Direct Evidence That the Proton Motive Force Inhibits Membrane Translocation of Positively Charged Residues within Membrane Proteins*

Tracy A. Schuenemanndagger , Vondolee M. Delgado-Nixon, and Ross E. DalbeyDagger

From the Department of Chemistry, Ohio State University, Columbus, Ohio 43210

    ABSTRACT
Top
Abstract
Introduction
References

The M13 phage procoat protein requires both its signal sequence and its membrane anchor sequence in the mature part of the protein for membrane insertion. Translocation of its short acidic periplasmic loop is stimulated by the proton motive force (pmf) and does not require the Sec components. We now find that the pmf becomes increasingly important for the translocation of negatively charged residues within procoat when the hydrophobicity of the signal or membrane anchor is incrementally reduced. In contrast, we find that the pmf inhibits translocation of the periplasmic loop when it contains one or two positively charged residues. This inhibitory effect of the pmf is stronger when the hydrophobicity of the inserting procoat protein is compromised. No pmf effect is observed for translocation of an uncharged periplasmic loop even when the hydrophobicity is reduced. We also show that the Delta Psi component of the pmf is necessary and sufficient for insertion of representative constructs and that the translocation effects of charged residues are primarily due to the Delta Psi component of the pmf and not the pH component.

    INTRODUCTION
Top
Abstract
Introduction
References

All membrane proteins must adopt their correct asymmetric orientations in the membrane in order to function correctly. This is achieved according to two major known parameters. The hydrophobic stretches within membrane proteins enable the protein to partition into the membrane and span the bilayer, whereas the hydrophilic regions are retained at the correct face of the membrane as dictated by the "positive inside" rule (1). This rule states that regions rich in positively charged residues are directed to the cytoplasmic face of the inner membrane of Escherichia coli (2, 3). The basis for the positive inside rule has not been pursued diligently until recently (4, 5). One hypothesis is that the proton motive force, Delta µH+ (pmf),1 which renders the cytoplasm basic and the periplasm acidic, favors a cytoplasmic location for positively charged amino acids. The electrical component of the pmf, Delta Psi , imposes an "uphill" barrier for positively charged residues to translocate into the periplasm, whereas negatively charged residues move downhill with the pmf (2). The pmf has been shown to drive the insertion of hydrophilic domains of both sec-dependent (6-8) and sec-independent proteins (9-11).

Evidence for an electrophoretic membrane insertion mechanism has been reported for mutants of the exported protein proOmpA (12) and for derivatives of leader peptidase and procoat (4, 13). It was shown that the pmf promotes the translocation of negatively charged residues within a short periplasmic loop (4, 13) and an amino-terminal tail (13) of a membrane protein. In addition, the pmf appears to impede translocation of an amino-terminal tail when it possesses a positive charge (13) since the tail inserts more efficiently in the absence of the potential. The pmf also contributes to the topology of the protein, since abolishing the pmf causes a partial topology inversion in various positively charged mutants (4). However, similar studies using the M13 procoat protein showed that a positively charged periplasmic loop inserted slightly less efficiently when the pmf was collapsed (13). Therefore, it is not clear whether the electrical potential plays a general role in influencing the membrane topology of proteins.

Another major factor that drives hydrophilic regions of membrane proteins across the phospholipid bilayer is the hydrophobicity of the transmembrane segments. It has been shown that the hydrophobic amino acids within these transmembrane domains are necessary for carboxyl- and amino-terminal translocation of hydrophilic regions (14, 15). Although the importance of hydrophobicity for protein translocation is well known (1, 16), the relative contributions of the hydrophobicity and the pmf for a type II membrane protein have not been examined.

In this study, the role of the pmf in the translocation of positively charged residues was tested. Also, the severity of the barrier of translocating positively charged residues caused by decreasing hydrophobicity of the inserting membrane protein was tested. In addition, we examined whether the pmf can play a primary role for translocation of negatively charged residues across the membrane when hydrophobicity is reduced. We fused an antigenic tag from leader peptidase to the carboxyl terminus of procoat, which enabled us to efficiently immunoprecipitate the proteins with leader peptidase antiserum (17). We have constructed a series of deletions within both the relatively hydrophobic signal sequence and the extremely hydrophobic membrane anchor. We found that translocation of basic residues within the periplasmic loop of procoat is increasingly hindered by the pmf as the hydrophobicity was incrementally reduced. This is in contrast to what is observed with negatively charged mutants. Here we observed that the pmf becomes increasingly critical for translocation as the hydrophobicity of the protein was reduced. This directional reaction to the pmf provides more evidence for an electrophoretic insertion mechanism and suggests that the electrical potential may be the basis for the positive inside rule of inner membrane proteins in agreement with Andersson and von Heijne (4). In addition, our results suggest that a pH gradient is not necessary for insertion of the procoat mutants, and the effects of charged residues on translocation can be attributed to the Delta Psi .

    EXPERIMENTAL PROCEDURES

Strains and Plasmids-- E. coli strain MC1061 (Delta lacX74, araD139, Delta (ara, leu)7697, galU, galK, hsr, hsm, strA) was from our laboratory stocks. XL-1 Epicurean coli (supE44, hsdR17, recA1, endA1, gyrA46, thi relA1, lac-) was from Stratagene. TolC- (lacY1 or lacZ4, gal-6, hisG1(Fs), Delta tolC5, uxaC201, rpsL8 or rpsL104 or rpsL17, malT1(lambda R), mtlA2) was from the E. coli Genetic Stock Center at Yale University. The pING plasmid (18), which contains the arabinose promoter and arabinose regulatory elements, was used to express procoat-lep mutants.

Materials-- Lysozyme, amino acids, phenylmethylsulfonyl fluoride, and CCCP were from Sigma. Proteinase K was from Boehringer Mannheim. The Sequenase version 2.0 kit was from U.S. Biochemical Corp. Tran35S-label, a mixture of 85% [35S]methionine and 15% [35S]cysteine was from ICN. Materials for Quikchange were from Stratagene. Deoxynucleotides were from Amersham Pharmacia Biotech. Qiagen Midi Plasmid Purification Kit was from Qiagen. PAGE-purified oligonucleotides were obtained from Integrated DNA Technologies.

DNA Manipulations-- Plasmid preps were accomplished using the Qiagen midi kit. Mutagenesis was carried out according to the Quikchange procedure, with modifications as described (19). Sequencing was done according to the methods of the Sequenase version 2.0 kit. Mutants verified by sequencing were transformed into MC1061 using the calcium chloride method of Cohen et al. (20).

Protease Mapping Assay-- One milliliter of M9 medium containing 0.5% fructose and 50 µg/ml each amino acid except methionine was inoculated with 20 µl of an overnight MC1061 cell culture bearing the plasmid of interest and allowed to grow for 3 h with shaking at 37 °C. Expression of procoat-lep mutants was induced for 1 h with a final concentration of 0.2% arabinose. The cells were metabolically labeled with 100 µCi of Tran35S-label for 1 min and subsequently transferred to prechilled microcentrifuge tubes. Where indicated, carbonyl cyanide m-chlorophenyl hydrazone (CCCP) was added 45 s prior to labeling at a final concentration of 50 µM. The cells were pelleted for 40 s at 14,000 rpm in a microcentrifuge and then resuspended in 250 µl of Solution I (Tris acetate, pH 8.2, 0.5 M sucrose, and 5 mM EDTA). Spheroplasts were formed by adding lysozyme to a final concentration of 80 µg/ml and 250 µl of ice-cold water immediately thereafter. After a 5-min incubation, MgSO4 was added to a final concentration of 40 mM. The spheroplasts were harvested by spinning again for 40 s and resuspended in 300 µl of Solution II (50 mM Tris-HCl, 0.25 M sucrose, 10 mM MgSO4). The spheroplasts were split into 3 aliquots. The first aliquot received no protease. 7.5 µl of 20 mg/ml proteinase K was added to the other two aliquots, and additionally, 30 µl of 10% Triton X-100 was added to the third aliquot. The spheroplasts were incubated on ice for 1 h and then the proteinase K was inactivated with 5 mM phenylmethylsulfonyl fluoride (final concentration). 250 µl of ice-cold 20% trichloroacetic acid was added, and immunoprecipitations were carried out (21). The proteins were then analyzed by SDS-PAGE using a 17% polyacrylamide gel and subjected to fluorography (22).

Signal Peptidase Processing Assay-- One milliliter of M9 media containing 0.5% fructose and 50 µg/ml each amino acid except methionine was inoculated with 20 µl of an overnight MC1061 cell culture containing the mutant plasmid of interest. Growth was allowed to proceed for 3 h, after which expression of the plasmid-encoded protein was induced with 0.2% arabinose. After a 1-h induction, the cells were metabolically labeled for 1 min with 50 µCi of Tran35S-label. Where indicated, CCCP was added at a final concentration of 50 µM, and after 45 s, Tran35S-label was added as before. The cells (500 µl) were transferred to prechilled microcentrifuge tubes containing 500 µl of 20% trichloroacetic acid. After a 30-min precipitation, the samples were analyzed by immunoprecipitation, SDS-PAGE, and fluorography as described above.

Delta pH Studies-- One-milliliter samples of M9 medium were inoculated with 20 µl of an overnight culture of TolC- cells harboring the plasmid of interest. After allowing growth for 3 h, the cells were resuspended in 1 ml of fresh M9 medium having a pH value of either 6.5 or 7.5. Ten microliters of 20% arabinose was added to induce synthesis of the plasmid-encoded proteins, and growth was allowed for 30 min more. The cultures were then labeled with 50 µCi of Tran35S-label for 1 min. Where indicated, 50 µM CCCP (final concentration) was added 45 s prior to labeling. The cells (500 µl) were aliquoted into tubes containing an equal volume prechilled 20% trichloroacetic acid, and immunoprecipitation was performed as before.

Quantitation of Insertion Data-- X-ray films were scanned using an AppleOne Scanner. The insertion efficiencies were then determined by quantitation of the appropriate bands on the film using NIH Image, a public domain program that can be found on-line.2 For the signal peptidase cleavage assays, the percent of insertion across the membrane was determined by calculating the percent of the processed band appearing on the gels. For full-length constructs, and leader sequence deletion mutants, two methionines were lost upon signal peptidase cleavage, so we used Equation 1 to determine membrane insertion.
<UP>Signal peptidase cleavage %</UP>=
  <FR><NU>(7/5 <UP>processed procoat-lep</UP>)</NU><DE><UP>7/5 processed procoat-lep</UP>+<UP>unprocessed procoat-lep</UP></DE></FR>×100 (Eq. 1)
For membrane anchor deletions (Delta 48-51, Delta 48-53, and Delta 48-55), two out of six methionines were lost, so we used Equation 2.
<UP>Signal peptidase cleavage</UP> %=
  <FR><NU>(6/4 <UP>processed procoat-lep</UP>)</NU><DE><UP>6/4 processed procoat-lep</UP>+<UP>unprocessed procoat-lep</UP></DE></FR>×100 (Eq. 2)
For the protease mapping assays, calculation of insertion efficiencies of procoat-lep was done in a similar manner as above with the following exceptions. The percentage of the protease-resistant fragment (prf) (slightly lower molecular weight than the mature band) was calculated and used as the percentage of membrane insertion. The percentage of cells that were converted to spheroplasts was determined for non-CCCP-treated cells with OmpA and ribulokinase controls and used to calculate the percent of protein that inserted (Equation 3).
<UP>Spheroplasts</UP> (%)=1−<FENCE><FR><NU><UP>OmpA remaining after proteinase K treatment</UP></NU><DE><UP>OmpA remaining after proteinase K treatment</UP>+<UP>untreated OmpA</UP></DE></FR></FENCE>×100 (Eq. 3)
<UP>Final insertion </UP>(%)=(<UP>insertion %/spheroplast</UP> %)×100.

Determination of Summed Hydrophobicity-- The summed hydrophobicity (H) of the transmembrane regions of procoat that were manipulated was determined as described (23) using the GES scale (24). We calculated H for the wild-type and mutant procoat-lep proteins by adding the hydrophobicity of the uninterrupted stretch of uncharged amino acids within the predicted transmembrane region.

    RESULTS

The Proton Motive Force Becomes Increasingly Important for Translocation of Negatively Charged Residues When the Hydrophobicity of Procoat Is Reduced-- The M13 coat protein is synthesized in a precursor form, termed procoat, with a 23-residue signal peptide. Procoat inserts into the membrane as a loop bordered by two hydrophobic domains (25). One hydrophobic domain is the signal peptide and the other is a membrane anchor in the mature coat protein. In our studies, the procoat protein contains a leader peptidase fragment of 103 residues fused to the carboxyl-terminal region of procoat, as previously published (13, 17). This protein is termed procoat-lep (Fig. 1A, lep is depicted by a zig-zag line). The wild-type periplasmic domain of procoat-lep (see Fig. 1A) contains the following charged residues: glutamic acid (position 25), aspartic acid (position 27), aspartic acid (position 28), lysine (position 31), and glutamic acid (position 43).


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Fig. 1.   Procoat-lep and mutants used to study the role of hydrophobic forces in membrane insertion. A, membrane topology of wild-type procoat-lep. The hydrophobic domains are represented by rectangles; the lep epitope by a zig-zag line. B, procoat-lep mutants with sequential deletions in both the signal sequence (Delta 9-10, Delta 9-12, Delta 9-14) and the membrane anchor (Delta 48-51, Delta 48-53, Delta 48-55). The total hydrophobicity of the native and mutant transmembrane segments was determined (23). The energy of transfer (kcal/mol) from an aqueous to a nonpolar environment for each amino acid side chains was summed and used as a measure of hydrophobicity lost. For simplicity, no charges are shown in the periplasmic loop in procoat-lep in B.

Previously, it had been shown that hydrophobic forces play a considerable role in membrane insertion of the wild-type procoat (25, 26). The pmf plays a secondary role for translocation of the acidic loop in the wild-type procoat and only stimulates the membrane insertion across the membrane (13). To test whether the pmf can play a primary role in membrane insertion if the hydrophobicity of the signal/membrane anchor is decreased, we first employed oligonucleotide-directed mutagenesis to change the lysine at position 31 and glutamic acid at position 43 to alanines. We termed the resulting construct PClep. All previous studies on the membrane insertion of procoat had utilized constructs that contained the wild-type residues at these positions in order to study the net charge of the loop (13). We next constructed a series of deletion mutants that are deficient in hydrophobic amino acids within either the leader sequence or the membrane anchor sequence, as shown in Fig. 1B. We deleted two, four, and six amino acids in the moderately hydrophobic signal sequence, and four, six, and eight residues in the extremely hydrophobic membrane anchor. The total hydrophobicity for the transmembrane segment from which amino acids were removed was determined (23). The hydrophobicity scale used was the transfer free energy (kcal/mol) of each amino acid side chain from water to a non-polar environment (24).

We used a protease accessibility assay to analyze the insertion of the procoat mutants into the membrane. We labeled 1 ml of exponentially growing E. coli cells with 100 µCi of Tran35S-label and then converted the cells into spheroplasts using the lysozyme/EDTA method previously described (27). These cells, or cells lysed with Triton X-100, were digested for 1 h with proteinase K. Aliquots were immunoprecipitated with antiserum to leader peptidase and analyzed by SDS-PAGE and fluorography. Digestion of inserted procoat-lep with the protease results in a protease-resistant fragment (prf) of slightly lower molecular weight than the mature band that is visible after immunoprecipitating with antibody to the carboxyl-terminal leader peptidase epitope, as published previously (13, 17). We quantitated the relative amount of the precursor form of procoat-lep that remained in the cytoplasm (see upper band, depicted by p in Fig. 2A, in protease-treated lanes) and compared this to the amount that had inserted across the membrane and was digested with protease (see the protease-resistant lower molecular weight fragment, prf). In addition, the protease-treated samples were compared with an aliquot of spheroplasts which were not subjected to digestion. We also performed a control in which we lysed an aliquot of spheroplasts with Triton X-100 to confirm that the periplasmically situated domains were not protease-resistant. Pulse-chase experiments and studies using IT41 (28), a temperature-sensitive signal peptidase strain, revealed which bands correspond to the mature and precursor form of the protein (data not shown). The upper band in Fig. 2A (non-protease-treated lanes) represents the precursor protein (p) in the cytoplasm, whereas the lower band (non-protease-treated lanes) corresponds to PClep that had inserted across the membrane and had been cleaved to the mature protein by signal peptidase. In the experiments where deletions were made in the signal sequence, we could not use the processing of procoat to coat by signal peptidase as an indication of insertion. This was due to the reduced efficiency of signal peptidase cleavage caused by the shortened signal peptide.


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Fig. 2.   Protease mapping to determine the insertion efficiencies of negatively charged procoat-lep mutants. A, insertion across the membrane of (-3) mutants with the pmf. MC1061 cells bearing plasmids encoding procoat-lep mutants were grown in M9 medium containing 50 µg/ml each amino acid except methionine and 0.5% fructose to mid-log phase. After an induction with 0.2% arabinose for 1 h, the cells were metabolically labeled with 100 µCi of Tran35S-label for 1 min. The cells were then converted to spheroplasts and analyzed by protease mapping, as outlined under "Experimental Procedures." After proteolysis, samples were immunoprecipitated with antisera to leader peptidase, run on 17% SDS-PAGE, and subjected to fluorography. p, precursor; prf, protease-resistant fragment (represents inserted material). B, membrane insertion of (-3) mutants in the absence of the pmf. MC1061 cells expressing the mutants containing three negatively charged amino acids were treated exactly as in A, except for the following. CCCP (final concentration of 50 µM) was added to collapse the pmf for 45 s prior to labeling with Tran35S-label. p, precursor; f, fragment (a possible proteolytic fragment that does not insert across the membrane). C, controls used to determine the degree of spheroplasting and lysis. Samples were immunoprecipitated with antisera to OmpA (an outer membrane protein that is potential dependent for translocation) and ribulokinase (Rib) (a cytoplasmic protein).

For negatively charged procoat-lep constructs, as the hydrophobicity of each transmembrane segment is incrementally decreased, insertion across the membrane also decreases in stepwise increments (see Table I for percent efficiencies and Fig. 2A for protease mapping data). PClep(-3), the full-length construct, inserts 94% efficiently into the membrane with the pmf present (-CCCP). As can be seen, PC-lep (-3) is efficiently processed by signal peptidase to the mature protein (band below the precursor form). The mature form of PClep (-3) was cleaved to a slightly lower molecular weight form (prf) by proteinase K. For the series of constructs Delta 9-10 (-3), Delta 9-12(-3), and Delta 9-14(-3), which contain increasingly larger deletions in the leader sequence, the amount of prf decreases as the size of the deletion increases (compare lanes 5, 8, and 11 in Fig. 2A). The series of three mutants possessing membrane anchor deletions, Delta 48-51(-3), Delta 48-53(-3), and Delta 48-55(-3) shows the same trend, with the largest deletion almost collapsing insertion at 15%. The major lower bands in the non-protease-treated lanes 1, 4, 7, 13, 16, and 19 are the result of processing by signal peptidase to the mature protein as the periplasmic loop of procoat crosses the membrane. The lowest band seen in lanes 7 and 16 is most likely the result of cleavage by an unknown protease. Testing these constructs in the temperature-sensitive signal peptidase strain, showed that the lowest band (f) in these lanes was not the result of signal peptidase cleavage. Fig. 2C shows representative controls for these experiments. We immunoprecipitated an equal portion of sample with antiserum raised against OmpA, a protein that is exported in a pmf-dependent manner to the periplasm (8). In the presence of the pmf, this control shows the degree to which the cells were converted to spheroplasts when comparing the non-protease-treated cells with those that had been digested by proteinase K (compare lanes 1 and 2). We also immunoprecipitated with antiserum to ribulokinase, a cytoplasmic protein. Intact ribulokinase in the protease-treated lanes (see lane 2) indicates that minimal lysis occurred when forming spheroplasts. We had a difficult time obtaining intact spheroplasts for Delta 48-55(-3), so the insertion efficiency, when corrected for lysis, is higher than that predicted from the gel (lanes 19 and 20).

                              
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Table I
Mutants containing negative charges in the periplasmic loop and their insertion efficiencies with and without the pmf

We next examined the effects of collapsing the pmf by adding 50 µM CCCP, a protonophore, 45 s before labeling the cells with Tran35S-label. Comparing lanes 1 and 2 in Fig. 2A with lanes 1 and 2 in Fig. 2B shows that the insertion of the full-length procoat-lep under these conditions decreases from 95 to about 69%, confirming that this protein is partially dependent on the pmf for insertion (10, 13), see also Table I. Interestingly, except for the Delta 9-10 and Delta 9-12 deletion mutants, membrane insertion of the mutants was abolished without a pmf (CCCP-treated cells), as shown by the small amounts of prf produced in the protease-treated lanes. The lower molecular weight band (depicted by a f) in lanes 4-5, 7-8, 10-11, 13-14, 16-17, and 19-20 is most likely the result of proteolysis by an unknown protease within the cytoplasm that occurs downstream of the signal peptidase-processing site. This in vivo proteolytic fragment of procoat does not contain a domain exposed across the membrane since its molecular weight does not shift upon treatment with proteinase K. Even a two-amino acid deletion in the leader sequence (Delta 9-10(-3)) has a measurable effect on procoat assembling across the membrane, at 20% (the prf in lane 5 can be discerned directly below the proteolytic fragment f described above). A six amino acid deletion within the signal sequence (Delta 9-14(-3)) completely abolishes insertion (lanes 10 and 11). Immunoprecipitating with OmpA antiserum served to confirm that the pmf was actually collapsed (see representative data in Fig. 2C). The precursor form of the protein (proOmpA), which is visible in lanes 4 and 5, is not digested by protease, indicating that this outer membrane protein remained in the cytoplasm. The spheroplasts were mostly intact as shown by immunoprecipitation with antiserum to ribulokinase, a cytoplasmic protein. These results show that the pmf is absolutely required to insert procoat across the membrane when the hydrophobicity is reduced, indicating that the pmf plays a primary role for inserting negatively charged residues across the membrane when the hydrophobic forces of the inserting procoat are compromised.

Fig. 3 shows the results of protease accessibility assays on mutants of procoat-lep containing no charges in the periplasmic loop. The glutamic acid residue at position 25 and the aspartic acid residues at positions 27 and 28 were changed to glutamine and asparagine residues, respectively. In addition, the hydrophobic amino acids in the signal and membrane anchor regions (refer to Fig. 1B) were deleted as in the (-3) series of mutants. Table II shows the insertion efficiencies of these seven mutants. As the hydrophobicity is decreased, there is a concomitant decrease in membrane insertion, as evidenced by a decrease in the amount of prf (compare in Fig. 3 lanes 4 and 5, and 7 and 8, etc.). These results show hydrophobic forces are important not only to insert a negatively charged region but also to insert an uncharged hydrophilic domain. Next we collapsed the pmf with CCCP. Here, in contrast to the (-3) mutants, the neutral mutants insert across the membrane with similar efficiencies as when the pmf is present. These results, when compared with the results for the (-3) mutants, confirm that the pmf only contributes to insertion of procoat when acidic amino acids are translocated.


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Fig. 3.   Pmf-independent translocation of uncharged loops when the hydrophobicity of procoat is reduced. A, insertion of neutral mutants of procoat-lep in the presence of the pmf. MC1061 cells bearing the plasmid of interest were treated exactly as in the legend to Fig. 2A. p, precursor; prf, inserted fragment. B, membrane insertion of neutral constructs in the absence of the pmf. MC1061 cells harboring the plasmid of interest were treated exactly as in Fig. 2B. C, controls as in Fig. 2.

                              
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Table II
Mutants containing no charges in the periplasmic loop and their insertion efficiencies with and without the pmf

Positive Charges in the Periplasmic Loop Impede Translocation in the Presence of the pmf-- We next investigated whether the translocation of positively charged residues would be hindered by the pmf, as postulated by an electrophoretic mechanism. For these experiments, we used the assay that relies on signal peptidase processing to ascertain whether membrane insertion has occurred. We labeled exponentially growing cells with Tran35S-label and immunoprecipitated procoat-lep with leader peptidase antiserum. We analyzed the processing efficiencies by signal peptidase cleavage of full-length procoat proteins and of mutants possessing two different deletions in the membrane anchor sequence, all containing positive charges inserted into the periplasmic loop (see Table III for positions of basic amino acid substitutions and insertion efficiencies). After labeling 1 ml of exponentially growing cells with 50 µCi of Tran35S-label for 1 min, we acid-precipitated the proteins with an equal volume of 20% trichloroacetic acid. The proteins were immunoprecipitated with antibody to leader peptidase and analyzed on 17% SDS-PAGE gels.

                              
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Table III
Mutants containing positive charges in the periplasmic loop and their insertion efficiencies with or without the pmf

Fig. 4A shows the signal peptidase processing results for mutants possessing a positively charged lysine residue near the center of the periplasmic loop at position 32. The top band in each lane represents the uninserted precursor protein (p), and the lower band corresponds to the mature form (m) which results from signal peptidase cleavage. This mature protein band was identified using a temperature-sensitive signal peptidase strain (28). The results (Fig. 4A) show that as the hydrophobicity of the membrane anchor was decreased, processing of the procoat protein was decreased. The positively charged procoat protein with an intact membrane anchor (PClep(+1)) was 61% processed in a 1-min pulse label, compared with 55 (Delta 48-51(+1)) and 23% (Delta 48-53 (+1)) processing with 4 and 6 residues deleted in the membrane anchor, respectively (compare lanes 1, 3, and 5). Strikingly, when the pmf is collapsed, the insertion of procoat with a positively charged residue becomes better, as indicated by increased processing of procoat (see lanes 2, 4, and 6). This enhanced translocation in the absence of the pmf increases as the hydrophobicity declines; there is a 20% increase in translocation when CCCP is added for PClep(+1) but a 37% increase for Delta 48-53(+1). The efficient translocation of a positive charge with high hydrophobicity of the membrane anchor shows that hydrophobic forces can overcome such a barrier even in the presence of the pmf.


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Fig. 4.   The pmf inhibits translocation of positively charged residues. A, translocation of one positive charge in the center of the loop of procoat mutants. MC1061 cells in M9 medium bearing the plasmid of interest were grown for 3 h, induced for 1 h with a final concentration of 0.2% arabinose. The cells were then metabolically labeled with 50 µCi of Tran35S-label for 1 min. For the pmf study, cells were treated 45 s prior to labeling with 50 µM CCCP (final concentration). The samples were then subjected to immunoprecipitation, SDS-PAGE, and fluorography. B, translocation of two spaced positive charges within procoat mutants. MC1061 cells were treated as before. C, translocation of two basic residues immediately after the signal sequence. D, translocation of two positive residues in the middle of the periplasmic loop of procoat mutants.

It should be noted that, in some cases, the addition of CCCP results in a decrease in protein synthesis of procoat-lep (see data for Delta 48-51 and Delta 48-53). This most likely is due to CCCP, which is an uncoupler of oxidative phosphorylation, lowering ATP levels in the cytosol.

Fig. 4B depicts the translocation results of constructs containing two lysine residues spaced within the loop at positions 25 and 32. These mutants are termed procoat-lep (+2S) with the S indicating that the charges are spaced apart. Again, as the hydrophobicity of the membrane anchor decreases, the processing of procoat decreases in the presence of the pmf (-CCCP) (compare lanes 1, 3, and 5). Procoat processing is 58, 34, and 0% for PClep (+2S), Delta 48-51 (+2S), and Delta 48-53(+2S), respectively (see Table III). When the pmf is collapsed with CCCP, the results are even more dramatic than for a single positive charge in the periplasmic loop. PClep(+2S) with two positively charged residues and an intact membrane inserts 17% better in the absence of the pmf. The positively charged mutant PClep Delta 48-51(+2S) (lanes 3 and 4), with a deletion of 4 residues in the membrane anchor, inserts across the membrane a dramatic 35% better when the pmf is collapsed compared with when the pmf is maintained. The Delta 48-53(+2S) mutant with a larger deletion within the membrane anchor cannot insert at all in the presence of the pmf, although it inserts to significantly high levels (44%) in the absence of the pmf (see lanes 5 and 6). This is approaching the insertion efficiency of the procoat mutant with two positively charged residues within an intact membrane anchor (PClep(+2S)) in the presence of the pmf. The processing results of procoat mutants possessing two positively charged lysine residues directly adjacent to the signal sequence (+2H1 constructs) at positions 25 and 26 are very similar to the findings for the (+2S) mutants (Fig. 4C). Finally, when we changed two adjacent residues at positions 32 and 33 in the middle of the loop to positively charged lysine residues (see PClep(+2M) constructs in Fig. 4D), we found that even the full-length as well as deletion mutant proteins were prohibited from insertion with or without the pmf present. This suggests that the center of the periplasmic loop cannot tolerate more than one positively charged residue for translocation. The data for Fig. 4, taken together, provide direct evidence that the pmf inhibits translocation of positive charges.

The Delta pH Is Not Required for the Insertion of Procoat-- We wanted to determine whether the Delta pH component of the pmf is necessary for the translocation effects of charged residues. We examined insertion of the procoat mutants under conditions where the transmembrane pH gradient was close to 0 or 1 by changing the pH of the periplasmic medium to 7.5 or 6.5, respectively. This was done using a TolC- mutant as described under "Experimental Procedures." In E. coli, the cytoplasmic pH is maintained at about 7.5 (29). The studies presented thus far in the paper were performed in a medium with a pH of 7.0, a pH gradient close to 0.5.

Fig. 5 shows the processing of procoat with either no charges in the periplasmic loop (Delta 48-53(0)), three negative charges adjacent to the leader sequence (Delta 48-51(-3)), or two positive charges spaced apart in the periplasmic loop (48-53(+2S)). We grew E. coli cells expressing these procoat mutants to mid-log phase in M9 medium and then resuspended the cells in minimal medium with a pH value of 7.5. After induction of the procoat mutants with 0.2% arabinose for 30 min, we labeled the cells with Tran35S-label for 1 min as in the studies shown in Fig. 4. To half of the cultures, we added CCCP for 45 s prior to labeling. Fig. 5 shows the signal peptidase processing results. As seen in Fig. 5A, the neutral and negatively charged procoat protein inserts across the membrane with a similar pmf requirement at pH 7.5 as it did at pH 7.0 (see Fig. 2, lanes 13 and 14 for Delta 48-51(-3), Fig. 3, lanes 16 and 17 for Delta 48-53(0)). The pmf requirement of procoat with negatively charged residues at pH 7.5 suggests that the pH gradient is not essential for the insertion of procoat across the inner membrane. Similarly, the positively charged procoat protein cannot insert at pH 7.5 as was observed at pH 7.0 (see Fig. 4B, lanes 5 and 6 for Delta 48-53(+2S)) but can when the pmf is dissipated with CCCP. These data are consistent with the transmembrane electrical gradient driving negatively charged residues across the membrane and hindering positively charged residues.


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Fig. 5.   Membrane insertion of positively charged, neutral, and negatively charged mutants at different pH values. A, insertion of a positively charged, a neutral, and a negatively charged construct at pH 7.5 TolC- cells harboring the plasmid of interest were grown for 2 h in M9 medium. The cells were then pelleted and resuspended in minimal medium with a pH of 7.5. Expression of procoat was induced for 30 min by the addition of 0.2% arabinose (final concentration). The cells were then metabolically labeled for 1 min with 50 µCi of Tran35S-label. Where indicated, CCCP was added 45 s before labeling at a final concentration of 50 µM. The samples were analyzed as in Fig. 4. B, insertion of the above constructs at pH 6.5. The same protocol was followed except, after growth in M9 medium at pH 7.0, the cells were resuspended in minimal medium at pH 6.5. The samples were precipitated by adding an equal volume of 20% trichloroacetic acid and subjected to immunoprecipitation, SDS-PAGE, and fluorography as before.

However, it is still possible that a pH gradient greater than the normal physiological level can assist in insertion. To test this idea, we performed an identical study to the one shown in Fig. 4A, except that we resuspended the cells in a minimal medium with a pH of 6.5. Fig. 5B shows that there may be a small contribution of this increased pH gradient for the Delta 48-51(-3) (lanes 3 and 4) and Delta 48-53(0) mutant (lanes 1 and 2). Insertion across the membrane of Delta 48-53(+2S) is still blocked with the increased Delta pH (lane 5). Interestingly, the positively charged mutant inserts more efficiently at this pH when CCCP is added to collapse the normal pmf (compare Fig. 5, A and B, lane 6). These studies with charged residues suggest that it is primarily the Delta psi component of pmf that affects that translocation of charged residues and not the pH component.

    DISCUSSION

The mechanism of membrane insertion of the M13 procoat protein has been studied in great detail (25, 30). Procoat inserts across the membrane in a sec-independent manner, since conditions that block the sec machinery have no effect on its insertion (31). It is thought that it inserts across the membrane by a spontaneous mechanism since procoat can assemble into protein-free liposomes (32). Translocation of its acidic loop to the periplasm is hypothesized to be driven by a synergistic entry of the two hydrophobic regions into the lipid bilayer (33). Although the pmf stimulates translocation, it is not absolutely required for insertion (13). The positively charged residues before the signal sequence and immediately after the membrane anchor remain in the cytoplasm. Thus, the topology of the procoat protein agrees nicely with the positive inside rule, which states that basic domains act as topological determinants because they are retained in the cytoplasm (for reviews see Refs. 1, 34, and 35).

Several factors have been hypothesized to contribute to the positive inside rule. First, anionic phospholipids are determinants of membrane protein topology. van Klompenburg and colleagues (5) showed that the membrane orientation of leader peptidase constructs depended on the anionic phospholipid content within the membrane. A second factor contributing to the positive inside rule is the internal dipole potential within the membrane that may be as large as 240 mV, interior positive (36, 37). This dipole potential would make it difficult for positive charges to enter the membrane. The anionic and dipole potential may explain why membrane proteins in the ER and thylakoid membranes obey the positive inside rule even though these membranes have a very low transmembrane potential. However, in large part, the available data in bacteria are consistent with the pmf (positive outside, negative inside) determining the asymmetry of the topology via an effect on positively charged residues (4). In this report, we provide direct evidence that charged amino acids in hydrophilic domains of membrane proteins are directed to the correct compartment of E. coli by the pmf.

We show here that the pmf plays a critical role in translocating negatively charged residues across the membrane when the hydrophobicity of the signal or membrane anchor is reduced. The pmf only stimulates translocation at most 30% for the wild-type protein (13) and a mutant containing only three negatively charged residues in the periplasmic loop (Fig. 2A). A small deletion of four amino acids in the membrane anchor or six in the signal sequence renders the negatively charged periplasmic loop translocation-incompetent in the absence of the pmf. This exemplifies the necessity for both hydrophobic and proton motive forces for translocating negative charges across the membrane.

The addition of the protonophore, CCCP, was used in this study at concentrations that dissipate the pmf to inhibit or block the translocation of the periplasmic region of procoat across the membrane. The CCCP treatment blocks the translocation step of the membrane biogenesis pathway of the M13 coat protein (10, 13). It is unlikely that CCCP treatment inhibits the folding of procoat in the cytosol or electrostatic binding of procoat to the surface of the membrane because treatment of the neutral mutants with CCCP shows no effect on translocation.

Whereas the pmf is important for translocation of negative charges across the membrane, our results so far with procoat do not reveal that negative charges play an active role in the translocation of the hydrophilic domain in the presence of the pmf. This is in striking contrast to the case of the Pf3 coat protein, which only moves its N-tail across the membrane in the presence of the pmf and when negatively charged residues are present (38). In the case of Pf3-leader peptidase, we found that one of the acidic residues in the amino-terminal tail plays an active role in protein translocation of the amino terminus of Pf3-leader peptidase (19). However, this occurred only when the hydrophobicity of apolar domain one of leader peptidase was decreased (19). Therefore, these recent results indicate that different sec-independent proteins, although requiring the pmf to translocate acidic amino acids when the hydrophobicity is low, may insert into the membrane by different mechanisms.

When no charges are present in the translocated domain, procoat can insert across the membrane rather efficiently with large deletions in the signal or membrane anchor (Fig. 3). For all the neutral mutants studied, the pmf did not stimulate translocation, suggesting that the pmf is required only in the presence of negatively charged residues. Translocation of an uncharged region is more efficient compared with translocation of a negatively charged region even in the presence of a pmf (compare Fig. 2 with 3). Thus, even with a pmf, negative charges can still act as a barrier. Intriguingly, even when most of the hydrophobic core of the signal is deleted, over 60% of the procoat protein can translocate when this region lacks charges. This is in contrast to translocation of three negatively charged residues with procoat with a signal peptide deletion in the presence of the pmf, which is only 19%. This confirms the results of Rohrer and Kuhn (39) in which they show the function of a leader peptide is to translocate a charged region. It is important to emphasize the significance of the pmf in membrane insertion for sec-independent proteins containing charged residues. The pmf appears to act directly on the polar domains of the proteins, since no protein translocation machinery has been found to be required. In contrast, for sec-dependent proteins, the pmf is required in a late stage of translocation for efficient export of periplasmic domains after entering the proposed SecYEG channel (40-42) and may act on the machinery itself (43). Moreover, it has been found that the pmf is required even for the sec-dependent secretion of an uncharged protein domain (44). Our work here illustrates the mechanistic differences between sec-independent proteins and proteins that are known to require this machinery.

Most intriguingly, we have also shown directly for the first time that the pmf (positive outside, negative inside) inhibits translocation of positively charged residues across the membrane. Our results provide additional evidence that the pmf can act as a determinant of the positive inside rule of bacterial membrane proteins. Moreover, we show that the inhibitory effect of the pmf on translocating positive charges is larger when the hydrophobicity of the membrane anchor is decreased (Fig. 4). This shows that hydrophobic forces assist in driving the charged residues across the membrane. When the hydrophobic forces are compromised, the charges become a barrier to insertion, especially in the presence of a positive outside potential. In previous studies that examined positive charges in the periplasmic loop of procoat, the negatively charged amino acid near the membrane anchor was always present, and thus complicated the results (13). In the present report, we examine the effects of just one or two positive charges in the absence of the acidic amino acid. Particularly compelling is the finding that two positively charged mutants with large deletions in the membrane anchor, Delta 48-53(+2S), and Delta 48-53(+2H1), did not insert across the membrane until the pmf was dissipated with CCCP. This is in contrast to the mutant Delta 48-53(+1), which partially inserts into the membrane in the presence of the pmf. Thus, the effects of the positive charges are accumulative, lending further evidence that an electrophoretic mechanism is in operation to help insert sec-independent membrane proteins. It is unclear why none of the (+2M) mutants inserted into the membrane, even without a pmf present.

The requirement for specific components of the pmf has been examined for several secreted proteins (45, 46) but for only one sec-independent protein, the E. coli melibiose permease (11). Those studies found that both components of the pmf could be utilized for secretion or insertion. Here we show for the first time that the Delta Psi is sufficient to hinder the insertion of basic residues and to promote the translocation of negatively charged amino acids (Fig. 5A). The finding that positive charges are able to better translocate when the pH of the periplasm is set at 6.5 and when CCCP is added was unexpected. This may be due to the fact that anionic phospholipids can act as determinants of membrane topology as well and prevent translocation of positively charged residues (5). It is possible that the positive charges in the periplasmic loop of procoat were able to move across the membrane more efficiently when CCCP abolishes the pH gradient. These positive charges might not be attracted as strongly to the anionic phospholipids (at pH 6.5) because of increased protonation of these anionic lipids on the cytoplasmic side of the membrane.

The significance of our studies may also extend into the eukaryotic realm. A subset of inner mitochondrial membrane proteins is sorted from the matrix by a process analogous to the sec-independent process found in bacteria. It has been found that certain amino-terminal region and loops of these proteins require the Delta Psi for insertion and that these domains are often enriched in acidic residues (for review see Ref. 47). In addition, the topology of mitochondrial inner membrane proteins conforms to the positive inside rule that is found in E. coli (48). Most importantly, the introduction of positively charged residues blocks amino-terminal translocation from the matrix (47, 49), thereby lending evidence that the pmf is one basis for the positive inside rule in mitochondria.

    FOOTNOTES

* This work was supported by National Science Foundation Grant MCB-9808843 (to R. E. D.).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.

Dedicated to the memory of Tracy A. Schuenemann.

dagger Deceased.

Dagger To whom correspondence should be addressed. Fax: 614-292-1532; E-mail: dalbey{at}chemistry.ohio-state.edu.

2 NIH Image, a public domain program, can be found on-line at the following address: http://rsb.info.nih.gov/nih-image/.

    ABBREVIATIONS

The abbreviations used are: pmf, proton motive force; CCCP, carbonyl cyanide p-chlorophenylhydrazone; PAGE, polyacrylamide gel electrophoresis; prf, protease-resistant fragment.

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Abstract
Introduction
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