From the Department of Microbiology and Molecular Genetics and the
Department of Cell Biology, Harvard Medical School,
Boston, Massachusetts 02115 and § Universität
Konstanz, Fakultät für Biologie, Postfach 5560, D606,
D-78434 Konstanz, Germany
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ABSTRACT |
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The assembly of integral membrane proteins is determined by features of these proteins and the protein translocation apparatus. We used alkaline phosphatase fusions to the membrane protein MalF to investigate the role of the protein translocation machinery in the arrangement of proteins in the cytoplasmic membrane of Escherichia coli. In particular, we studied the effects of prlA mutations on membrane protein topology. These mutations lie in the secY gene, which encodes a core component of the protein translocation apparatus. We find that the topology of some of the fusion proteins is changed and, in one case, is completely inverted in prlA mutants. We discuss the mechanism of prlA-mediated export and the role of the protein translocation apparatus in contributing to membrane protein topology.
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INTRODUCTION |
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Assembly of cytoplasmic membrane proteins depends on features of the protein itself and on the cell's protein translocation machinery. Long hydrophobic stretches in membrane proteins, averaging around 20 amino acids, can act as export signals promoting the translocation of hydrophilic domains across the membrane. These stretches themselves remain embedded in the membrane, acting as anchors and sometimes contributing to the protein's function. Such hydrophobic stretches also act as stop transfer sequences when they follow a hydrophilic domain that has been translocated across the membrane. Thus, these transmembrane sequences can be oriented with either their amino terminus or their carboxyl terminus in the cytoplasm.
Features of transmembrane proteins that determine their membrane topology include 1) basic amino acids in hydrophilic domains that result in a cytoplasmic location for that domain (1-3); 2) amphipathic helices in hydrophilic domains that may also contribute to anchoring those domains in the cytoplasm (4); 3) rapid folding of a hydrophilic domains (folding in the cytoplasm may prevent export of a domain, while folding of a domain in the periplasm may ensure the location of that domain (5, 6)); 4) salt bridges (or other linkages) between transmembrane segments may maintain their transmembrane configuration (7); and 5) the lipid composition of the membrane may influence topology.1
The efficient assembly of many membrane proteins also depends on the cell's protein translocation machinery. The assembly of the E. coli leader peptidase into the membrane and the translocation of a large hydrophilic domain of the cytoplasmic membrane protein, MalF, are defective in sec mutants that alter the translocation machinery (8, 9). For MalF, effects on assembly occur only when the defects in the secretion apparatus are severe. This stringent requirement for sec defects may be due to high affinity of the very hydrophobic transmembrane stretches of MalF for the secretory apparatus compared with much shorter hydrophobic regions of cleavable signal sequences (9).
This dependence for membrane protein assembly on sec gene products has led us to examine further the effects of mutant sec genes on this process. In particular, we have studied the prlA mutations of E. coli that lie in the secY gene, encoding a core membrane component of the bacterial protein translocation apparatus. The prlA mutations alter SecY so as to allow the export of proteins with defective signal sequences. In prlA mutants, alkaline phosphatase (AP)2 carrying point mutations or a complete deletion of the signal sequence can be exported relatively efficiently (10). Moreover, prlA mutations allow the export of AP when it is fused to the cytoplasmic domains of membrane proteins (11-14). We were interested in studying the mechanism that allows AP fused to a cytoplasmic domain of a membrane protein to be translocated across the membrane in prlA strains.
Here we show, using a set of fusions of alkaline phosphatase to cytoplasmic domains of the MalF protein, that the topology of a number of these fusions is altered in prlA mutants. Further, we present evidence that, in one case, prlA mutations result in the inversion of the topology of a membrane protein. These results are discussed both in terms of the role of the Sec proteins in contributing to the topology of membrane proteins and the mechanism of prlA-mediated export.
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EXPERIMENTAL PROCEDURES |
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Strains and Plasmids-- Strains and plasmids are listed in Table I. Media are as described in Ref. 15.
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Screen for Mutants That Increase the AP Activity of the MalF-AP M Fusion-- DHB5181 was mutagenized with nitrosoguanidine as in Ref. 15. After mutagenesis, cells were plated on NZ-amine-A plates containing 40 µg/ml 5-bromo-3-chloro-3-indolyl phosphate (a chromogenic substrate for AP). Plates were incubated for 1 day at 37 °C and 1 day at 4 °C, and dark blue colonies were picked. Elevated AP activity in the mutants was assayed. The mutants were mapped by P1 cotransduction.
Mutants mapping near secY were tested in two ways to see if they contained prlA mutations. The mutants' ability to export MalE18-1 (a version of MalE with a defective signal sequence) was assessed by transducing malE18-1 into the mutants from WP144 and plating on maltose tetrazolium medium (15); malE18-1, prlA mutants are white on this medium; prlA+ cells are red. The mutants' ability to export APAlkaline Phosphatase Assays-- Cells were grown in NZ-amine-A plus 200 µg/ml ampicillin to an optical density at 600 nm of approximately 0.4. The production of the MalF-AP fusions was induced for 30 min by the addition of IPTG at a final concentration of 5 mM. The cells were harvested, and alkaline phosphatase activity was assayed in duplicate as in Ref. 10 with less than 5% variation.
Proteolysis of the MalF-AP Fusions-- 1.5-ml cultures were grown at 37 °C in M63 minimal medium containing 0.2% glucose, 50 µg/ml each of all amino acids except cysteine and methionine, and 5 mM IPTG. After growth to an optical density of approximately 0.4 at 600 nm, the cultures were labeled for 1 min with 45 µCi/ml [35S]methionine and chased with an excess of cold methionine for 30 min. 1 ml of cells was placed at 0 °C for 20 min, pelleted, and resuspended in cold spheroplast buffer containing 40% sucrose, 33 mM Tris, pH 8, 2.5 mM EDTA, and 5 µg/ml lysozyme. After 15 min at 0 °C, the spheroplasts were divided into 0.5-ml portions that were either left untreated or proteolyzed with proteinase K at a final concentration of 500 µg/ml for 20 min at 0 °C. Proteolysis was stopped with the addition of phenylmethylsulfonyl fluoride at 0.4 µg/ml. The spheroplasts were separated from cell envelope proteins by pelleting (7 min at 14,000 rpm in a microcentrifuge at 4 °C), resuspended in spheroplast buffer, and disrupted by freezing and thawing three times. Immunoprecipitation with antisera against AP and glucose-6-phosphate dehydrogenase, SDS-PAGE, and autoradiography were performed as described (16).
Urea Extraction--
Cultures were grown in MOPS minimal medium
(17) at 37 °C with 0.2% glucose and a 50 µg/ml concentration each
of all amino acids except cysteine and methionine. They were induced
with 0.04 mM IPTG for 15 min, pulse-labeled with 45 µCi/ml [35S]methionine for 1 min, and chased for 2 min
with an excess of cold methionine. Fractions of 0.5 ml were put on ice,
and the following was added: 1 ml of 8 M urea, 0.2 ml of 1 M iodoacetamide, 0.02 ml of 0.5 M EDTA, and
0.02 ml of 0.1 M phenylmethylsulfonyl fluoride. The samples
were then incubated on ice for 10 min and centrifuged at 14,000 rpm in
a microcentrifuge at 4 °C for 15 min. The supernatant was taken as
the urea-extractable fraction. The pellet was taken up in an equal
volume of a urea solution of the same composition as the supernatant.
The fractions were made 10% in trichloroacetic acid, incubated 15 min
on ice, and then centrifuged. The supernatants were discarded, and the
pellets were washed twice with cold 100% acetone. Immunoprecipitation was carried out as described (18). Samples were released from IGSorb by
suspension in 0.05 ml of SDS sample buffer without 2-mercaptoethanol and separated into two fractions. One fraction was reduced by incubation at 80 °C for 5 min with volume of 0.2 M dithiothreitol, and then
volume of 1 M iodoacetamide was added to each fraction followed by a 5-min incubation at 80 °C. Equal volumes of each fraction were loaded on SDS-PAGE gels.
Kinetics of D1 Export--
Cultures were grown in MOPS medium
as described above, induced with 0.1 mM IPTG for 15 min,
and pulse-labeled as described above. At 1, 2, 4, 8, and 12 min of
chase, samples were removed to tubes on ice with
volume of
1 M iodoacetamide. After at least 5 min on ice,
trichloroacetic acid precipitation, immunoprecipitation, sample
preparation, and SDS-PAGE were carried out as described above.
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RESULTS |
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prlA-mediated Export of Alkaline Phosphatase in MalF-AP Fusions-- Initially, we set out to study the mechanism whereby basic amino acids act to anchor the cytoplasmic domains of membrane proteins. To do this, we sought suppressor mutations that would reduce the anchoring activity of these amino acids and allow export of a cytoplasmic domain of a membrane protein. Such suppressors might be expected to alter the mechanism that responds to the presence of the basic amino acids. Their analysis, thus, might yield insights into this mechanism.
We employed a strain in which alkaline phosphatase is fused to a cytoplasmic domain of MalF (the M fusion, Fig. 1). The AP portion of this fusion is stably anchored in the cytoplasm by the basic aminoacyl residues in the MalF cytoplasmic domain that precedes it. In the cytoplasm, AP does not assemble into an active enzyme, since the the two essential intrachain disulfide bonds in AP cannot form (19, 20). Thus, when AP is fused to cytoplasmic domains of membrane proteins, it exhibits low enzymatic activity (21). In contrast, when AP is fused to a periplasmic domain of a membrane protein, it is exported to the periplasm and becomes enzymatically active. Selecting mutants that increase AP activity of the M fusion should yield strains in which the AP is exported to the periplasmic space. Such mutants could include those that no longer recognize the basic amino acids as signals to anchor the AP in the cytoplasm. We chose the M fusion because we have previously shown that eliminating the basic aminoacyl residues in the MalF cytoplasmic domain that precedes the AP portion of this fusion results in increased export of AP (22).
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Does a prlA Mutant Alter the Topology of the MalF-AP Fusion Protein or Does It Export Cleaved AP?-- We considered two explanations for the export of the AP moiety of the MalF-AP M fusion protein in prlA strains (Fig. 2). First, the prlA mutation may result in a change in the topology of the membrane protein so that AP, still attached to MalF, is now in the periplasm rather than the cytoplasm (Fig. 2B). Alternatively, the AP moiety in the cytoplasm may be cleaved from the fusion protein, and then the cleaved AP, lacking any export signal, is exported by the altered secretion machinery (Fig. 2A). This latter explanation represents a reasonable alternative, since 1) many of the MalF-AP fusion proteins are known to be unstable (21) and 2) AP without a signal sequence is exported in certain prlA mutants (10).
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Reversal of the Topology of a Membrane Protein Promoted by the
prlA-altered Secretion Machinery--
For these studies, we chose the
MalF-AP D1 fusion protein (Fig. 1). The D fusion protein has AP
fused to the second cytoplasmic domain of MalF and exhibits very low AP
activity (Table II). The D
1 fusion protein was derived from the D
fusion by the deletion of the first membrane-spanning segment of MalF
(MSS1) and the short periplasmic domain that follows it (Fig.
1B; Ref. 23). The construct was made in such a way that MSS2
is now bounded by two hydrophilic domains that correspond to the first
two cytoplasmic domains of MalF. The second cytoplasmic domain is then
followed by AP. Both of these hydrophilic domains are enriched for
basic amino acids. Since each of these domains could, in principle, act
as a cytoplasmic anchor, it was not immediately obvious what the
topology of this protein would be. However, analysis of the protein
showed 1) that the AP moiety is in the cytoplasm, thus exhibiting very
low enzymatic activity and 2) that the amino terminus of the fusion
protein is exposed to proteolytic attack on the periplasmic surface of
the cytoplasmic membrane (23). These findings led to the conclusion
that the D
1 fusion protein has the topology indicated in Fig.
1B. We proposed that the net positive charge in the second
cytoplasmic domain predominated in orienting the protein in the
membrane (23).
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SecB Dependence of Inversion of Membrane Protein
Topology--
SecB is a chaperone required for the efficient
export of certain secreted proteins. AP is not one of these proteins
(25). However, in a prlA strain that exports AP lacking its
signal sequence, the export is SecB-dependent
(10). This change in SecB dependence is thought to be due to the slower
post-translational export of alkaline phosphatase that occurs in
prlA-suppressed signal sequence mutants. We asked whether,
in a prlA4 mutant, the translocation of the AP portion of
the M and D1 fusions is SecB-dependent. Whereas a
secB+ prlA4 strain expressing the M
fusion has 10 units of AP activity (Table II), an isogenic
secB strain has only 1.7 units, indicating that export of
the AP moiety of the M fusion requires SecB. In contrast, the
translocation of AP in the D
1 fusion is largely independent of SecB
(Fig. 4). The kinetics of OmpA export are slowed in the cells missing
SecB (Fig. 4).
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DISCUSSION |
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Membrane Protein Topology and the Sec Machinery--
Our results
show that the topology of a membrane protein can be altered by
mutations that alter the bacterial protein export machinery. The AP
moiety of the MalF-AP M and D1 fusions is localized to the cytoplasm
in wild-type cells and the periplasm in prlA mutants. For
both fusions, the export of AP caused by the prlA mutation
was not due to cleavage of AP from the hybrid protein in the cytoplasm
and subsequent translocation of the free signal sequenceless AP; the
exported AP is still part of the hybrid protein. Thus, in both cases,
the arrangement of the hybrid protein in the membrane of
prlA mutants must be at least partly altered from the
arrangement it assumes in wild-type cells.
How Does the prlA Alteration of the Export Machinery Promote Export
of AP in a Protein Such as D1?--
To consider this question, we
take into account studies with the E. coli cytoplasmic
membrane protein leader peptidase, which suggest the following
properties of the assembly system for membrane proteins (27). When a
hydrophilic domain of a membrane protein is less than 25 amino acids,
the translocation of the domain across the membrane does not depend on
the sec gene products. However, for domains larger than 25 amino acids, translocation does require the bacterial export machinery.
Such long hydrophilic domains simply cannot pass through the lipid
bilayer.
What Happens to D1 in the prlA Mutant Strains?--
Two
possibilities are as follows.
The Role of SecB in the Export of MalF-AP Fusions--
Export of
wild-type alkaline phosphatase is not SecB-dependent (25).
However, the export of signal sequenceless AP or AP in the M fusion in
a prlA mutant is SecB-dependent (10). SecB is
required to maintain certain exported proteins in a
translocation-competent conformation (33). We suggest that export of
wild-type AP is so rapid that SecB is not required; translocation
outpaces folding. However, in the signal sequenceless AP or in the M
fusion, the export is so slow that SecB is needed to maintain the
cytoplasmically accumulating form in a conformation that can be
exported. If this explanation is correct, it is the rapid export of AP
seen in the D1 fusion in a prlA background that avoids
the requirement for SecB.
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FOOTNOTES |
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* This work was supported by Merit Grant GM38922 from NIGMS, National Institutes of Health (to J. B.) and a grant from Deutsche Forschungsgemeinschaft (to M. E.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ Supported in part by an American Cancer Society Research Professorship and by D. Pette and Grant SFB156 from the University of Konstanz. To whom all correspondence should be addressed: Dept. of Microbiology and Molecular Genetics, Harvard Medical School, 200 Longwood Ave., Boston, MA 02115. Tel.: 617-432-1921; Fax: 617-738-7664; E-mail: jbeckwit{at}warren.med.harvard.edu.
1 W. Dowhan, personal communication.
2
The abbreviations used are: AP, alkaline
phosphatase; MOPS, 3-(N-morpholino)propanesulfonic acid;
IPTG, isopropyl-thio--D-thiogalactopyranoside; PAGE,
polyacrylamide gel electrophoresis; MSS, membrane-spanning segment.
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REFERENCES |
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