From the Department of Molecular and Cell Biology, The University of Connecticut, Storrs, Connecticut 06269
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ABSTRACT |
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Many integral membrane proteins contain an
amino-terminal segment, often referred to as an N-tail, that is
translocated across a membrane. In many cases, translocation of the
N-tail is initiated by a cleavable, amino-terminal signal peptide. For
N-tail proteins lacking a signal peptide, translocation is initiated by
a transmembrane segment that is carboxyl to the translocated segment.
The mechanism of membrane translocation of these segments, although
poorly understood, has been reported to be independent of the protein
secretion machinery. In contrast, here we describe alkaline phosphatase
mutants containing artificial transmembrane segments that demonstrate
that translocation of a long N-tail across the membrane is dependent
upon SecA, SecB, and the electrochemical potential in the absence of a
signal peptide. The corresponding mutants containing signal peptides
also use the secretion machinery but are less sensitive to inhibition
of its components. We present evidence that inhibition of SecA by sodium azide is incomplete even at high concentrations of inhibitor, which suggests why SecA-dependent translocation may not
have been detected in other systems. Furthermore, by varying the charge around the transmembrane segment, we find that in the absence of a
signal peptide, the orientation of the membrane-bound alkaline phosphatase is dictated by the positive inside rule. However, the
presence of a signal peptide is an overriding factor in membrane orientation and renders all mutants in an
Nout-Cin orientation.
Membrane integration and assembly of integral membrane proteins
are complex processes. Integral membrane proteins have been classified
according to their transmembrane topography and mode of membrane
translocation (1). Type I consists of proteins with a cleavable signal
peptide and a translocated amino terminus. In Escherichia
coli, the signal peptide engages the Sec translocation machinery,
which is responsible for the translocation of the amino-terminal periplasmic segment across the inner membrane. However, many integral membrane proteins are synthesized without signal peptides. These proteins are divided into two types. Type II proteins have a
nontranslocated amino-terminal segment and are membrane integrated in
an Nin-Cout orientation. Type III proteins have
a translocated amino-terminal segment sometimes referred to as an
N-tail that results in an Nout-Cin orientation.
Thus, type I and type III membrane proteins have the same final
orientation but the mechanism of membrane insertion and translocation
might be expected to be different because of the involvement of the
signal peptide in one case but not the other.
The mechanism of N-tail translocation in the absence of a signal
peptide is particularly intriguing. There are many proteins without
signal sequences that bear translocated amino-terminal segments,
including the majority of known G protein-coupled receptors (2). For
these proteins, how is membrane insertion initiated by a transmembrane
segment that ultimately resides in the membrane with the opposite
directionality of a signal peptide? What role do flanking charged
residues have in the mechanism of insertion and final orientation?
It has been proposed that the amino-terminal portions of type III
integral membrane proteins are translocated in a SecA-independent fashion. Cao and Dalbey (3) have reported efficient SecA-independent translocation of amino-terminal segments of mutant bacteriophage Pf3
coat proteins of up to 38 amino acids. Whitley et al. (4) reported that translocation of the 100-residue-long N-tail of the
E. coli inner membrane protein ProW is SecA-independent.
These observations have led to the proposal of an electrophoresis-like translocation mechanism (5, 6) for amino-terminal segments in which the
electrochemical potential of the membrane is utilized by the charged
amino acids flanking the transmembrane segment. These residues, with a
"positive inside" preference (7), influence the orientation of the
transmembrane segment and translocation of the tail.
SecB is a cytosolic chaperone that facilitates translocation of some
membrane proteins, including PhoE and LamB, by forming a complex with
them and maintaining them in an unfolded state. Little is known about
the potential involvement of SecB in translocation of type III membrane
proteins. It is possible that SecB or other chaperones are necessary to
maintain amino-terminal tails in an unfolded, translocation competent
state until translocation can be initiated via the transmembrane segment.
To investigate the characteristics of the translocation of
amino-terminal segments, we have utilized a protein with an artificial transmembrane segment. The conversion of alkaline phosphatase, a
normally transported, water-soluble protein into an integral membrane
protein by use of an artificial transmembrane segment has been
previously described (8). This approach offers several advantages: the
wild type form is translocated to the periplasm, which suggests that
there is no bias against membrane translocation; because it is not a
membrane protein, there is no intrinsic topogenic preference; and
alkaline phosphatase has an amino-terminal signal peptide, the deletion
of which can invert the membrane orientation of the transmembrane
segment-containing protein. Consequently, this provides an ideal model
system with which to elucidate the distinguishing characteristics of
membrane integration of type III relative to type I proteins.
In contrast to previous findings, we describe alkaline phosphatase
mutants containing artificial transmembrane segments that demonstrate
that translocation of a long amino-terminal segment across the membrane
is SecA-dependent, SecB-dependent, and
electrochemical potential-dependent, regardless of the
presence or absence of a signal peptide. Mutants lacking signal
peptides obey the positive inside rule. Those with more positive
regions flanking the amino terminus of the transmembrane segment orient
in an Nin-Cout manner. By varying the charge in
the regions flanking the transmembrane segment to make the carboxyl
flanking region more positive than the amino flanking region, a
reversion of orientation occurs that results in an
Nout-Cin orientation in which a long
amino-terminal segment is translocated. Surprisingly, mutants
containing a signal peptide have an Nout-Cin
orientation regardless of flanking charge. Further, these mutants
demonstrate that when signal peptide-mediated translocation is
compromised by SecA inhibition, transmembrane segment-mediated
translocation also does not occur.
Bacterial Strains and Media--
E. coli AW1043
( DNA Manipulations and Mutagenesis--
Mutants were generated
from a previously described construct, CN-LA2 (8, 11), a derivative of
alkaline phosphatase containing an artificial transmembrane segment
composed of 5 leucine and 16 alanine residues. Sequences coding for
various flanking residues were introduced on either side of that coding
for the transmembrane segment. Deletion of signal sequences was
performed as described (8), resulting in mutants that have the
initiating methionine, lysine, glutamine, serine, and threonine
followed by the sequence of the mature region of alkaline phosphatase.
All sequences were confirmed by restriction enzyme analysis and DNA
sequencing (12).
Induction of Alkaline Phosphatase Expression and
Labeling--
The preparation of bacterial cultures and alkaline
phosphatase expression was described previously (13). Following
induction, cells were radiolabeled with 40 µCi/ml
L-[35S]methionine for 30 s at 37 °C
and then chased with 4 mg/ml cold methionine for 30 s. Cells were
then put on ice for further manipulations.
NaOH Treatment--
Following labeling, an aliquot was removed
to an ice-cold tube containing an equal volume of ice-cold NaOH at a
final concentration of 0.1 N (14). The mixture was vortexed
hard and centrifuged at 4 °C for 15 min in a microcentrifuge to
separate the soluble fraction from the membrane-associated fraction.
The protein in each fraction was precipitated with 5% trichloracetic
acid, and alkaline phosphatase was immunoprecipitated as described
(13).
Cell Fractionation--
Membrane association was assayed by cell
fractionation as described (8). Spheroplasts were stabilized with 10 mM MgCl2 and lysed in 10 mM HEPES,
2 mM EDTA. Membrane and cytoplasmic fractions were
separated by centrifugation at 4 °C for 5 min.
Protease Protection Analysis--
The transmembrane topography
of all mutants was assayed by protease protection analysis as described
(8).
Sodium Azide Treatment--
To assess the involvement of SecA,
cells were treated with sodium azide essentially as described by Oliver
et al. (15). Cells were incubated with sodium azide ranging
in final concentration from 2 to 10 mM at 37 °C for 1 min prior to labeling as described above. Translocation was assayed by
protease protection analysis.
Carbonyl Cyanide 3-Chlorophenylhydrazone Analysis--
To
dissipate the proton motive force, the procedure of Kuhn and Wickner
(16) was used. Cells were incubated at 37 °C in 100 µM
CCCP1 in dimethyl sulfoxide
or with an equal volume of dimethyl sulfoxide alone for 1 min prior to
labeling. Translocation was assayed by protease protection analysis.
Electrophoresis and Quantitation of
Proteins--
Immunoprecipitated proteins were separated by
electrophoresis on 7.5% or 10% Laemmli SDS-polyacrylamide gels (17).
Patterns were visualized by autoradiography and quantified by
phosphorimage analysis using a Bio-Rad phosphorimager and Molecular
Analyst version 1.4.1 software.
To examine the requirements for stable membrane integration and
orientation, an artificial transmembrane segment was inserted into
E. coli alkaline phosphatase. Position 278 was chosen for insertion of the transmembrane segment because it is in a loosely folded region removed from the core and the catalytic site of the
enzyme (18, 19) and can accommodate large insertions. In all mutants,
the hydrophobic segment of the transmembrane region consists of 21 amino acids, 5 leucines, and 16 alanines. This composition was chosen
because previous studies (8) suggested that it offered a balance
between being stably membrane-associated yet not so highly hydrophobic
that effects due to tandem charges would be obscured. To analyze the
role of charged residues within the regions flanking the transmembrane
segment, the degree of charge and the charge balance of the region
flanking the termini of the transmembrane segment were varied. On the
amino-terminal side of the transmembrane segment, the charge was varied
from 0 to +5 using various combinations of lysine and glycine residues. In the region immediately flanking the carboxyl terminus of the transmembrane segment, the charge was the wild type charge of +0.5 or
+5.5, the latter generated by the insertion of five lysine residues.
Parallel constructs were made in which the amino-terminal signal
peptide was deleted. Fig. 1 shows the
amino acid sequences of the mutants.
INTRODUCTION
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Abstract
Introduction
References
EXPERIMENTAL PROCEDURES
lac gal U gal K
(leu-ara) phoA-E15 pro C::Tn5) was
used for the generation and replication of mutant forms of alkaline
phosphatase and for translocation and membrane association analyses.
For general propagation of AW1043, LB medium (9) was used. E. coli CK1953 (MC4100 secB::Tn5; gift from
C. A. Kumamoto) was used for SecB-dependent
translocation analysis. CK1953 was propagated in M63 medium (10). All
media were supplemented with 50 µg/ml kanamycin and 250 µg/ml ampicillin.
RESULTS
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Fig. 1.
Amino acid sequences of the relevant regions
of mutant alkaline phosphatase constructs. All constructs
designated SP contain a wild type signal sequence.
Constructs with deleted signal sequences are designated DS.
The first number in the construct name designates the
positive charge amino-terminal to the TMS. The final W or
5 indicates whether the wild type sequence or an insertion
of 5 lysines flanks the TMS on the carboxyl side. The downward
facing arrow marks the signal peptide cleavage site, and the
numbers above the sequences indicate the position of the
amino acid in the mature wild type alkaline phosphatase sequence. The
signal peptide and TMS are designated above the sequence.
To ascertain the ability of the inserted sequences to function as transmembrane segments, NaOH extractions were performed by the method of Russel and Model (14). The extent of membrane association of the signal peptide-containing mutants is shown in Fig. 2A. All of these mutants show membrane association, and little difference was observed as a result of varying the charge surrounding the transmembrane segment. Membrane association is further confirmed by cell fractionation (Fig. 2B). By treating spheroplasts with proteinase K, the transmembrane topography of these mutants was determined. Unlike the tightly folded native alkaline phosphatase, which is transported across the membrane to the periplasm and is resistant to proteolysis, the periplasmic segments of membrane-bound alkaline phosphatase mutants are very sensitive to proteolysis. Conversely, the fraction of the mutant that remains in the cytoplasm is protected from proteolysis. Due to the position of the transmembrane segment at approximately two-thirds the length of the protein away from the amino terminus, the membrane orientation can be determined by the size of the fragment protected. If the protein inserts in an Nout-Cin orientation, the carboxyl-terminal one-third of the protein will be protected. In the Nin-Cout orientation, the amino-terminal two-thirds of the protein will be protected. Treatment of the spheroplasts with 0.2% CHAPS to solubilize the membrane results in the previously protected cytoplasmic fragments becoming susceptible to proteolysis. All signal peptide-containing mutants exhibited an Nout-Cin orientation regardless of flanking charge as indicated by the protection of a fragment corresponding to the carboxyl-terminal one-third of the protein (C1/3) (Fig. 2C). Addition of CHAPS resulted in the loss of this protected fragment, consistent with its cytoplasmic location. It is remarkable that clustered positive charges are translocated across the membrane in mutants containing five lysines (SP5TMW) and three lysines (SP3TMW) immediately amino-terminal to the transmembrane segment. The positive inside rule (7) would predict that the amino termini of the transmembrane segment in these constructs would not be translocated because they are more positively charged than the carboxyl termini. However, in this context, the presence of a wild type signal sequence seems to override the positive inside rule and dictate that the orientation of the transmembrane span be Nout-Cin.
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The extent of membrane association of mutants with deleted signal peptides is shown using NaOH extraction (Fig. 3A) and cell fractionation (Fig. 3B). Like the signal peptide-containing mutants, all deleted signal peptide mutants demonstrate membrane association. To determine the orientation of these mutants, protease protection analysis was performed (Fig. 3C). In the absence of a signal peptide, the orientation of the protein is dependent upon the charge state of the regions flanking the transmembrane segment. Mutants with amino-terminal flanking regions that are more positive than the carboxyl-terminal flanking regions (DS5TMW and DS3TMW) exhibited an Nin-Cout orientation as indicated by protected fragments corresponding to the amino-terminal two thirds of the protein (N2/3). No fragments corresponding to the carboxyl-terminal one third of the protein (C1/3) were evident in the DS5TMW or DS3TMW mutants, indicating that no significant fraction of the expressed protein is in the opposite orientation of Nout-Cin. This suggests that in the absence of the signal peptide, a transmembrane segment with amino-terminal positive charges behaves like a signal anchor, i.e. it functions to initiate translocation of the carboxyl-terminal one-third of the protein. In contrast, the mutants with a more positive carboxyl flanking region (DS0TMW and DS0TM5) exhibited the opposite orientation of Nout-Cin as evidenced by the presence of a fragment corresponding to the carboxyl-terminal one-third of the protein plus the transmembrane segment. Hence they contain a translocated amino-terminal segment of approximately 288 residues that is accessible to proteolysis. As with the signal peptide-containing mutants, addition of CHAPS resulted in the loss of all protected fragments. Thus, in the absence of a signal peptide, the charge balance surrounding the transmembrane segment influences orientation, and the positive inside rule is followed, even though it necessitates translocation of a long N-tail. The fraction of full-length protein remaining in the presence of protease indicates that some of the population of labeled protein is not fully translocated. Thus the transmembrane segment is less efficient at initiating translocation than the signal peptide. However, that protein that is translocated is oriented in accordance with the positive inside rule.
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The DS0TM5 mutant was chosen to investigate the mechanism of translocation of long amino-terminal segments. A concentration course of sodium azide treatment was used to ascertain the involvement of SecA. In this experiment inhibition of translocation was demonstrated by a decrease in the amount of protected fragment (C1/3) and a concomitant increase in the amount of full-length protein that was protected from proteloysis with increasing azide concentration. As shown in Fig. 4, sodium azide treatment blocked the translocation of the amino-terminal segment of DS0TM5 as demonstrated by the near absence of the protected small fragment at high azide concentrations. Across the concentration course from low azide concentrations to high, the fraction of total labeled protein that is represented in the small fragment decreases, and the fraction of full-length, fully protected protein increases to about 100% at 10 mM, indicating that SecA-mediated transport is inhibited.
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The SecA dependence of amino-terminal segment translocation is also evident from azide treatment of mutant SP0TM5 (Fig. 5). There are two possible initiators of translocation in this mutant, the signal peptide and the transmembrane segment. The presence of the mature form in the samples not treated with protease indicates that signal-peptide mediated translocation has occurred, because the signal peptide is cleaved upon translocation. If translocation via the signal sequence does not occur, it is possible that the transmembrane segment can initiate translocation, resulting in retention of the precursor form. The different fates of these forms when treated with protease are indicative of the extent to which translocation, initiated via either the signal peptide or possibly by the transmembrane segment, is inhibited. The fraction of total labeled protein that is present as precursor increases across the azide concentration course, to about 30% at 10 mM azide, indicating that signal peptide-mediated translocation is inhibited. Interestingly, even at high azide concentrations there remains significant signal peptide-mediated translocation, as evidenced by the presence of full-length mature protein. Such significant levels of translocation are not only indicative of the high efficiency of the signal peptide but also that protein synthesis and transport occur at 10 mM azide. Virtually none of this mature population remains intact upon protease treatment. This demonstrates that virtually all of the mature protein, the population for which the signal peptide has functioned, is translocated and accessible to protease in an Nout-Cin orientation. In contrast the precursor form is totally protected from protease, demonstrating that when the signal sequence is inhibited, there is no transmembrane segment-mediated translocation. Were transmembrane segment-mediated translocation SecA-independent, one would expect the precursor form to be susceptible to proteolysis, because it too would be translocated. Even if the signal peptide itself remained associated with the membrane due to its hydrophobicity, the bulk of the amino-terminal segment should be translocated and protease sensitive. This experiment further indicates that the signal peptide is more resistant to compromise of the Sec machinery by sodium azide than the transmembrane segment; substantial levels of signal peptide-mediated translocation readily occur at concentrations of azide at which no transmembrane segment-mediated translocation occurs for both the DS0TM5 and SP0TM5 mutants.
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Because only about 30% of the expressed SP0TM5 is in precursor form even at high azide concentrations, a pulse-chase analysis following azide treatment was performed. We found that following treatment with 10 mM azide and a 30-s pulse labeling followed by a 30-s cold methionine chase that 74% of the labeled protein was in the precursor form compared with 6% in the untreated control (data not shown). This suggests that a larger population was inhibited in the azide treatment experiment described above (Fig. 5) but that much of the precursor chased through to the mature form during the time taken for protease treatment.
The SecB dependence of SP0TM5 and DS0TM5 mutants was assayed by protease protection analysis of translocation in the SecB null strain CK1953 (Fig. 6). Two previously described signal peptide-containing proteins were used as controls (20). WT-MCS(dl), a modified form of wild type alkaline phosphatase, is SecB-independent, and WT-K5L5 is SecB-dependent. Both proteins exhibited this behavior under the conditions used in the present study. SP0TM5 is readily translocated in the absence of SecB because most of the population of labeled protein is present in the mature form in the sample not treated with protease. The mature form is susceptible to proteolysis, giving rise to the C1/3 fragment, further indicating that the amino-terminal segment has been translocated even in the absence of SecB. Only a small fraction of the SP0TM5 population is not translocated and accumulates as precursor. In contrast, DS0TM5 is translocated poorly in the SecB null strain. Virtually all of the labeled protein is protected from proteolysis, indicating that DS0TM5 is largely untranslocated and is thereby very sensitive to the loss of SecB. This is consistent with the notion that dependence on SecB is more pronounced for proteins that are translocated slowly and post-translationally (21).
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Dissipation of the electrochemical potential by CCCP also inhibits translocation of the amino-terminal segment (Fig. 7). Treatment with 100 µM CCCP greatly affects the efficiency of translocation in DS0TM5 as evidenced by the near absence of a protected fragment and a concomitant increase in the amount of full-length protected protein. SP0TM5 is less affected; the amount of protected fragment present under CCCP treatment is comparable with that of the untreated parallel sample. Thus, translocation of DS0TM5 is virtually entirely inhibited at 100 µM CCCP, whereas that of SP0TM5 is somewhat less inhibited, suggesting that transmembrane segment mediated translocation is more sensitive to loss of the electrochemical potential than signal peptide-mediated translocation. These results parallel the sensitivities of DS0TM5 and SP0TM5 to azide treatment. This trend was previously observed for several signal peptide (22, 23) and SecB-dependent (20) mutants and points to the intriguing possibility that the requirement for SecA and the electrochemical gradient are directly related.
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DISCUSSION |
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Mutants that do not have a signal peptide obey the positive inside rule (7), which stipulates that the orientation of a transmembrane segment is determined by the flanking charge, with the more positively charged flanking region remaining in the cytoplasm. As demonstrated in Fig. 3C, mutants with deleted signal peptides have an Nin-Cout orientation when the more positively charged flank is at the amino terminus of the transmembrane segment. However, mutants with reduced amino-terminal charge or increased carboxyl-terminal charge exhibit an Nout-Cin orientation.
In contrast, the presence of a wild type signal peptide is an overriding factor in membrane orientation. Regardless of amino-terminal flanking charge, all signal peptide-containing mutants have an Nout-Cin orientation (Fig. 2C). Prima facie, the positive inside rule would predict that the orientation of the mutants would be determined by the charge balance surrounding the transmembrane segment, as it does correctly in the deleted signal peptide mutants. However, the signal peptide seems to dictate assembly, when necessary initiating the translocation of as many as five lysines at the amino terminus of the transmembrane segment with little effect upon extent of membrane integration. Perhaps the reason that translocation of a high density of basic residues occurs in this system and not others (24) is because of the considerable length of polypeptide between the signal peptide and the transmembrane segment. Upon translocation initiation by the signal peptide, the long N-tail may be translocated before the cluster of basic residues is encountered, and at that point the system is poised strongly in favor of the passage of the basic residues and completing translocation.
DS0TM5 also gives rise to a translocated N-tail of approximately 288 amino acids. The energy required to translocate such a long hydrophilic segment cannot be explained by membrane partitioning. Although it is reasonable that very short hydrophilic segments may be translocated as a result of spontaneous insertion of a nearby transmembrane segment (25), the free energy barrier for translocating longer segments is too high to be overcome by the free energy gained through insertion of a transmembrane segment. Thus, the SecA dependence of N-tail translocation may result from the need for active translocation.
In the reported examples of SecA-independent N-tail translocation that utilize sodium azide as a SecA inhibitor, the concentration of sodium azide used was typically 2-3 mM. In our study, mutant DS0TM5 is largely translocated at 2 mM azide (Fig. 4); significant inhibition of translocation is not observed until higher azide concentration. Treatment of cells with sodium azide can be expected to be leaky; the concentration of azide used may not be sufficient to completely inhibit all SecA in these experiments. Under such conditions, sequences with high affinities for SecA will be preferentially translocated. Such a possibility has been suggested to account for differences in the sensitivity of alkaline phosphatase signal peptide mutants to azide treatment (21, 22) and underscores the potential difficulties in using proteins that differ from the protein whose transport is being studied as reporters for the loss of SecA function. Similarly, MalF, an integral membrane protein, was reported to be SecA-independent using azide treatment (26) but later shown to be SecA-dependent by Traxler and Murphy (27).
It has been proposed that the Sec machinery has an amino-to-carboxyl
directionality (6). The reported examples of SecA independence of
N-tail translocation would seem to confirm this because the
transmembrane segment-mediated translocation has the opposite
directionality and apparently does not utilize the Sec machinery.
However, our results indicate that amino-terminal segment translocation
is SecA-dependent and can be initiated by a transmembrane segment that ultimately resides in an Nout-Cin
orientation. In our system, there are no other segments between the
amino terminus and the transmembrane segment that even remotely
resemble a signal sequence and that might be expected to fullfill this
role even transiently. Therefore, it is interesting to speculate how
the interaction of a transmembrane segment with SecA occurs. The
interaction may be relatively nonspecific, involving hydrophobic
surfaces and perhaps the flanking charged region. These features could be contributed by the amino acid side chains irrespective of the directionality of the peptide bonds. Such interactions involving overall amino acid side chain properties have been demonstrated for
other systems including -endorphin (28) using peptides composed of D
rather than L amino acids. Alternatively, SecA may interact with
polypeptides exclusively in the amino-to-carboxyl direction with the
final orientation of the transmembrane region being subsequently driven
by the balance of charged flanking residues. However, such a model
would require that the transmembrane segment undergo a reversion of
orientation at some point during the translocation process.
The SecA-dependent translocation of an artificially produced amino-terminal tail suggests that it is very likely the case that many N-tails are translocated in the same fashion. Further, it is possible that many sequences shorter than the one described here are SecA-dependent as well but that translocation of those tails has been observed under insufficiently impaired SecA conditions. Future studies will need to consider the affinity of SecA for various signal sequences and transmembrane segments. If truly SecA-independent translocation does occur, it is probable that the partitioning of transmembrane segments into the membrane would provide only enough energy to translocate very short N-tails.
Mutant DS0TM5 is considerably more reliant on the presence of SecB for translocation than is SP0TM5. It may be the case that SecB is required to maintain the amino-terminal two-thirds of the protein in an unfolded, translocation competent state until the transmembrane segment can be synthesized and then recognized by the translocation machinery. The folding state of the amino-terminal domain of a type III protein has been shown to affect translocation (29). In that study, rapidly folding domains inserted into the amino-terminal segment hindered or altogether prevented translocation of the amino-terminal segment. Thus, the involvement of SecB in translocation of membrane proteins may be a common phenomenon.
In contrast to previous reports of amino-terminal tail translocation
that is independent of the translocation machinery, we show that the
amino-terminal tail in our study requires SecA and SecB for efficient
membrane translocation. Moreover, the lack of a signal peptide in our
type III membrane protein confers greater susceptibility to compromises
in transport machinery function and electrochemical gradient disruption.
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ACKNOWLEDGEMENTS |
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We thank Jinoh Kim and Sharyn Rusch for helpful discussions of these studies.
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health Grant GM37639 (to D. A. K.).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.
To whom correspondence should be addressed: Dept. of Molecular and
Cell Biology, Box U-44, The University of Connecticut, Storrs, CT
06269. E-mail: kendall{at}uconnvm.uconn.edu.
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ABBREVIATIONS |
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The abbreviations used are: CCCP, carbonyl cyanide 3-chlorophenylhydrazone; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propane-sulfonate; TMS, transmembrane segment.
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