Journal of General Microbiology (1984), 130, 1673-1682.
Printed in Great Britain
1673
Detection of a Membrane-associated Protein on Detached Membrane
Ribosomes in Staphylococcus aweus
By L A R S - A K E A D L E R * A N D S T A F F A N A R V I D S O N
Department of Bacteriology, Karolinska Institutet, S-104 0 I Stockholm, Sweden
(Received 9 November 1983; revised 14 February 1984)
Membrane ribosomes from Staphylococcus aureus which were detached from the membrane by
extraction with the nonionic detergent Triton X-100 retained a protein (MBRP) with a
molecular weight of 60000, which was absent from cytoplasmic ribosomes. MBRP was detected
and quantified by immunological methods. When membrane ribosomes were dissociated into
50s and 30s subunits, MBRP remained associated with the 50s particle. MBRP was found both
on membrane ribosomes and in the cytoplasm in roughly equal amounts. When added to Triton
X-100-solubilized protoplasts, antibodies to MBRP produced immunoprecipitates which contained a complex of MBRP and three other proteins with molecular weights of 71 000,46000 and
41000. Four proteins with the same molecular weights as those of the MBRP complex were
found associated with membrane ribosomes. The proteins of molecular weight 7 1 000, 60000,
46000 and 41 000 seemed to be present in stoichiometrically equivalent amounts in the complex.
INTRODUCTION
Secreted proteins in both eukaryotic (Palade, 1975) and prokaryotic (Baty et al., 1981;
Randall & Hardy, 1977; Varenne et al., 1978) cells are synthesized on membrane-associated
ribosomes. Part of a specific secretion apparatus has been demonstrated in eukaryotic cells,
consisting of a cytoplasmic protein-RNA complex (signal recognition particle, SRP), which
binds to the signal sequence of the nascent secretory protein and induces a translational arrest
Walter & Blobel, 1980, 1982), and a membrane-bound docking protein, which releases the
translation arrest when the polysome reaches the membrane (Meyer et al., 1982). SRP could also
direct the secretion of a prokaryotic exoprotein in a eukaryotic in vitro translation/secretion
system, indicating that a similar mechanism is operating in prokaryotic cells (Muller et al.,
1982). Indirect evidence for a protein-secretion apparatus in bacteria also comes from the
isolation of several classes of secretion-deficient mutants from Escherichia coli, mapping at
different locations on the E. coli chromosome (Wanner et al., 1979; Dassa & Boquet, 1981). The
postulation of a bacterial SRP is also consistent with results recently obtained by Hall et al.
(1983), who found a mutation within the hydrophilic segment of the lambda receptor signal
sequence, which resulted in an early stop in the translation of the lambda receptor RNA.
Recently Horiuchi et al. (1983a, b) and Marty-Mazars et al. (1983) demonstrated a protein with a
molecular weight of 64000 in ribosome-bearing membranes that was covered on the cytoplasmic
surface by the ribosomes. This protein, which was found both in the cytosol and in the
membranes, was suggested to participate in the initiation of protein secretion, as has been
shown for SRP (Horiuchi et al., 1983b).
In the present study the association of membrane proteins with membrane-bound (i.e. exoprotein-synthesizing) ribosomes has been investigated using Staphylococcus aureus. This
organism secretes large amounts of extracellular proteins, which together constitute over 30% of
Abbreviarions: MBRP, membrane-bound ribosome protein ; SRP, signal recognition particle.
0022-1287/84/000l-1589$02.00 0 1984 SGM
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L.-A. ADLER A N D S. ARVIDSON
the total protein synthesis (Abbas-Ali & Coleman, 1977), and it should therefore have a
relatively large number of membrane-associated ribosomes. Our concept was that membranebound ribosomes which were gently dissociated from the membrane with a nonionic detergent
might retain one or more of the membrane proteins involved in the binding of exoproteinsynthesizing ribosomes to the membrane. A similar approach was used by Kreibich et al. (1978),
when they identified two ribosome-binding proteins (ribophorines) in the endoplasmic
reticulum.
METHODS
Bacterial strain and cultivation conditions. Staphylococcus aureus V8 was used throughout this study. Precultures
were grown overnight in Tryptic Soy Broth (Difco). Bacteria from 50 ml of the preculture were used to inoculate
250ml CCY-medium (Arvidson et al., 1972) in a 5-1 baffled shake-flasks, which were incubated for 4 h at 37 "C on
a rotary shaker. At this time 200 pg chloramphenicol ml- (Parke Davis, Pontypool, Gwent, UK) was added and
the culture was poured onto 50ml frozen buffer A (10 mM-Tris/HCl, pH 7.4; 10 mM-magnesium acetate; 50 mMN H,CI) .
All further handling, except for protoplasting, was at 4 "C.
Preparation of protoplasts. Cells were centrifuged at 7000g for lOmin, resuspended in buffer A with 200 pg
chloramphenicol ml- and repelleted. Protoplasts were prepared by incubating the bacteria at 37 "C for 15min in
buffer A (1/10 the original culture volume) containing 1.1 M-sucrose, 50 pg chloramphenicol ml-' and 100 pg
lysostaphin ml- (Schwarz-Mann, Orangeburg, NY, USA). Whole cells and debris were removed by
centrifugation at 5000g for 10 min. Protoplasts were sedimented at 20 OOOg for 30 min and washed once in buffer
A containing 1 . 1 M-Sucrose and 50 pg chloramphenicol ml-l.
Preparationof cytoplasmic and membrane-bound ribosomes. The protoplasts were lysed by dilution in 30 ml10 mMNa2HP0,, 10 mM-magnesium acetate, 50 mM-KC1, pH 7.4 (pH was adjusted by adding 1 M-HCI).
The membrane suspension was sonicated twice for 10 s with an MSE 100 W ultrasonic disintegrator, and the
membranes were pelleted at 27000g for 30min. The supernatant was centrifuged at 150000g for 2 h in a
Beckman SW 50.1 rotor to sediment the cytoplasmic ribosomes. The membrane pellet was washed twice in
phosphate buffer and suspended in buffer A containing 4%(w/v) Triton X-100 (Sigma) to solubilize the membrane
and release the membrane-associated ribosomes. After centrifugation at 15 OOOg for 10 min to remove large
particles the ribosomes were collected by ultracentrifugation at 150 OOOg for 2 h. These ribosomes will be referred
to as membrane ribosomes.
Both membrane and cytoplasmic ribosomes were further purified by chromatography on Sepharose 2B
(Pharmacia) as described by Smith et al. (1978). Fractions of the ribosome peak were pooled and pelleted at
150000g for 2h.
Preparation of membrane antigen. Protoplasts were prepared as described, except for the omission of
chloramphenicol. The protoplasts were lysed by suspension in buffer B (10 ~ M - N ~ ~ H 10
P mM-magnesium
O~,
acetate, pH 7.4), containing 1 pg DNAase ml- (Sigma) and 1 pg RNAase ml- (Sigma). Membranes were
0 ~mM-Na2-EDTA,
,
pH 7.4;
washed twice in buffer B, then twice in phosphate/EDTA buffer ( 1 0 m ~ - N a ~ H P 10
pH was adjusted by adding 1 M-NaOH) and finally once in phosphate/EDTA buffer containing 0.5 M-KCI.
Preparation of antisera. Freund's complete adjuvant containing 5 mg protein was injected into rabbits
subcutaneously in the neck in five portions. After 3 weeks, and then at 5 week intervals, the same amount of
protein in Freund's incomplete adjuvant was injected intramuscularly, and 10 d after each intramuscular injection,
30-40ml of blood was collected from the ear vein.
Antisera towards individual antigens were prepared by injecting isolated immunoprecipitates from crossed
immunoelectrophoresis gels. The precipitates were cut out and homogenized in adjuvant with a Teflon pestle
homogenizer. The precipitates from four gels were injected each time, as described above.
Antibodies were purified according to Harboe & Ingild (1973), except for omission of the final chromatography
step. Concentration was performed in a Minicon-B cell (Amicon Corp., Lexington, Mass., USA) to a protein
concentration of 50-100 mg ml-I.
SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Proteins to be analysed were denatured by boiling for
2 min in 2% (w/v) sodium dodecyl sulphate (SDS), 0.1 M-DL-dithiothreitol, 0.08M-Tris/sulphate buffer, pH 6.1,
10% (w/v) glycerol and 0.005% (w/v) bromophenol blue. Electrophoresis was performed at 35 mA for 4 h, in a slab
gel apparatus described by Studier (1973) in a 7.5-15% (w/v) polyacrylamide gradient gel using a discontinuous
buffer system according to Neville (1971), with the separation gel buffer at pH 9.18.
Alternatively, electrophoresis was done in a Protean dual-slab cell (Bio-Rad) using the buffer system of Laemmli
(1970). Samples were electrophoresed for 5 h at 15 mA in a 12% (w/v) polyacrylamide gel at pH 8.8.
Crossed immunoelectrophoresis (CIE).CIE was done as described by Owen & Salton (1975) in 1 % (w/v) Sea Kern
Me agarose (Marine Colloids Div., FMC Corporation, Rockland, Me., USA) containing 0.024 M-barbital buffer,
pH 8-6,and 1 % (w/v) Triton X-100. Gels (3.5 ml) were cast on 5 x 5 cm glass plates. Samples were applied to wells
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with 4 mm diameter and electrophoresis was at 5 V cm- for 80 min in a watercooled cell. An agarose strip of
about 10 x 50 mm was retained, and the rest of the gel was replaced with antibody-containing gel. Electrophoresis
in the second dimension was performed at 1.5 Vcm-l for 14-18 h. The gels were stained for protein with
Coomassie brilliant blue R-250.
SDS-PAGEICIE. The electrophoresis was done essentially as described by Chua & Blomberg (1979). Polyacrylamide strips (5 x 180 mm) with separated proteins were cut out from SDS-PAGE gels and placed on a glass plate
(100 x 200 mm). The proteins in the polyacrylamide gel were electrophoresed through an agarose gel containing
sodium deoxycholate and Lubrol PX (Sigma) into an antibody-containing gel with 4% (w/v) polyethyleneglycol.
Electrophoresis was run at 1.5 V cm-' overnight, and gels were stained as before.
Quant8cation of antigens by rocket immunoelectrophoresis.The amount of MBRP in various subcellular fractions
was determined by rocket immunoelectrophoresis according to Weeke (1973), with the gel containing 1% (w/v)
Triton X-100. Each sample was quantified by cutting out paper copies of rockets and weighing them.
Analysis of MBRP on rapidly prepared membrane ribosomes. Protoplasts from 50 ml culture were suspended in
50 ml buffer A with 0.2 pg DNAase ml- (RNAase-free, Worthington), and centrifuged through a 10 ml cushion
of 1 M-sucrose in buffer A at 27000g for 40 min. The pellet was solubilized in 5 ml buffer A with 2% (w/v) Triton
X-100, and insoluble material was removed by centrifugation at 27000g for 20 min. The supernatant was further
centrifuged at 150000g for 45 min and 0.1 ml of the suspended pellet was run in a 15-30% (w/v) sucrose gradient at
150000g for 80min. Fractions of 12 drops were collected from the bottom of the tube, and absorption was
measured at 260 and 280 nm. Fractions were dialysed against distilled water, lyophilized and analysed by rocket
immunoelectrophoresis against MBRP-antiserum, as described.
Radiolabelling of total protein. To obtain satisfactory incorporation of radiolabelled amino acids, cells were
grown in a synthetic medium. Cells from 10 ml preculture were inoculated into 50 ml AAM-medium (Rudin et al.,
1974), with the following modifications: the pH was 7.2, and glycine and serine were added to a final
concentration of 500 pg ml- I each. After 4 h cultivation, 25 pCi (925 kBq) [U-14C]protein hydrolysate
(Amersham) was added, and the culture was grown for another 2 h.
Immunoprecipitation. Radiolabelled protoplasts were solubilized in PBS buffer (0-015 M-phosphate, 0.83 MNaCl, pH 7.2) with 4% (w/v) Triton X-100. Debris was removed by centrifugation at 20000g for 20min.
Antibodies (200 pl) were coupled to 50 pl protein-A Sepharose CL-4B (Pharmacia) in PBS buffer for 1 h at room
temperature with agitation. The Sepharose was washed three times in PBS buffer before adding the solubilized
protoplasts. The mixture was agitated for 1 h at 4°C in the presence of 1 mhl-phenylmethylsulphonylfluoride
(Sigma). After washing eight times with 1 ml PBS, the pelleted sample was denatured in 40 1.11 preparation buffer
for SDS-PAGE electrophoresis as described.
Autoradiography. Fluorography with sodium salicylate (Merck) was performed as described by Chamberlain
(1979). Gels were subjected to autoradiography for 1 week using Kodak XAR-5 film.
Assays. RNA was determined by the orcinol method (Herbert et al., 1971) using yeast RNA (Sigma) as
standard. Protein concentrations were determined by the method of Lowry, with bovine serum albumin as
standard. Malate dehydrogenase was assayed as oxaloacetate-dependent oxidation of NADH by the method of
Yoshida (1965). Succinate dehydrogenase was determined as described by Hederstedt et al. (1979) using the
synthetic electron acceptor 2,6-dichloroindophenol with phenazine methosulphate as electron carrier.
RESULTS
Comparison of cytoplasmic and membrane ribosomes by CIE
The antiserum raised against the highly purified membranes gave a maximum of
approximately 40 visible immunoprecipitates when run against Triton X-100-solubilized S .
aureus membranes (Fig. 1 c). The antigen contained less than 1 % RNA relative to protein and
was therefore considered as essentially free of ribosomes. Membrane ribosomes which were
released from the membranes by Triton X-100 and then purified by repeated ultracentrifugation
and Sepharose 2B chromatography were shown to retain at least one membrane antigen
detected by CIE against the membrane antiserum (Fig. 1b). This antigen (MBRP) was released
from the ribosomes by 0.5 M-KClor with 1% (w/v) deoxycholate in buffer A. No MBRP was
found on cytoplasmic ribosomes purified by the same procedures (Fig. 1 a). A monospecific antiserum against MBRP was obtained by immunizing rabbits with the immunoprecipitates from
CIE gels similar to those seen in Fig. 1(b). CIE of Triton X-100-solubilized protoplasts against
this antiserum gave only one visible immunoprecipitate as seen in Fig. 1 ( d ) . This precipitate
was identical to that obtained with the high-salt or deoxycholate wash from the membrane
ribosomes, as revealed by CIE of a mixture of both antigens against the MBRP antiserum (data
not shown).
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L . - A . ADLER A N D S. ARVIDSON
Fig. 1 . CIE of proteins released by 1 % sodium deoxycholate from equal amounts of (a) cytoplasmic and
(b)membrane ribosomes, using an antiserum against ribosome-free membranes. (c, d) CIE of Triton X100-solubilized protoplasts against the membrane antiserum (c) and the monospecific MBRP
antiserum (4.
Sucrose gradient Centrifugation of membrane ribosomes
Membrane ribosomes were subjected to sucrose gradient centrifugation in ribosomepreserving buffer A, and each fraction was analysed for MBRP by rocket immunoelectrophoresis against the monospecific MBRP antiserum. MBRP closely followed the sedimentation
pattern of the ribosomes. Because the gradient was deliberately overloaded in order to be able to
detect MBRP, a typical ribosome/polysome sedimentation profile was not obtained (data not
shown). However, that the A,,,-absorbing material in the peak fractions represented ribosomes
was shown in parallel experiments where the starting material was treated with RNAase before
the gradient centrifugation (data not shown) or where the magnesium concentration during
sucrose gradient centrifugation was lowered, thereby dividing the ribosomes into their subunits
(Fig. 2). Dissociation of the ribosomes into 50s and 30s subunits resulted in a change in the
position of MBRP which now sedimented together with the 50s subunit (Fig. 2). This indicated
that MBRP was really bound to the ribosomes and did not simply co-sediment with them. The
fact that MBRP sedimented slightly faster than the 50s peak indicated that MBRP was not
bound to every 50s particle, and that particles associated with MBRP were heavier (see below).
Analysis of cytoplasmic and membrane ribosomes by SDS-PAGE
SDS-PAGE of membrane ribosomes and cytoplasmic ribosomes revealed several qualitative
and quantitative differences. At least four proteins were missing from the cytoplasmic ribosome
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Membrane ribosomes in S . aureus
0.8
1677
50s
il
0.7
0.6
0.5
0
m
pc" 0.4
0.3
0.2
0.1
Top of tube
Bottom of tube
Fraction no.
Fig. 2
-Fiu
- ~ 3-..
-----__-- --_-_-
Fig. 3
------ -.--..-..---
Sedimentatinn n a t t e r n nf memhrane-hniind
rihncnmec and MRRP in .....---..ciicrncc D
uradient
*------------1
.
1
-
I--_-_----_----_-
1.-1--*
1
.
1
centrifugation: 0,
absorbance at 280 nm; @, relative amount of MBRP; 8 A280units of ribosomes
were suspended and centrifuged in buffer A with a low Mg2 concentration (1 mM).
+
Fig. 3. SDS-PAGE of (a) cytoplasmic and (b)membrane ribosomes. Samples containing 40 pg protein
were applied to the gel, and electrophoresis was performed according to Neville (1971). Molecular
weight markers were bovine serum albumin (68 000), ovalbumin (43000), carbonic anhydrase (29000),
trypsin inhibitor (20 100) and lysozyme (14300), indicated by pointers.
preparation, and three proteins were present in significantly lower amounts (Fig. 3). From this
experiment it could not be determined which protein was MBRP. SDS-PAGE/CIE of
membrane ribosomes against the MBRP antiserum showed one single immunoprecipitate of a
protein with an apparent molecular weight of 60000 (see Fig. 4). No immunoprecipitate was
seen with proteins from cytoplasmic ribosomes (data not shown). The molecular weight of
MBRP was confirmed by immunoprecipitation of a high-salt wash of radiolabelled membrane
ribosomes. One single band with a molecular weight of 60000 was obtained (data not shown).
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L.-A. ADLER A N D S. ARVIDSON
I
I
43 000
68 000
I
29 000
I I
20 100
14 300
Fig. 4. SDS-PAGE/CIE of membrane ribosomes (60 pg protein) against the MBRP antiserum. The
separation in the first dimension was in a 7.5-15% SDS-PAGE gel according to Neville (1971).
Molecular weight standards were as in Fig. 3.
Immunoprecipitation of MBRP from Triton X-100-solubilized radiolabelled protoplasts
When 4C-labelled protoplasts were solubilized with Triton X- 100 and immunoprecipitated
with protein A-coupled antibodies against MBRP, four peptides with apparent molecular
weights of 71 000,60000 (=MBRP), 46000, and 41 000 were recovered (Fig. 5a). Since [U-14C]protein hydrolysate was used for radiolabelling, it could be concluded from the autoradiogram
that these proteins appeared at approximately equal amounts in the precipitate. The same four
proteins seemed to be present on rapidly prepared membrane ribosomes. (Fig. 5 b). As judged
from the staining, these proteins also appeared in approximately equimolar amounts.
Cellular distribution of MBRP
The distribution of MBRP between the cytoplasm and the membrane is represented in Table
1. Of the total MBRP, 30-32% was found in the cytoplasmic fraction after osmolysis in a
ribosome-preserving buffer and 40-43 % in the membrane fraction. Sonication of the
membranes in a buffer with a low Mg2 concentration (1 mM) resulted in the release of 20-33 %
of the total MBRP, leaving only 10-2074 in the membranes. This treatment also resulted in the
release of most of the ribosomes, measured as RNA. Sonication released only 7% of the
membrane-bound enzyme succinate dehydrogenase, indicating that the integrity of the
membranes was not seriously disturbed. After sonication, the membrane fraction was almost
completely free of malate dehydrogenase, which is a cytoplasmic enzyme.
Centrifugation of the cytoplasmic fraction at 105000g for 2 h to sediment the ribosomes
revealed that 15-18% of the MBRP in the cytoplasm sedimented together with the ribosomes.
The total recovery of MBRP in the subcellular fractions was only about 75% of that found in
Triton X-100-solubilized protoplasts.
+
DISCUSSION
Membrane-bound ribosomes which were detached by solubilization of the membrane with
Triton X-100 retained one protein (MBRP) which was detected with an antiserum against ribosome-free membranes (Fig. lb). This supported the hypothesis that this protein was a
component of a protein-secretion apparatus that remained associated with the ribosomes after
gentle extraction of the membrane with nonionic detergent.
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Membrane ribosomes in S . aureus
Fig. 5. (a) Autoradiogram of 14C-labelled proteins precipitated with the MBRP antiserum. The
immunoprecipitates were separated by SDS-PAGE according to Laemmli (1970). (b) SDS-PAGE of
the peak fraction from sucrose gradient centrifugation of rapidly prepared membrane ribosomes.
Electrophoresis was performed according to Neville (1971). Molecular weight standards were as in
Fig. 3.
Table 1. Distribution of MBRP relative to a cytoplasmic enzyme, malate dehydrogenase, and to a
membrane enzyme, succinate dehydrogenase
The amount of MBRP is expressed relative to the total amount in Triton X-100-solubilizedprotoplasts.
Cytoplasmic fraction
Membrane fraction
Membranes after sonication
at 1 mM-Mg2+
Malate
dehydrogenase
Succinate
dehydrogenase
0.86
0.14
0.02
<0.01
0.99
0.92
MBRP
0.30-0-32
0-40-0.43
0*10-0*20
Marty-Mazars et al. (1 983) recently identified six proteins in the ribosome-bearing domains of
Bacillus subtilis membranes, which all sedimented together with the ribosomes after extraction
of the membranes by Triton X-100, and at least one of which was considered to be involved in
protein secretion (Horiuchi et al., 1983b). MBRP, which had an apparent molecular weight of
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L.-A. ADLER A N D S. ARVIDSON
60000 in SDS-PAGE, was attached to the membrane ribosomes even after extensive washing
with Triton X-100 and subsequent Sepharose 2B chromatography and sucrose gradient
centrifugation. Since MBRP was not found on equally purified cytoplasmic ribosomes we
assumed that MBRP was specifically associated with membrane ribosomes and might therefore
be part of a protein-secretion apparatus. Sucrose gradient centrifugation of dissociated
ribosomes indicated that MBRP was bound to the 50s subunit (Fig. 2). This sedimentation
profile also indicated that 50s subunits with MBRP sedimented slightly faster than the 50s
subunit alone. This was consistent with the finding that MBRP was associated with three other
proteins in roughly equimolar amounts (Fig. 5 b), which together would increase the mass of the
50s particle by approximately 10%. The association of MBRP with the 50s particle further
supports the idea that it may be engaged in protein secretion, since the nascent peptide exit site
and also the membrane-binding site are located on the 50s subunit (Bernabeu & Lake, 1982).
The fact that MBRP was originally detected on membrane ribosomes with an antiserum
raised against ribosome-free membranes suggested that MBRP was a membrane protein. However, when the subcellular distribution was investigated we found that large amounts of MBRP
were present both in the cytoplasm and in complexed membranes (i.e. membranes including
ribosomes). Most of the MBRP in the membranes was associated with the ribosomes and was
released together with them upon sonication at low Mg2 concentration (Table 1) or extraction
with Triton X-100 (quantitative data not shown). In contrast, MBRP in the cytosol was not
associated with the ribosomes, although a minor part (15-18 %) sedimented together with the
ribosomes at 150000g (Table 1). Whether this portion of MBRP was really bound to the
ribosomes was not tested. However, MBRP sedimenting with cytoplasmic ribosomes
disappeared when the ribosomes were purified by Sepharose chromatography, indicating a
much weaker association than with membrane ribosomes.
The presence of MBRP both on membrane ribosomes and in the cytoplasm suggests that
MBRP may participate in protein secretion in a way similar to that of the signal recognition
particle (SRP) of eukaryotic cells which oscillates between the ribosomes and the membranes
(Walter & Blobel, 1982). Horiuchi et al. (1983a, b) have recently found a protein (CM-protein,
mol. wt. 64000) in B. subtilis membranes which was protected from proteolysis by the ribosomes,
and which was also present in large amounts in the cytoplasm. A similar distribution was also
found for a protein (SecA, mol. wt 92000) involved in secretion in E. coli (Oliver & Beckwith,
1982).
A very interesting finding was that antibodies against MBRP precipitated a complex of four
different proteins from Triton X- 100-solubilized protoplasts (Fig. 5 a). Since the antiserum
against MBRP was prepared by immunizing rabbits with immunoprecipitates cut out from CIE
gels (see Fig. 1b), it was possible that the antigen (i.e. immunoprecipitate) contained all these
four proteins, and hence the MBRP antiserum was not monospecific. However, immunoprecipitation of the antigen, used for preparation of the CIE gels, with the final MBRP antiserum
yielded only one protein band on SDS-PAGE with an apparent molecular weight of 60000 (data
not shown). This antiserum was raised against the material released by sodium deoxycholate
from purified membrane-ribosomes. It could thus be concluded that the MBRP antiserum was
monospecific, and that most of the MBRP in the cell was complexed with three other proteins.
Analysis of rapidly prepared membrane-bound ribosomes by SDS-PAGE revealed the
presence of four proteins in roughly equimolar amounts with the same molecular weight as those
of the MBRP-complex (Fig. 5b). Since the polypeptides of molecular weight 71 000,46000 and
41 000 were absent on extensively purified membrane ribosomes (Fig. 3b) it may be concluded
that MBRP was more firmly bound to be ribosomes than to the other three proteins of the
MBRP-complex, and that the complex was probably bound to the ribosomes via the MBRP.
Horiuchi et al. (1983a) reported that antibodies to a membrane protein (mol. wt 64000) that
was covered by secreting ribosomes immunoprecipitated a complex of several proteins. Antibodies to the staphylococcal MBRP (mol. wt 60000) gave, with Triton X-100-solubilized
protoplasts of B. subtilis, an immunoprecipitate which contained four proteins with apparent
molecular weights of 64000,62000,43 000 and 40000 (Adler & Arvidson, 1984). We believe that
the protein of molecular weight 64000 studied by Horiuchi et al. (1983a, b) corresponds to the
+
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MBRP described here, and that the complex they observed is analogous to the MBRP-complex.
Further work will be required to determine the function of this complex in protein secretion.
We thank Berit Lindholm and Agneta Wahlquist for their expert technical assistance. This work was supported
by the Swedish Medical Research Council, grant no. 4513.
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