Polarity and Charge of the Periplasmic Loop Determine the YidC and Sec Translocase Requirement for the M13 Procoat Lep Protein*

Background: Procoat protein mutants require the Sec translocase in addition to YidC. Results: Subtle changes in the polarity of the translocated region alter the mode of insertion. Conclusion: The translocase requirement increases as the energy transfer expense of the translocated region increases. Significance: Selection of the translocase is determined by the energy barrier of translocation. During membrane biogenesis, the M13 procoat protein is inserted into the lipid bilayer in a strictly YidC-dependent manner with both the hydrophobic signal sequence and the membrane anchor sequence promoting translocation of the periplasmic loop via a hairpin mechanism. Here, we find that the translocase requirements can be altered for PClep in a predictable manner by changing the polarity and charge of the peptide region that is translocated across the membrane. When the polarity of the translocated peptide region is lowered and the charged residues in this region are removed, translocation of this loop region occurs largely by a YidC- and Sec-independent mechanism. When the polarity is increased to that of the wild-type procoat protein, the YidC insertase is essential for translocation. Further increasing the polarity, by adding charged residues, switches the insertion pathway to a YidC/Sec mechanism. Conversely, we find that increasing the hydrophobicity of the transmembrane segments of PClep can decrease the translocase requirement for translocation of the peptide chain. This study provides a framework to understand why the YidC and Sec machineries exist in parallel and demonstrates that the YidC insertase has a limited capacity to translocate a peptide chain on its own.

In Escherichia coli, two main translocases have been identified to date, and they are involved in inserting inner membrane proteins (1)(2)(3). The Sec translocase is the major translocase of E. coli and is responsible for inserting the majority of the inner membrane proteins as well as translocating exported proteins across the inner membrane into the periplasmic space. The second is the YidC membrane insertase, which plays a role in inner membrane protein insertion and assembly.
The Sec translocase is a heterotrimeric protein composed of the SecYEG protein-conducting channel, the accessory SecDF-YajC trimeric complex, and the peripheral subunit SecA. SecA is mainly involved in the insertion of proteins with large periplasmic domains (4 -6) and has been shown to be a motor ATPase involved in driving the protein through the translocation channel. It is believed to insert ϳ20 -30 residues for every ATP hydrolysis cycle (7). The precise function of SecDF-YajC remains unclear, but it is important for the insertion of some membrane proteins (8). SecDF is believed to be involved posttranslationally in translocation and helps prevent backward movement of the preprotein via interaction of the substratetranslocated domain with the large periplasmic P1 domain on SecD (9).
In the year 2000, a new inner membrane protein, YidC, was discovered to be involved with membrane protein insertion (10,11). It is believed to function in concert with the Sec translocase to integrate proteins into the lipid bilayer and can also act, in certain cases, as a chaperone assisting membrane proteins to attain their correct membrane-embedded fold within the membrane (3). YidC can also function as an insertase, independent of the Sec translocase (12,13). In this pathway, YidC promotes the membrane insertion of the M13 procoat and Pf3 coat protein, which were previously thought to insert into the membrane "spontaneously" (11,14). At this time, YidC has been found to be required for the membrane insertion of several endogenous membrane proteins such as subunit C of F 1 F 0 -ATPase (13,(15)(16)(17), subunit II of cytochrome bo oxidase (18 -20), TatC (21), and MscL (22).
All of the YidC-only pathway substrates identified have a small periplasmic domain. Because of this common feature, it has been suggested that YidC is only capable of acting as an insertase for proteins with small periplasmic domains, whereas larger periplasmic domains require the Sec pathway for translocation. Interestingly, several studies have shown that modification of the primary structure of different YidC-only substrates can alter their insertion pathway to a Sec-dependent mechanism. The first observation was that an extension of the periplasmic loop of the M13 procoat by 174 residues (from OmpA) 2 switches the protein from a Sec-independent pathway * This work was supported by National Science Foundation Grant MCB-to the Sec pathway (5). Many other changes to the membrane protein have been found to alter the insertion pathway from Sec-independent to Sec-dependent (23,24). Subtle changes such as increasing the number of charged residues in the periplasmic loop, the length of the periplasmic loop, or changing the location of charged residues have been shown to change the membrane biogenesis pathway of the M13 procoat from Sec-independent to Sec-dependent (8,23,24). Roos et al. (25) demonstrated that certain mutations in the procoat periplasmic loop result in the preprotein binding to the SecA protein, which then engages the SecYEG machinery. In contrast, with a single-spanning Pf3 TMLep model protein, the addition of a positively charged residue to the N-tail makes its insertion SecYEG-dependent but not SecA-dependent (28). Although these studies showed that mutations in the membrane proteins' translocated region could change the translocase requirement, the reason for these results was not understood.
In this study, we present the polarity/charge hypothesis, which proposes that the membrane transfer expense of the translocated region determines the translocase requirement. We show that the insertion mechanism of Procoat lep (PClep) can be altered in a predictable way by changing the charge and polarity of the peptide region that is translocated. Although translocation of a peptide of low polarity can occur in a YidC/ Sec-independent manner, YidC is required for translocation when the periplasmic loop is made more polar. Further increases in the polarity, by the substitution of positively charged residues or inserting negatively charged residues in PClep, change the mechanism such that both the YidC and Sec translocases are needed. However, substitution of apolar amino acids, with charged residues in the transmembrane segment of PClep to decrease its hydrophobicity, can also increase the requirements for a translocase.

EXPERIMENTAL PROCEDURES
Materials-Sodium azide and lysozyme were purchased from Sigma. Proteinase K (PK) was purchased from Qiagen; isopropyl 1-thio-␤-D-galactopyranoside was from Research Products International Corp.; PMSF was purchased from United States Biochemical (Affymetrix). Tran 35 S-label, a mixture of 85% [ 35 S]methionine and 15% [ 35 S]cysteine, 1000 Ci/mmol, was from PerkinElmer Life Sciences. Antisera to leader peptidase (anti-Lep) and outer membrane protein A (anti-OmpA) were from our own laboratory collection.
Strains, Plasmids, and Growth Conditions-JS7131, the E. coli YidC depletion strain, and MC1060 are from our collection. CM124, the SecE depletion strain, was obtained from Beth Traxler and is described in Ref. 26. The yidC and secE genes in the JS7131 and CM124 strains, respectively, are under the control of the araBAD promoter. JS7131 cells were cultured at 37°C for 3 h in LB media with 0.2% arabinose (YidC expression conditions) or 0.2% glucose (YidC depletion conditions) (11). The SecE depletion strain CM124 was cultured in M9 media supplemented with 0.2% arabinose plus 0.4% glucose (SecE expression conditions) or 0.4% glucose (SecE depletion conditions) (26) for 8 -9 h. Prior to induction of the plasmid-encoded proteins in JS7131 and CM124, the cells were exchanged into fresh M9 media (27) and shaken for 30 min at 37°C. To express the PClep mutants in CM124, JS7131, and MC1060, the genes were cloned into the pLZ1 vector under the control of the lacUV5 promoter (28).
Protease Accessibility Studies-Expression of the PClep proteins encoded on the vector was induced by 1 mM isopropyl 1-thio-␤-D-galactopyranoside (final concentration) for 5 min. Cells were labeled with [ 35 S]methionine for 1 min and converted to spheroplasts (29). Briefly, the pulse-labeled cells were collected by centrifugation and resuspended with spheroplast buffer (33 mM Tris-HCl, pH 8.0, 40% (m/v) sucrose). The resuspended cells were treated with 1 mM EDTA and 10 g/ml lysozyme on ice for 30 min. An aliquot was then digested by the proteinase K (0.75 mg/ml) for 1 h on ice, and the reaction was quenched by the addition of 5 mM phenylmethylsulfonyl fluoride (PMSF) for 5 min. An equal volume of 20% trichloroacetic acid (TCA) buffer (m/v) was then added to the sample and incubated on ice for another 1 h. The total protein was then spun down at 14,000 ϫ g for 10 min and washed with 1 ml of ice-cold acetone. The protein pellet was then solubilized with Tris-SDS buffer (10 mM Tris-HCl, pH 8.0, 2% (m/v) SDS). The samples were immunoprecipitated with antiserum to leader peptidase (Lep) to precipitate the PClep derivatives or with antiserum to OmpA for a control. For the SecA inhibition studies, the cells were pretreated with sodium azide (3 mM final concentration) for 5 min prior to labeling of the cells. The samples were analyzed by SDS-PAGE and phosphorimaging.
Mutagenesis of PClep to Create Mutants-All the positively and negatively charged mutants, neutral polar mutants, as well as the hydrophobic derivatives were constructed by site-directed mutagenesis using the QuikChange mutagenesis kit (Stratagene). Mutations were verified by DNA sequencing.

RESULTS
PClep and PClep Mutants-The M13 coat protein is synthesized as a precursor called procoat with a cleavable signal sequence and a mature region consisting of a 20-amino acid periplasmic loop and a transmembrane segment (Fig. 1). The signal sequence of procoat is proteolytically removed by signal peptidase 1 (SP1, also known as leader peptidase) to generate the mature coat protein after membrane insertion. In our study, we used PClep in which the cytoplasmic region is extended by 103 amino acids of the P2 domain of SP1 (30). This extension makes the protein easier to detect by using an antibody.
Increased Sec Requirement with Substituted Positive Charges to the Loop of Procoat-Wild-type PClep has a total of five charged amino acids in the periplasmic loop between the signal peptide and the membrane anchor. It contains four negatively charged and one positively charged residue, giving it a predicted net charge of Ϫ3 (Fig. 1). The YidC dependence of wildtype PClep was assayed using the YidC depletion strain, JS7131, which has yidC under the control of the araBAD promoter (11). Membrane insertion can be studied under YidC depletion conditions by growing the bacteria in growth media supplemented with glucose for 3 h, as described under "Experimental Procedures." JS7131 cells expressing different protein constructs were labeled with [ 35 S]methionine for 1 min under YidC expression (0.2% arabinose) and YidC depletion conditions (0.2% glucose). They were then analyzed by the protease acces-sibility assay (31). Briefly, JS7131 cells were converted to spheroplasts, by the addition of lysozyme and EDTA to allow the protease to have access to the inner membrane. A portion of the cells was treated with PK for 60 min to probe membrane insertion of PClep.
When the 35 S-labeled PClep was expressed under YidC expression conditions, it was processed to the mature form ( The Sec dependence of the wild-type PClep and respective mutants was studied in the SecE depletion strain, CM124. Cells bearing the respective mutants on a plasmid were back-diluted in M9 minimal media supplemented with 0.4% glucose (SecE depletion conditions) or 0.4% glucose ϩ 0.2% arabinose (SecE expression conditions) for 8 -9 h. The cells were then transferred to fresh M9 medium and grown for 30 min before induc-tion and 35 S labeling of the protein as described under "Experimental Procedures." Depletion of SecE had no effect on the membrane insertion of procoat because the mature Clep is detected under both SecE expression and depletion conditions (Fig. 2B, center panel). This mature Clep was efficiently digested by PK to give rise to the fragment (Fig. 2B, f) confirming that what we are seeing is indeed the transmembrane segment Clep. Outer membrane protein A was used as a control for efficient SecE depletion (Fig. 2F) because it requires the SecA/ SecYEG to translocate across the inner membrane. Under SecE depletion conditions, OmpA accumulated as a cytoplasmic precursor, ProOmpA, as evidenced by the slower migration on the gel. Similarly, addition of sodium azide to the cells prior to pulse labeling causes an inhibition of the SecA ATPase activity (32). Insertion of PClep into the membrane was unaffected by inhibition of SecA function. Once again, OmpA was used as a control to confirm efficient SecA inhibition (Fig. 2F). OmpA controls were carried out for every experiment performed in this study, but are just being shown once for representative purposes.
To investigate if the charges in the loop of PClep play a role in determining the mechanism of membrane insertion, we studied a series of mutants with a progressively increasing number of positively charged residues at the ϩ2, ϩ4, and ϩ5 positions (30). The amino acid sequences of the mutants are depicted in Fig. 2A. We first studied the ARGNN PClep mutant with an arginine at the ϩ2 position, giving the periplasmic loop a net charge of ϩ1. Fig. 2C shows that this procoat mutant is strictly dependent on YidC because membrane insertion of ARGNN PClep was completely blocked under YidC depletion conditions. Interestingly, this mutant now appears to be slightly SecE-dependent, as well as SecA-dependent for insertion. This is evident by the slight accumulation of precursor observed under SecE depletion conditions and SecA inhibition conditions. Fig. 2D shows that ANGRR PClep with 2 arginines at the ϩ4 and ϩ5 positions and a net charge of ϩ2 in the loop is also completely YidC-dependent for membrane insertion (Fig. 2D). However, in this case, the extent of SecE and SecA dependence was stronger as compared with the ARGNN PClep mutant. A significant portion of the protein accumulated as a cytoplasmic precursor under SecE depletion/SecA inhibition conditions.
Membrane insertion of ARGRR PClep, with 3 arginine residues at ϩ2, ϩ4, and ϩ5 positions of PClep, became dramatically more SecE/SecA-dependent for membrane insertion as compared with the other positively charged mutants, but it remained YidC-dependent for insertion. Fig. 2E shows the majority of the protein accumulated in the cytoplasm under SecE depletion conditions and almost all of the protein accumulated when SecA was inhibited, showing that this protein was strongly dependent on the Sec translocase for insertion.
Thus, it appears that there is a linear relationship between the number of positively charged residues in the loop of PClep and the extent of Sec dependence (see supplemental Table S1 for quantification of the membrane insertion efficiency under the various depletion conditions).
Increased Sec Translocase Requirement When Charged Residues Are Added to the Signal Peptide or to the Transmembrane Segment of PClep-To examine if the addition of a positively charged residue to the transmembrane segment of PClep and to FIGURE 1. Membrane topology of procoat lep and mutants. Sequence of procoat is shown highlighting the amino acid residues that were modified in this study. The precursor form of procoat is made with a cleavable N-terminal signal peptide and a transmembrane segment in the mature region (42). The PClep used in this study has a P2 epitope fused at the C terminus of procoat corresponding to residues 220 -323 of SP1. The residues in the signal peptide are denoted with a Ϫ, and residues in the mature domain are denoted with a ϩ with respect to the cleavage site. SP1 cleaves PClep between the two Ala residues at the Ϫ1 and ϩ1 positions. The gray circles indicate where substitutions were made. The Ͼ indicates the site of insertion of additional amino acids for the Ϫ4 and Ϫ5 PClep mutants.
the H-region of the signal peptide had a similar effect on Sec recruitment as the addition of a positive charge in the loop, we proceeded to substitute the methionine at the Ϫ5 position with an arginine in the signal sequence of PClep (see Fig. 3A for construct). This mutant, designated L-R PClep, remained YidC-dependent for insertion, as the precursor form was resistant to PK in the JS7131 strain (Fig. 3B). The positively charged residues in the signal peptide made the protein slightly dependent on the Sec translocase for insertion, as judged from the small accumulation of preprotein under SecE depletion conditions. Similarly, membrane insertion was affected when SecA was inhibited by the addition of sodium azide. Interestingly, substitution of a negatively charged residue (L-D PClep) in place of the positively charged residue also caused the protein to be slightly SecE/SecA-dependent for insertion, while remaining completely YidC-dependent (Fig. 3C).
PClep with a positively charged residue substituted for the glycine at ϩ23 in the transmembrane segment of PClep (TM-R PClep) strictly required YidC for membrane insertion, but both SecE as well as SecA made insertion more efficient (Fig. 3D). Similar results were observed with an aspartic acid (TM-D PClep) introduced at the same position (ϩ23) within the TM segment (Fig. 3E).
To study whether the addition of multiple charges had a cumulative effect on the Sec requirement of PClep, we incorporated arginine residues into both the signal sequence and the transmembrane segment of the protein (L/TM-R PClep). As seen in Fig. 3F, this protein showed a dramatic increase in the Sec dependence with more than half of the protein being accumulated in the cytoplasm under SecE depletion conditions or when SecA was inhibited. Substitution of aspartic acid residues at both positions in the protein (L/TM-D PClep) caused the protein to be SecA-and SecE-dependent for insertion, in addition to being completely YidC-dependent (Fig. 3G).
Next, we examined whether the substitution of a neutral hydrophilic residue would have the same effect as a charged residue when added to the same positions as the charged residues in the signal peptide or TM sequence. The mutants L-Q, TM-Q, and L/TM-Q showed no Sec requirement, while remaining completely dependent on YidC for insertion (Fig. 3, H-J, respectively). Taken together, these studies show that Sec requirement was enhanced only when a charged residue was added and not with a neutral polar residue.
Requirement for YidC and Sec Increases with Increased Polarity of the Periplasmic Loop-Previously, we found that the Sec dependence of insertion was increased by adding two to three negatively charged residues to the wild-type PClep (8). Fig. 4C shows the Ϫ5 PClep with 2 glutamic acid residues added after the ϩ2 position of PClep was strongly SecYEG-and SecA-dependent for its insertion, whereas Ϫ4 PClep with 1 glutamic acid added after the ϩ2 position was largely unaffected (Fig.  4B). This shows that YidC on its own can translocate a periplasmic region with one additional negative charge added to the wild-type periplasmic loop, but the Sec translocase is required, when the polarity of the translocation region is increased by introducing an additional negative charge. To test whether we can reduce the YidC requirement for membrane insertion by decreasing the polarity of the periplasmic loop, we studied the ANGNN PClep mutant with the negatively charged residues at positions ϩ2, ϩ4, and ϩ5 of wildtype PClep substituted with neutral residues (30). Fig. 4D shows that ANGNN PClep inserted into the membrane in a less YidCdependent manner with a small portion of the protein inserting into the membrane even under YidC depletion conditions compared with a complete block for the wild-type PClep. Decreasing the polarity further, by substituting alanine instead of the uncharged but still polar asparagine residues at the ϩ2, ϩ4, and ϩ5 positions (AAGAA PClep), led to a similar insertion efficiency as the ANGNN mutant under YidC-depleted conditions (Fig. 4E).
To further test whether the YidC requirement is correlated with the translocation of a loop that is highly polar, we decreased the polarity of the loop even more by changing the lysine at the ϩ8 position and the glutamic acid residue at ϩ20 position to alanines, in addition to the AAGAA mutations. Fig. 4F shows that AAGAA/AA PClep mutant inserted into the membrane almost completely independent of YidC. All the uncharged mutants studied in this section, namely the AAGAA, ANGNN, and the AAGAA/AA mutants, inserted independent of the Sec translocase.
Increasing the Hydrophobicity of the TM Segment Can Decrease the YidC Requirement of Insertion-The TM4L PClep mutant has a more hydrophobic TM segment as compared with WT PClep due to the substitution of the residues at positions ϩ24 to ϩ27 with leucines (from tyrosine, alanine, tryptophan, and alanine, respectively) ( Figs. 1 and 5A). This mutant, TM4L PClep, inserted into the membrane in a largely YidC-independent manner with roughly half of the mutant being membraneinserted in the JS7131 strain. The insertion of this protein was completely unaffected by SecE depletion or SecA inhibition (Fig. 5B). Possibly, the increased hydrophobicity of the mutant allowed a direct interaction with the hydrophobic core of the membrane, driven by its own hydrophobicity.
Next, we tested whether combining the TM4L PClep mutant with the ANGNN mutation in the loop of PClep would result in a lower dependence on YidC on insertion. Fig. 5C shows the TM4L/ANGNN PClep protein inserts also largely independent of YidC as well as SecE and SecA. A completely YidC-and Sec-independent mechanism of insertion is seen with the TM4L/AAGAA mutant, which contains alanine residues at the ϩ2, ϩ3, and ϩ5 position instead of the more polar asparagine residues (Fig. 5D).
With the increased hydrophobic driving force of the TM4L PClep construct, it may be possible that the loop containing the ARGRR could be translocated with less dependence on the Sec translocase. Indeed, TM4L/ARGRR PClep inserted across the membrane in a largely SecYEG-independent manner, although the protein still required YidC for insertion (Fig. 5E). Therefore, the energy barrier for translocation due to the positively charged arginines was still high, even with the increased hydrophobic TM segment of PClep. To test whether the TM4L segment could drive translocation of the Ϫ5 loop with less dependence on the Sec machinery, we tested the TM4L/-5 PClep mutant under SecE depletion conditions or when the SecA function was inhibited. Fig. 5F shows this construct inserted almost completely independent of the Sec machinery, suggesting that the negatively charged periplasmic loop is easily translocated when the hydrophobic driving force is increased by the 4-leucine substitution and can largely do this without requiring SecA and SecYEG.

DISCUSSION
Our hypothesis is that the polarity and charge of the periplasmic loop determines the Sec and YidC requirements for M13 procoat translocation. The translocase requirement of a protein is thermodynamically determined because the higher the polarity of the loop, the higher the translocation barrier for moving the peptide chain through the lipid phase. What we found is compelling. By increasing the polarity of the loop region, either by incorporating negatively or positively charged residues, the Sec requirement for insertion of PClep was increased linearly. In contrast, decreasing the polarity of the translocated loop region from that of the wild-type protein decreased the degree to which YidC is needed for insertion.
Starting with PClep, which is YidC-dependent, we found that when two negatively charged residues were added, membrane insertion was completely Sec/YidC-dependent (Ϫ5 PClep), whereas adding only one negatively charged residue did not change the insertion mechanism (Ϫ4 PClep). Thus, YidC on its own can translocate the PClep periplasmic loop with one additional negatively charged residue but cannot with two added charged residues. This latter Ϫ5 PClep mutant required SecYEG and also SecA. Interestingly, previous studies with the MscL protein (33), which spans the membrane twice, found that adding negatively charged residues to the periplasmic region made the protein SecYEG-dependent for membrane insertion but did not affect its SecA independence for insertion. However, in this study, we found a remarkable correlation between the level of SecE dependence with the extent of SecA dependence for the Sec-dependent PClep mutants.
YidC has difficulty transporting the periplasmic loop with a small number of positively charged residues added to the loop showing that positive charges have a bigger requirement for Sec assistance than negative charges. This is seen most clearly by comparing the wild-type PClep containing AEGDD with the ARGRR PClep mutant, where the positively charged mutant has arginines at the same position as the wild-type negative charges (ϩ2, ϩ4, and ϩ5); otherwise, the periplasmic sequence is identical. The triple arginine mutant was completely YidC/ SecE-and SecA-dependent, whereas the wild-type protein was Sec-independent. Interestingly, adding one arginine to the sequence with the other neutral residues caused the protein (ARGNN PClep) to insert into the membrane in a slightly SecEand SecA-dependent manner while maintaining its dependence on YidC. Substituting another arginine led to a further increased Sec dependence of insertion (as in PClep ANGRR). The ARGRR PClep mutant, like Ϫ5 PClep, was completely Secdependent, indicating that three positively charged residues lead to a Sec dependence similar to 5 negatively charged residues. Our results with the positively charged PClep mutants are different from recent results obtained with a single span Pf3-Lep protein (28). With this Pf3-Lep model protein, the addition of a single positively charged residue to the periplasmic region switched the insertion pathway from Sec-YidC-independent to Sec-YidC-dependent, and insertion was only weakly SecA-dependent. This difference may have to do with the fact that pro-coat inserts with two hydrophobic domains, whereas the Pf3-Lep model protein inserts with only one hydrophobic domain (34).
We found that by decreasing the polarity of the wild-type PClep periplasmic loop by changing the negatively charged residues at ϩ2, ϩ4, and ϩ5 to either asparagines (ANGNN PClep) or alanines (AAGAA PClep) (Fig. 4, D and E), we were able to decrease the YidC dependence of insertion to ϳ40 -50% YidCindependent insertion. Further decreasing the polarity of the loop (AAGAA/AA PClep), by substituting all the charged residues with alanines, led to an even further decrease in YidC dependence providing support for our hypothesis that the polarity of the loop of PClep is a major determinant for pathway selection.
Increasing the hydrophobicity of the TM regions should provide more thermodynamic force to drive the translocation of the PClep periplasmic loop. The ⌬G app for the wild-type membrane anchor of PClep is Ϫ20.2 kJ/mol as compared with Ϫ32.4 kJ/mol for the TM4L mutant (35). Indeed, we observed membrane insertion was over 50% independent of YidC with the TM4L mutant ( Fig. 5; Table 1). Moreover, translocation of the less hydrophilic periplasmic loop of the AAGAA PClep mutant, in combination with the TM4L, was completely YidC-independent as the hydrophobic force was increased with the presence of the more hydrophobic TM segment (TM4L ϩ AAGAA). Intriguingly, the very hydrophobic TM segment was insufficient to drive insertion of a loop independent of a translocase when it contained a number of positively charged residues as seen with the TM4L/ϩARGRR PClep mutant. We assume that the translocation barrier was too high to go by the YidC-only pathway, and therefore, this positively charged mutant remained slightly Sec-dependent. This is to be compared with the ARGRR PClep mutant (without the 4 leucines substituted in TM1) that was almost completely dependent on the SecYEG machinery, in addition to YidC, for insertion. Similar results were observed with the TM4L/Ϫ5 mutant with a number of negatively charged residues in the translocated region. Remarkably, the more hydrophobic TM segment reduces the Sec translocase requirement for translocation of both negatively and positively charged residues ( Table 1).
As mentioned above, our results indicated that positively charged residues added to the periplasmic region have a larger effect on the requirements for a translocase than negatively charged residues. Therefore, not only polarity, but also the charge of the peptide chain, needs to be taken into consideration because of the membrane potential. The membrane potential (ϩ side periplasmic) favors the transfer of negatively charged residues across the membrane but hinders the translocation of positively charged residues (23,36). Table 1 summarizes the predicted standard free energy needed (not considering the contribution of the membrane potential) for transfer of the periplasmic region across the membrane for the PClep mutants. The values for the membrane transfer expense were determined using the GES scale for each amino acid and include the contribution of the peptide bond (37). As expected, as the GES values decrease from the wild-type PClep to the ANGNN PClep, AAGAA PClep, to the AAGAA/AA PClep mutant, membrane insertion becomes less dependent on YidC. Intriguingly, as the number of the positively charged residues is increased (going from ARGNN f ANGRR f ARGRR), both the Sec dependence and the GES values of the periplasmic domain increase. The insertion of two negatively charged residues leads to a Sec-dependent insertion (Ϫ5 PClep), although one added negatively charged residue (Ϫ4 PClep) did not change the YidC-only insertion mechanism. Because the protonmotive force (ϩ side on the periplasm) supports the membrane transfer of negatively charged residues but restricts the transfer of positively charged residues, this might account for the Sec independence of Ϫ4 PClep that has a ϩ278 GES value, whereas the ANGRR that has a ϩ251 GES value is slightly Secdependent. The periplasmic region poses a bigger barrier for the ANGRR over the Ϫ4 PClep mutant because the transfer of positive charge increases the standard free energy due to the membrane potential, whereas a negative charge decreases the standard free energy.
The mutants that show a low energy barrier for translocation of their periplasmic domain and possess an increased hydrophobic TM segment require neither YidC nor Sec translocase. Although insertion could be facilitated by another not yet identified component, it is conceivable that in this case the TM regions directly insert into the lipid bilayer, and even the periplasmic domain is translocated through the lipid bilayer directly. This unassisted mechanism has also been observed for proteins that insert into the thylakoid membrane (38). Moreover, a recent in vitro study has shown that the Foc subunit inserts into liposomes with substantial efficiency (39). Also, Koch and co-workers (40) have shown that a minor amount of MtlA was inserted into liposomes in the presence of SRP and FtsY but without YidC and Sec translocase. The addition of YidC, SecYEG, or both improved the insertion efficiency. In agreement with these studies, our results indicate that small periplasmic domains with low polarity and charge can insert without assistance. However, when YidC is present, the hydrophobic TM segment of PClep will readily interact with the TM regions TM1, TM3, TM4, and TM5 of YidC, which contain the substrate contact sites (41). Most likely, during the translocation process, the periplasmic domain is passively dragged along with the TM region into the bilayer by strong interactions between the TM segments of YidC and the substrate. Polar and charged residues within the periplasmic region need further assistance of the Sec translocase that likely shields these residues during their translocation process (Fig. 6).
In conclusion, we found that if the energy barrier of translocation is low, insertion can occur largely by an unassisted mechanism requiring neither YidC nor the Sec translocase. If the barrier is increased due to a more hydrophilic loop and below a certain polarity/charge threshold, then YidC can catalyze the translocation step by itself. However, if the barrier exceeds a certain threshold due to a higher polarity and charge of the periplasmic loop, then both YidC and Sec are required for insertion (Fig. 6). Exactly how YidC and the Sec machinery work together to drive insertion is still not known. We favor the The membrane transfer expense for translocation of the periplasmic region of the PClep constructs calculated using the GES scale for each amino acid (37) The standard free energy contribution of the membrane potential is not considered here for the transfer of charged residues. ϩϩϩ indicates a strict translocase requirement for insertion; ϩϩ indicates a partial translocase requirement; ϩ indicates a weak translocase requirement; Ϫindicates no translocase requirement. idea that PClep inserts at a YidC/SecYEG interface such that both translocases can facilitate translocation simultaneously. In addition, the work presented here shows that subtle changes throughout the procoat protein, i.e. to the leader sequence, the TM segment, or periplasmic loop of the M13 procoat can change the insertion pathway from YidC-only to YidC/Sec or to YidC/Sec-independent.