
(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
is in the hairpin loop conformation. We have
therefore analyzed the relative susceptibilities of the COOH terminus
of cytochrome b
toward proteolysis by trypsin, in
the absence of microsomes as well as for membrane-bound cytochrome b
. The pattern of the radioactive 4-5 kDa
hydrophobic tryptic peptide should be the same for cytochrome b
digested in the absence of microsomes and for
cytochrome b
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
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
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
and its degradation products and is expressed even in the absence
of mRNA coding for cytochrome b
, 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
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
translated in the absence of microsomes this peptide contains all
of the radioactivity originally present in the intact cytochrome b
, indicating that both Met
and
Met
of cytochrome b
inserted in
microsomes are protected from digestion by trypsin and that the
membrane-anchor of cytochrome b
is in the
transmembrane conformation. Digestion of non-microsomal bound
cytochrome b
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
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
to trypsin upon treatment
of the microsomal membrane with this detergent.
Figure 5:
Digestion of free and membrane-bound
cytochrome b
with trypsin. Cytochrome b
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
. PROT. p., protected
peptide.
It should be pointed
out that, when cytochrome b
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
. In contrast, the
reticulocyte lysate proteins, including hemoglobin, are not present
during the digestion of membrane-bound cytochrome b
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
since the proteases cannot digest the COOH terminus of
membrane-bound cytochrome b
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
(not shown).
Insertion of Cytochrome b
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
molecule,
canine pancreas microsomes were pretreated with trypsin and then tested
for their ability to post-translationally incorporate cytochrome b
. Treatment of microsomes with trypsin
inactivates the SRP-dependent translocation machinery (Meyer and
Dobberstein, 1980) as seen in Fig. 6. Control microsomes process
factor to its mature form (Panel A, lane 2) whereas
trypsin-pretreated microsomes are incompetent in the translocation of
factor (lane 3). The trypsin-pretreated microsomes can,
however, still effectively incorporate cytochrome b
in a topology which is indistinguishable from the topology
resulting from the incorporation of cytochrome b
in control microsomes. Cytochrome b
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
incorporated into control microsomes (Fig. 6B, lanes 1-6). Also the susceptibility of
cytochrome b
to digestion by CPY (Fig. 6C, lanes 1-6) is unchanged compared to
that for cytochrome b
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
.
Figure 6:
Post-translational insertion of cytochrome b
into microsomes pretreated with trypsin.
Microsomes were treated with trypsin and assayed for their ability to
incorporate
factor into the microsomal lumen and to bind
cytochrome b
as described under
``Experimental Procedures.'' A,
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-
fac, processed
factor; u-
fac, unprocessed
factor. B, cytochrome b
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
was incubated post-translationally
with control (mic: lanes 1-3) or trypsin-pretreated
microsomes (t-mic: lanes 4-6). The cytochrome b
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
into Microsomal Membranes
Pro
which is in
the middle of the putative transmembrane domain of cytochrome b
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
-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
to
become incorporated into microsomes, Pro
was mutated to
an alanine. The P115A mutant should have a rigid uninterrupted
hydrophobic
-helical membrane anchor. The wild type and mutant
cytochrome b
were synthesized in the reticulocyte
cell-free system for 90 min in the presence of canine pancreas
microsomes and the cytochrome b
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,
factor was
translated in the same reaction mixture with wild type or P115A mutant
cytochrome b
. 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
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
into microsomes. The mRNAs coding for
factor (lanes 1-4) and either wild type (WT, lanes 1 and 2) or P115A mutant (P115A, lanes 3 and 4) cytochrome b
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
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
and the P115A Mutant Protein into a Waveguide-supported Planar
Lipid Bilayer
The spontaneous binding of the wild type and P115A
mutant cytochrome b
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
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
. The soluble form of cytochrome b
lacking the membrane anchor does not significantly bind to the
bilayer. (
)The apparent plateau of the binding of cytochrome b
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
per cytochrome b
molecule, a deposited mass of 0.01 µg/cm
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
(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
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
? Cleavage of membrane-bound cytochrome b
, 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
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
since it would include
both Met
and Met
. With cytochrome b
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
. 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
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
in the absence of
microsomes can be compared. The size of the radioactive peptide
resulting from digestion of membrane-bound cytochrome b
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
digested in the absence of microsomes, the
proteolytic radioactive peptide of membrane-bound cytochrome b
contains all of the radioactivity originally
present on undigested cytochrome b
. These
observations are consistent with the pattern expected for the digestion
of cytochrome b
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
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
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
population. In summary the
proteolysis studies strongly favor a transmembrane topology of
cytochrome b
but the possibility cannot be totally
excluded that the carboxyl terminus of cytochrome b
is extraluminal and is sterically hindered from interacting with
the proteases unless detergent is present (Jennings, 1989).
Mode of Insertion of Cytochrome b
In
keeping with the two modes of binding of cytochrome b
to vesicles and to microsomes (see Introduction), we have asked
whether the COOH terminus of cytochrome b
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
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
result in the same membrane topology as judged by the
susceptibility of the protein to CPY. The COOH terminus of cytochrome b
can therefore be translocated
post-translationally into the lumen of microsomes.Intracellularly
cytochrome b
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
to insert into trypsin-pretreated microsomes
establishes that cytochrome b
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
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
.
Function of Pro
Pro
in
the middle of the transmembrane peptide of cytochrome b
does not determine the topology of the protein in the membrane
since the P115A mutant cannot be distinguished from wild type
cytochrome b
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
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
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
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
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
(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
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
into the microsomal membrane would be that cytochrome b
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.