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J Biol Chem, Vol. 273, Issue 14, 8419-8424, April 3, 1998
,
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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Top
Abstract
Introduction
Procedures
Results
Discussion
References
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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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Top
Abstract
Introduction
Procedures
Results
Discussion
References
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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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Top
Abstract
Introduction
Procedures
Results
Discussion
References
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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 AP
2-22 (a signal sequenceless version of AP) was assessed by
transforming the mutants with pAID135 and plating on media containing
40 µg/ml 5-bromo-3-chloro-3-indolyl phosphate and 5 mM
isopropyl-thio-
-D-thiogalactopyranoside (IPTG) to induce
production of AP2-22; prlA mutants are dark blue on these plates, while prlA+ cells are light
blue.
Alkaline 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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Top
Abstract
Introduction
Procedures
Results
Discussion
References
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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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5) that exhibited
increased alkaline phosphatase activity (see "Experimental
Procedures"). P1 cotransduction experiments showed that 24 of 41 mutants tested carried suppressor mutations unlinked to the gene
fusion. The remainder were due to alterations within the gene fusion
that increased alkaline phosphatase activity. The unlinked suppressors
were further mapped and all shown to lie in or close to the
secY gene. Since it had already been shown that
prlA mutations can promote the export of cytoplasmic
alkaline phosphatase in membrane protein fusions (11-14), we guessed
that our set of mutations might also be prlA mutations. To
test this explanation, we examined the effect of the suppressor
mutations on the export of two proteins with defective signal
sequences, the maltose-binding protein (MalE) with the signal sequence
alteration 18-1 (M18R) and AP missing its entire signal sequence. All
of the secY-linked mutants restored export to these proteins
(data not shown), suggesting that they contained typical
prlA mutations that suppress a variety of alterations of
signal sequences. Thus, the effects of these mutations were not limited
to reversing the interference with export by basic amino acids, as we
had hoped.
To further study the suppression by prlA mutants, we
transferred the following MalF-AP fusion constructs into the
prlA4 mutant background: B, C, D, J, M, O, P, C1, and
D1 (Fig. 1). For fusions C, M, O, P, and D1, we found a
significant increase in alkaline phosphatase activity, indicating
export of AP to the periplasmic space (Table
II). The absence of any increase for all
of the remaining fusions, except for D, was expected, since the
alkaline phosphatase is already efficiently exported to the periplasm.
The D fusion, on the other hand, allows little export of alkaline
phosphatase and is unaffected by the prlA mutation. This
refractory behavior may be due to the strength of the cytoplasmic
anchoring signal in this fusion, to the stabilization of the topology
resulting from interactions between the two transmembrane segments, or
to a combination of both.
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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 D1 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 D1 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).
1 fusion protein now exhibits high levels of AP activity (Table II). We asked whether the exported AP in this strain was still attached
to the membrane protein or was the result of export of the AP moiety
after cytoplasmic cleavage. This strain showed an added complexity in
that, as we describe below, the exported AP still attached to MSS2 was
slowly cleaved in the periplasm to release free soluble AP. This
property made it difficult to evaluate the mechanism of export of D1
using the same technique that we used to assess the M fusion. Instead,
we made use of the fact that AP can only form disulfide bonds when it
is in the periplasm but not in the cytoplasm. These disulfide bonds
provide an accurate measure of the cellular location of AP.
Experiments on the kinetics of export of AP as assayed by disulfide
bond formation are presented in Fig. 4.
There, we show 1) that the initial export of AP is very rapid, with a
substantial proportion of the protein becoming oxidized (disulfide
bonded) after a 1-min pulse-label and 1-min chase and 2) that the
disulfide-bonded AP is still attached to MSS2. Thus, as with the M
fusion, the export of the AP in the D1 fusion is not due to export
of an AP moiety that was cleaved from the fusion protein in the
cytoplasm. However, it is also clear that the exported AP is slowly
cleaved in the periplasm from the fusion protein. The cleaved material is marked with an asterisk in Fig. 4. Note that the reduced
breakdown product and oxidized full-length protein run with the same
mobility. In addition, two forms of mature OmpA are seen, because a
fraction of this protein retained some secondary structure.
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1 fusion in the prlA background. We imagined two
possible topologies for this protein that would depend on the mechanism of AP export (Fig. 5). In the first, the
fusion protein initially inserts into the membrane in the same way as
in a wild-type background, but the altered protein translocation
machinery promotes export of AP. This export might lead to the
structure depicted in Fig. 5A, in which both amino terminus
and carboxyl terminus of the hybrid protein are on the periplasmic face
of the membrane. Such a protein would not be expected to be stably
inserted in the membrane. In the second, the fusion protein, before any
interaction with the membrane, is recognized by the
prlA-altered secretion machinery differently than in the
wild-type situation so that MSS2 is seen as an export signal allowing
translocation of the AP moiety that follows it. The D1 protein, in
this case, would end up inserted in the membrane with the opposite
topology from that which it exhibits in a wild-type background (Fig.
5B).
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1 fusion protein in the prlA
background is resistant to urea extraction, suggesting that it is
stably anchored in the membrane via the full insertion of its MSS in
the membrane (Fig. 6). Control
experiments showed that the conditions we used extracted 97% of
wild-type AP, while OmpA was resistant to extraction (data not shown).
Longer exposures of the gel in Fig. 6 revealed small amounts of
AP-sized breakdown product in the supernatant of the prlA4
strain expressing the D1 fusion. This probably reflects the slow
cleavage of AP from D1 after D1 is inserted into the membrane
(Fig. 4). Other fractionation methods, including extraction of
spheroplasts or membranes with urea or extraction with 0.1 N NaOH gave similar results; all full-length fusion protein
was associated with the membrane fraction. Extraction with octyl
glucoside, on the other hand, solubilized about half of the full-length
fusion protein, as is seen with other integral membrane proteins under
similar conditions (data not shown).
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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 D1 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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Top
Abstract
Introduction
Procedures
Results
Discussion
References
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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.
1, our results suggest that the topology of the
protein has been completely and efficiently inverted. A transmembrane segment that in the wild-type protein and in the D1 fusion acts as a
stop-transfer sequence now appears to be acting as an export signal.
These results suggest that the protein translocation apparatus
contributes to determining the topology of the D1 fusion; the
prlA-altered Sec machinery may be unable to recognize some of the topogenic determinants in D1. The mechanism of
prlA-mediated export of D1 is discussed below.
An alternative explanation of our results is that the D1 fusion is
targeted for export in prlA mutants by its AP moiety, since
the mature domain of AP is known to contain targeting information that
allows it to be exported in prlA mutants (10, 26). However, there is a noticeable difference in the kinetics of
prlA-mediated export of D1 (Fig. 4) and that of AP with
an altered or missing signal sequence. In the latter case, the export
is quite slow, with half-times of export of about 5 min. In contrast,
the AP moiety of the D1 fusion is translocated quite rapidly, with
about 50% being localized to the periplasm within a minute. Thus, it seems unlikely that the AP portion of D1 is responsible for
determining the topology of this fusion in prlA mutants.
These findings raise the question of the role of the Sec machinery in
the topological arrangement of membrane proteins under wild-type
conditions. In most cases, the protein translocation machinery, if it
is involved, would be only one of the several factors that contribute
to the assumption and stability of membrane protein topology we have
outlined in the introduction. Thus, alteration of this machinery by
prlA mutations may not affect topology for most proteins.
However, it could be that for membrane proteins of simple structure,
for instance with a single transmembrane segment, some confusion of
signals could take place in prlA strains, leading to a mixed
population of topological structures. An effect of the altered
secretion machinery on topology would then suggest that this machinery
may be a contributor to the orientation of transmembrane segments of
membrane proteins, in general. Such experiments may be limited by the
folding properties of the normal cytoplasmic domains of these
proteins.
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.
1, we picture the following steps in its
assembly in a wild-type background. The positive charges at the
carboxyl terminus of the hydrophobic MSS prevents D1 from being
engaged by the export machinery or being properly oriented in the
machinery. Instead, this hydrophobic sequence inserts into the lipid
bilayer as a result of its natural affinity for the membrane.
Ordinarily, the orientation of this MSS is determined by the
"positive-inside rule," whereby positively charged hydrophilic domains of membrane proteins tend to be localized to the cytoplasm. However, in D1, there are positively charged residues at both ends
of the MSS. Our previous studies have shown that the N terminus of
D1 is translocated into the periplasm, despite its net positive charge (23). This translocation may not be
sec-dependent, since it has been suggested that
N-terminal export of this sort does not utilize the sec
machinery (31, 32).
What Happens to D1 in the prlA Mutant Strains?--
Two
possibilities are as follows.
1 still
maintains an intact highly hydrophobic sequence, which may add to the
facilitation of export by contributing to the activation of the
apparatus already primed by the prlA mutation.
2) Alternatively, the prlA-altered machinery may act to
export AP after D1 has assumed the orientation in the membrane found in the wild type. If this is the case, the translocation of the AP to
the periplasmic face of the cytoplasmic membrane will bring with it at
least a portion of the carboxyl terminus of the MSS. This would lead to
the likely membrane-unstable structure shown in Fig. 5A.
Our results with urea extraction and other fractionation techniques
support the first model, in which the D1 protein is stably inserted
in the membrane, with its amino terminus in the cytoplasm (Fig. 6).
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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Abstract
Introduction
Procedures
Results
Discussion
References
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) or treated (+) with proteinase K. Glucose-6-phosphate
dehydrogenase (G6PD) and AP were immunoprecipitated and
separated with SDS-PAGE. WP148 and WP149 are two of the mutants that
were isolated in the screen described under "Experimental
Procedures." WP148 expresses a mutant version of the M fusion, which
has increased AP activity.
