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• W2095078885 abstract "The Escherichia coli TolQ protein is a 230-amino acid integral cytoplasmic membrane protein required for the import of group A colicins, for infection by the filamentous phage, and for maintenance of the integrity of the bacterial envelope. TolQ is a polytopic protein with three membrane-spanning regions. The first membrane-spanning region has a 19-residue periplasmic NH2-terminal tail, while the second and third membrane-spanning segments are separated by a short 17-amino acid periplasmic loop. To study the membrane assembly of TolQ, fusions of different membrane-spanning regions were examined for their ability to insert in the absence of functional SecA or the membrane potential. Fusions containing the first membrane-spanning region plus the adjacent cytoplasmic domain and a construct containing the “hairpin loop,” formed by the second and third membrane-spanning regions, insert in the absence of functional SecA. The fusion containing the second and third membrane-spanning regions required the membrane potential for insertion while the first membrane-spanning region was able to insert even in the absence of a membrane potential. Taken together, these results suggest that insertion of intact TolQ is not dependent on the Sec system, but does require the membrane potential. The Escherichia coli TolQ protein is a 230-amino acid integral cytoplasmic membrane protein required for the import of group A colicins, for infection by the filamentous phage, and for maintenance of the integrity of the bacterial envelope. TolQ is a polytopic protein with three membrane-spanning regions. The first membrane-spanning region has a 19-residue periplasmic NH2-terminal tail, while the second and third membrane-spanning segments are separated by a short 17-amino acid periplasmic loop. To study the membrane assembly of TolQ, fusions of different membrane-spanning regions were examined for their ability to insert in the absence of functional SecA or the membrane potential. Fusions containing the first membrane-spanning region plus the adjacent cytoplasmic domain and a construct containing the “hairpin loop,” formed by the second and third membrane-spanning regions, insert in the absence of functional SecA. The fusion containing the second and third membrane-spanning regions required the membrane potential for insertion while the first membrane-spanning region was able to insert even in the absence of a membrane potential. Taken together, these results suggest that insertion of intact TolQ is not dependent on the Sec system, but does require the membrane potential. INTRODUCTIONThe TolQRA proteins are components of a bacterial system that is required for infection by filamentous phage and entry of the group A colicins (1Webster R.E. Mol. Microbiol. 1991; 5: 1005-1011Google Scholar). In addition, mutations in the tolQRA genes render the cells sensitive to detergents and permeable to periplasmic enzymes suggesting a loss of integrity in the outer membrane (2Lazzaroni J.C. Fognini-Lefebvre N. Portalier R. Mol. Gen. Genet. 1989; 218: 460-464Google Scholar, 3Fognini-Lefebvre N. Lazzaroni J.C. Portalier R. Mol. Gen. Genet. 1987; 209: 391-395Google Scholar). TolR and TolA are type II bitopic inner membrane proteins with most of the polypeptide comprising soluble periplasmic domains (4Muller M.M. Vianney A. Lazzaroni J.C. Webster R.E. Portalier R. J. Bacteriol. 1993; 175: 6059-6061Google Scholar, 5Kampfenkel K. Braun V. J. Bacteriol. 1993; 175: 4485-4491Google Scholar, 6Levengood S.K. Webster R.E. J. Bacteriol. 1989; 171: 6600-6609Google Scholar). TolQ is a 230-amino acid protein which crosses the inner membrane three times. Its membrane topology was recently determined by protease accessibility and the analysis of TolQ fusion proteins (5Kampfenkel K. Braun V. J. Bacteriol. 1993; 175: 4485-4491Google Scholar, 7Vianny A. Lewin T.M. Beyer Jr., W.F. Lazzaroni J.C. Portalier R. Webster R.E. J. Bacteriol. 1994; 176: 822-829Google Scholar). The first 19 amino-terminal residues and a short loop from amino acids 157–174 are exposed to the periplasm, while the carboxyl terminus and a large loop between the first and second membrane-spanning region are cytoplasmic (see Fig. 1). Amino-terminal sequence analysis of overexpressed TolQ purified from the inner membrane revealed the presence of the initiating formylmethionine, which suggests that TolQ is synthesized without a signal sequence and is rapidly inserted into the cytoplasmic membrane (7Vianny A. Lewin T.M. Beyer Jr., W.F. Lazzaroni J.C. Portalier R. Webster R.E. J. Bacteriol. 1994; 176: 822-829Google Scholar). In addition, overexpressed TolQ properly localizes to the cytoplasmic membrane and has no effect on cell growth. These characteristics raise the question whether the Sec system is required for TolQ membrane insertion.The role of Sec proteins in translocation of periplasmic proteins has been well characterized (reviewed in 8Schatz P.J. Beckwith J. Annu. Rev. Genet. 1990; 24: 215-248Google Scholar and 9Wickner W. Driessen A.J.M. Hartl F.U. Annu. Rev. Biochem. 1991; 60: 101-124Google Scholar). A number of intrinsic membrane proteins such as Lep, Tsr, and MtlA (10Wolfe P.B. Rice M. Wickner W. J. Biol. Chem. 1985; 260: 1836-1841Google Scholar, 11Gebert J.F. Overhoff B. Manson M.D. Boos W. J. Biol. Chem. 1988; 263: 16652-16660Google Scholar, 12Werner P.K. Saier Jr., M.H. Muller M. J. Biol. Chem. 1992; 267: 24523-24532Google Scholar) have also been shown to require the Sec system for proper insertion into the cytoplasmic membrane. However, other membrane proteins such as LacY, SecY, and M13 coat protein (10Wolfe P.B. Rice M. Wickner W. J. Biol. Chem. 1985; 260: 1836-1841Google Scholar, 13Yamato I. J. Biochem. (Tokyo). 1992; 111: 444-450Google Scholar, 14Swidersky U.E. Rienhofer-Schweer A. Werner P.K. Ernst F. Benson S.A. Hoffschulte H.K. Muller M. Eur. J. Biochem. 1992; 207: 803-811Google Scholar) do not require the Sec proteins for membrane insertion. On the basis of these and other observations, it has been proposed that inner membrane proteins which form “hairpin loops,” that is two membrane-spanning segments separated by a periplasmic domain of less than 60 residues, insert Sec independently (15von Heijne G. FEBS Lett. 1994; 346: 69-72Google Scholar). If the two membrane-spanning regions are separated by a larger periplasmic region, then the Sec system appears to be required for translocation. Additional observations suggest that the Sec system is not required for insertion of membrane-spanning segments which have unprocessed periplasmic amino termini (16Rohrer J. Kuhn A. Science. 1990; 250: 1418-1421Google Scholar, 17Cao G. Dalbey R.E. EMBO J. 1994; 13: 4662-4669Google Scholar, 18Whitley P. Zander T. Ehrmann M. Haardt M. Bremer E. von Heijne G. EMBO J. 1994; 13: 4653-4661Google Scholar).The topology of the TolQ protein (Fig. 1) would suggest that both the first membrane-spanning region and the hairpin loop formed by the second and third membrane-spanning regions are inserted into the membrane independent of the Sec proteins. Experiments presented in this study demonstrate that all membrane-spanning regions of TolQ are translocated into the membrane independent of active SecA protein. In contrast, insertion of the second and third membrane-spanning regions requires the membrane potential, while the first inserts even in the absence of a membrane potential. These results are discussed in relation to the membrane assembly of TolQ.RESULTSTolQ is synthesized without a signal sequence and, once inserted into the cytoplasmic membrane, has very little of its sequence exposed to the periplasm (Fig. 1). One of these periplasmic regions is the amino-terminal 19 residues that still retains the initiating formylmethionine group (7Vianny A. Lewin T.M. Beyer Jr., W.F. Lazzaroni J.C. Portalier R. Webster R.E. J. Bacteriol. 1994; 176: 822-829Google Scholar). This observation suggests that the amino terminus and perhaps the other periplasmic region (residues 157–174) might be translocated across the membrane in a manner independent of the Sec system. To test this hypothesis, a number of TolQ fusion proteins were constructed (Fig. 1) and tested for their ability to be inserted into the membrane in the absence of functional SecA protein.Insertion of the First Membrane-spanning Region Is Sec-independent and Does Not Require the Membrane PotentialThe TolQ128 fusion protein has the amino-terminal 128 amino acids of TolQ fused to alkaline phosphatase (Fig. 1). This places alkaline phosphatase 81 residues to the carboxyl side of the first transmembrane region. Therefore the membrane-spanning region of the fusion protein might be expected to be presented to the membrane for insertion in the same manner as for the intact TolQ protein. A cellular fractionation experiment showed that approximately 65% of the fusion protein is localized to the inner membrane (Table I). The absence of alkaline phosphatase activity in cells synthesizing TolQ128 (Table I) confirms the cytoplasmic localization of the alkaline phosphatase moiety.Table I.Characteristics of TolQ fusion proteinsFusion proteinPlasmidAP activityaAlkaline phosphatase activity of intact cells (average from five experiments) corresponds to units of p-nitrophenylphosphate cleaved/min/cells/ml. − is <5; ++ is 20 to 40; +++ is 40 to 60; N/A = not applicable.LocalizationbBacteria were fractionated into cytoplasm (C), periplasm (P), inner membrane (IM), and outer membrane plus inclusion bodies (OM) in the presence or absence of 2 M NaBr. Each fraction, containing material from the same number of cells, was analyzed by Western blot and quantitated by phosphorimaging as described under “Materials and Methods.”CPIMOM%TolQ36pTLQ36++50—50—TolQ128pTLQ128−35—65—TolQ155pTLQ155+++35—65—TolQIIpTLQ020++40—60—TolQII/IIIpTLQ023N/A30—5020a Alkaline phosphatase activity of intact cells (average from five experiments) corresponds to units of p-nitrophenylphosphate cleaved/min/cells/ml. − is <5; ++ is 20 to 40; +++ is 40 to 60; N/A = not applicable.b Bacteria were fractionated into cytoplasm (C), periplasm (P), inner membrane (IM), and outer membrane plus inclusion bodies (OM) in the presence or absence of 2 M NaBr. Each fraction, containing material from the same number of cells, was analyzed by Western blot and quantitated by phosphorimaging as described under “Materials and Methods.” Open table in a new tab To test the necessity of the Sec sytem for insertion of TolQ128, inner membranes were isolated from K17 and K17secA bacteria following expression of TolQ128 for 10 min in the presence and absence of 1 mM azide. This low concentration of azide selectively inhibits the ATPase activity of SecA (19Oliver D.B. Cabelli R.J. Dolan K.M. Jarosik G.P. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 8227-8231Google Scholar). Equal amounts of protein from each inner membrane preparation were subjected to quantitative Western blot analysis (Fig. 2A). Quantitation of the radioactivity in the bands shows approximately 20% more TolQ128 is detected in the membranes isolated from the azide treated K17 cells (Fig. 2A, lane 2) as compared to that observed in untreated bacteria (Fig. 2A, lane 1). The TolQ128 present in azide-treated or untreated membranes was not removed by treatment with 2 M NaBr (data not shown), suggesting that TolQ128 is an integral membrane protein and spans the membrane with the proper orientation. The ability of TolQ128 to insert into the K17 cytoplasmic membrane when expressed in the presence of 1 mM azide, indicates that the first membrane-spanning region of TolQ does not require SecA for insertion. The presence of 1 mM azide inhibited normal SecA function as shown by the appearance of precursor to ribose binding protein in azide treated K17 bacteria (Fig. 2B, compare lanes 1 and 2). Ribose binding protein is constitutively expressed and any precursor produced was the result of synthesis only during the 10-min period of azide treatment.Fig. 2Effect of azide and CCCP on TolQ128 insertion.Top, K17 (lanes 1 and 2) and K17secA (lanes 3 and 4) containing plasmid pTLQ128 were induced in the absence (lanes 1 and 3) and presence (lanes 2 and 4) of 1 mM azide. A, cytoplasmic membranes were isolated from an isopycnic sucrose gradient, and equal amounts of total protein from each membrane sample were subjected to 11% SDS-PAGE and Western blot analysis with antibody to alkaline phosphatase. B, a sample of the harvested bacteria was subjected to 11% SDS-PAGE and Western blot analysis using antibody to ribose binding protein (RBP). Bottom, K17 containing plasmid pTLQ128 were induced in the absence (lane 1) and presence (lane 2) of 50 µM CCCP. C, cytoplasmic membranes were isolated from an isopycnic sucrose gradient, and equal amounts of total protein from each membrane sample were subjected to 11% SDS-PAGE and Western blot analysis with antibody to alkaline phosphatase. D, a sample of the harvested bacteria was subjected to 11% SDS-PAGE and Western blot analysis using antibody to ribose binding protein (RBP).View Large Image Figure ViewerDownload (PPT)The control strain, K17secA, carries a point mutation in secA (azi4) which allows for normal SecA ATPase activity in the presence of 1 mM azide (19Oliver D.B. Cabelli R.J. Dolan K.M. Jarosik G.P. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 8227-8231Google Scholar). This mutant secA bacteria also exhibits normal growth in the presence of 1 mM azide. Membranes isolated from azide treated K17secA bacteria contained 92% of the TolQ128 detected in membranes from the untreated cells (Fig. 2A, compare lanes 3 and 4). This shows that azide alone does not affect membrane insertion. Azide treatment of K17secA bacteria also does not affect normal protein secretion as demonstrated by the absence of precursor to ribose binding protein in K17secA bacteria treated with 1 mM azide (Fig. 2B, lane 4). Ribose binding protein requires both an intact membrane potential (see below; Fig. 2D) and the ATPase activity of SecA for proper translocation.To examine the requirement for the membrane potential in the insertion of the first membrane-spanning region of TolQ128, half the culture was treated with 50 µM CCCP at the time of induction. Inner membranes were isolated and equal amounts of protein from each inner membrane sample were analyzed by Western blot with antibody to alkaline phosphatase. Quantitation of the blot shows that membranes from CCCP treated cells contain 83% of the amount of full length TolQ128 detected in membranes from untreated cells (Fig. 2C, compare lanes 1 and 2). The presence of 50 µM CCCP disrupted the membrane potential as shown by the appearance of precursor to ribose binding protein (Fig. 2D, compare lanes 1 and 2). Since TolQ128 can insert into the cytoplasmic membrane in the presence of CCCP, then the membrane potential is not an absolute requirement for insertion of the first membrane-spanning region of TolQ.The First Membrane-spanning Region Can also Function as a SecA-dependent Signal SequenceThe positively charged amino acids on the cytoplasmic side of the membrane-spanning region of TolQ128 might be responsible for the proper orientation and insertion of this region in the membrane. Removal of the cytoplasmic basic amino acids as in TolQ36 (Fig. 1) makes the NH2 terminus and first membrane-spanning region of TolQ look like a typical signal sequence. Expression of TolQ36 results in the bacteria exhibiting alkaline phosphatase activity (Table I). This activity is not due to translocation of the entire TolQ36 fusion to the periplasm, since no anti-alkaline phosphatase reactive species is detected in the periplasmic fraction (Table I). Since 50% of TolQ36 is localized to the inner membrane (Table I), it would seem that some TolQ36 can be inserted into the membrane in the opposite orientation to that determined for the NH2 terminus of wild type TolQ or TolQ128, resulting in the alkaline phosphatase moiety being exposed to the periplasm.The alkaline phosphatase moiety in TolQ36 is translocated in a SecA-dependent manner since the alkaline phosphatase activity observed in K17 bacteria is dramatically reduced in the presence of 1 mM azide (Fig. 3A, compare bars 1 and 2). Azide alone does not inhibit alkaline phosphatase activity, since in K17secA bacteria treated with 1 mM azide, the alkaline phosphatase activity only decreases 10% (Fig. 3A, bars 3 and 4). The loss of alkaline phosphatase activity observed in the presence of azide in K17 indicates that the translocation of alkaline phosphatase fused to residue 36 of TolQ proceeds in a SecA-dependent manner. However, TolQ36 is still inserted into the membrane in the presence of azide. Western blot analysis of K17 samples shows the same amount of TolQ36 is detected in the cytoplasmic membrane following treatment with azide (Fig. 3B, lane 2) or without azide (Fig. 3B, lane 1). Treatment with 2 M NaBr does not affect the amount of TolQ36 detected in inner membranes following induction in the presence or absence of azide (data not shown), suggesting that TolQ36 spans the membrane.Fig. 3Effect of azide on TolQ36 activity and insertion. K17 (bars and lanes 1 and 2) and K17secA (bars and lanes 3 and 4) containing plasmid pTLQ36 were induced in the absence (bars and lanes 1 and 3) and presence (bars and lanes 2 and 4) of 1 mM azide. A, alkaline phosphatase activity corresponds to units of p-nitrophenylphosphate cleaved/min/cells/ml. B, cytoplasmic membranes were isolated from an isopycnic sucrose gradient and equal amounts of total protein from each membrane sample were subjected to 11% SDS-PAGE and Western blot analysis with antibody to alkaline phosphatase.View Large Image Figure ViewerDownload (PPT)The Second Membrane-spanning Region Can Function as an Internal Signal SequenceTolQ155 has alkaline phosphatase fused to residue 155 which lies at the COOH terminus of the second membrane-spanning region (region B in Fig. 1). Cellular fractionation shows that TolQ155 is found primarily (65%) in the inner membrane (Table I). Bacteria expressing TolQ155 have alkaline phosphatase activity (Table I) indicating a periplasmic localization for the alkaline phosphatase moiety.The SecA dependence for the insertion of the second membrane-spanning region in TolQ155 was examined by monitoring alkaline phosphatase activity following induction of TolQ155 in the presence and absence of 1 mM azide. In K17 bacteria, no alkaline phosphatase activity is observed when TolQ155 is expressed in the presence of 1 mM azide (Fig. 4A, bar 2), suggesting that SecA is required for the translocation of the alkaline phosphatase moiety into the periplasm.Fig. 4Effect of azide on TolQ155 activity and insertion. K17 (bars and lanes 1 and 2) and K17secA (bars and lanes 3 and 4) containing plasmid pTLQ155 were induced in the absence (bars and lanes 1 and 3) and presence (bars and lanes 2 and 4) of 1 mM azide. A, alkaline phosphatase activity corresponds to units of p-nitrophenylphosphate cleaved/min/cells/ml. B, cytoplasmic membranes were isolated from an isopycnic sucrose gradient and equal amounts of total protein from each sample were subjected to 11% SDS-PAGE and Western blot analysis with antibody to alkaline phosphatase.View Large Image Figure ViewerDownload (PPT)We also examined the role of the membrane potential in translocation of the alkaline phosphatase moiety of TolQ155. No alkaline phosphatase activity is observed when TolQ155 is expressed in the presence of 50 µM CCCP (data not shown). The lack of alkaline phosphatase activity is not due to a block in protein synthesis, as analysis of whole bacteria shows that CCCP treated bacteria synthesize 80% of the amount of TolQ155 detected in untreated cells (data not shown). These results suggest that the membrane potential is required for the translocation of the alkaline phosphatase moiety of TolQ155 into the periplasm.The data presented above suggest that the Sec system is used for the insertion of the second membrane-spanning region of TolQ155 into the cytoplasmic membrane. These experiments do not address the role of the first membrane-spanning region in this process. To test whether the first membrane-spanning region affects the insertion of the second membrane-spanning region, an amino-terminal truncation of TolQ155 was made, generating TolQII (Fig. 1). Cellular fractionation shows that 60% of the TolQII synthesized is detected in the inner membrane (Table I). TolQII also has alkaline phosphatase activity (Table I) indicating a periplasmic localization for the alkaline phosphatase moiety.In K17 bacteria, induction of TolQII results in a two fold increase in alkaline phosphatase activity above the uninduced level. When TolQII is induced in the presence of 1 mM azide no increase in alkaline phosphatase activity above the uninduced level is observed (data not shown). The absence of additional alkaline phosphatase activity following expression of TolQII in the presence of 1 mM azide, indicates that SecA is required for the translocation of the alkaline phosphatase moiety into the periplasm. This result is consistent with the result obtained for TolQ155 and suggests that the absence of the first membrane-spanning region of TolQ has little effect on the insertion of the second membrane-spanning region fused to alkaline phosphatase.Although the above data are consistent with SecA being required for insertion of the second membrane-spanning region of TolQ155 into the membrane, it does not address the question of whether insertion of this transmembrane domain affects the mode of insertion of the first membrane-spanning region. Therefore, we analyzed the cellular location of TolQ155 produced in K17 bacteria in the presence or absence of 1 mM azide. A Western blot of K17 inner membrane samples shows that 45% less TolQ155 is present following azide treatment (Fig. 4B, lane 2) as compared to that detected in the untreated sample (Fig. 4B, lane 1). Azide treatment did not affect the expression of TolQ155 as measured by Western blot analysis of whole cell samples (data not shown). Therefore, the decrease in the amount of inner membrane TolQ155 detected following azide treatment suggests that efficient insertion of the first membrane-spanning region may depend on the insertion of the second membrane-spanning region. In the control strain, K17secA, 15% more TolQ155 is detected in the inner membrane in the presence of 1 mM azide (Fig. 4B, lane 4) than in the absence of azide (Fig. 4B, lane 3).The Second and Third Membrane-spanning Regions Insert in a Sec-independent MannerThe experiments with TolQ155 suggest that the second membrane-spanning region behaves as a Sec-dependent internal signal sequence. However, the TolQ155 construct does not mimic the actual membrane topology for TolQ, which have the second and third membrane-spanning segments (Fig. 1, regions B and C) connected by a short (17-amino acid) periplasmic loop. It has recently been shown that Sec-independent insertion of such hairpin loops requires both membrane-spanning regions (26Cao G. Cheng S. Whitley P. von Heijne G. Kuhn A. Dalbey R.E. J. Biol. Chem. 1994; 269: 26898-26903Google Scholar). To test the insertion characteristics of the combined second and third membrane-spanning regions of TolQ, amino acids 113–197 were inserted into the middle of EcoRI endonuclease to create TolQII/III (Fig. 1). Cellular fractionation indicated that at least 50% of TolQII/III is inserted into the cytoplasmic membrane (Table I). Treatment with 2 M NaBr does not remove TolQII/III from the membrane suggesting that TolQII/III spans the inner membrane. The 20% found in the outer membrane fraction was the result of inclusion body formation.The membrane topology of TolQII/III was verified by a trypsin accessibility experiment. Previous studies have shown that TolQ is only partially susceptible to trypsin at very high concentrations (>100 µg/ml) (5Kampfenkel K. Braun V. J. Bacteriol. 1993; 175: 4485-4491Google Scholar, 7Vianny A. Lewin T.M. Beyer Jr., W.F. Lazzaroni J.C. Portalier R. Webster R.E. J. Bacteriol. 1994; 176: 822-829Google Scholar) and that EcoRI endonuclease is trypsin sensitive (22Horabin J.I. Webster R.E. J. Biol. Chem. 1988; 263: 11575-11583Google Scholar). Therefore, if TolQII/III adopts a hairpin loop conformation in the membrane it should be resistant to digestion at low concentrations of trypsin. To test this hypothesis, bacteria expressing TolQII/III were either plasmolyzed or lysed, and then treated with 10 µg/ml trypsin. A Western blot with antibody against EcoRI endonuclease shows that trypsin was unable to digest TolQII/III in plasmolyzed cells (Fig. 5, lane 2), while trypsin completely digested the fusion protein in lysed cells (Fig. 5, lane 3). As a control, C1S (22Horabin J.I. Webster R.E. J. Biol. Chem. 1988; 263: 11575-11583Google Scholar), an EcoRI fusion protein with a single membrane-spanning region (Fig. 1), was subjected to the same trypsin digestion. Trypsin treatment of plasmolyzed cells resulted in digestion of the periplasmically exposed COOH terminus of the 51-kDa C1S protein to yield a 30-kDa protected fragment (Fig. 5, compare lanes 4 and 5). These results suggest that TolQII/III adopts a membrane conformation similar to that predicted for the second and third membrane-spanning regions of TolQ.Fig. 5Trypsin digestion of TolQII/III. K91 containing plasmid pTLQ023 (lanes 1–3) or plasmid pC1S (lanes 4 and 5) were induced for the synthesis of TolQII/III or C1S, respectively. Cells were plasmolyzed (lanes 1, 2, 4, and 5) or lysed (lane 3) and incubated in the absence (lanes 1 and 4) or presence (lanes 2, 3, and 5) of 10 µg/ml trypsin. Equal amounts of cellular material were subjected to 11% SDS-PAGE and Western blot analysis with antibody against EcoRI endonuclease. The open arrow indicates the position of the 30-kDa C1S protected fragment.View Large Image Figure ViewerDownload (PPT)TolQII/III was used to examine the SecA-dependence of the insertion of the second and third membrane-spanning regions of TolQ. Quantitative Western blot analysis of inner membranes from azide treated K17 cells, showed that they contained 90% of the amount of TolQII/III detected in membranes from untreated cells (Fig. 6A, compare lanes 1 and 2). Membrane-associated TolQII/III in azide-treated bacteria was not removed by treatment with 2 M NaBr (data not shown) and is trypsin-resistant in plasmolyzed bacteria. This suggests that TolQII/III expressed in the presence of azide adopts the correct membrane topology. In azide-resistant cells, azide treatment results in only a 20% decrease in the amount of inner membrane TolQII/III detected (Fig. 6A, lane 4). The ability of TolQII/III to insert properly in the absence of functional SecA indicates that the second and third membrane-spanning regions of TolQ insert in a SecA-independent manner.Fig. 6Effect of azide and CCCP on TolQII/III insertion.A, K17 (lanes 1 and 2) and K17secA (lanes 3 and 4) containing plasmid pTLQ023 were induced for the synthesis of TolQII/III in the absence (lanes 1 and 3) and presence (lanes 2 and 4) of 1 mM azide. Cytoplasmic membranes were isolated from an isopycnic sucrose gradient and equal amounts of total protein from each membrane sample were subjected to 11% SDS-PAGE and Western blot analysis with antibody to EcoRI endonuclease. B, K17 containing plasmid pTLQ023 was induced in the absence (lane 1) and presence (lane 2) of 50 µM CCCP. Cytoplasmic membranes (lanes 1 and 2) were isolated from an isopycnic sucrose gradient and equal amounts of total protein from each membrane sample were subjected to 11% SDS-PAGE and Western blot analysis with antibody to EcoRI endonuclease. A sample of the harvested bacteria (lanes 3 and 4) was also subjected to Western blot analysis.View Large Image Figure ViewerDownload (PPT)K17 bacteria expressing TolQII/III were also treated with 50 µM of the protonophore CCCP. Inner membranes were isolated and equal amounts of protein from each inner membrane preparation were subjected to Western blot analysis. No TolQII/III was detected in the CCCP treated sample (Fig. 6B, lane 2). CCCP treatment did not affect the expression of TolQII/III as illustrated by the presence of a band corresponding to TolQII/III in a whole cell sample (Fig. 6B, lane 4). The absence of inner membrane TolQII/III following CCCP treatment indicates that the membrane potential is required for the insertion of the hairpin loop formed by the second and third membrane-spanning regions.Overexpression of TolQ Does Not Affect Correct Protein LocalizationOur experiments with the TolQ fusions suggest that TolQ is inserted into the cytoplasmic membrane independently of functional SecA protein. To assess the effects of TolQ overexpression on the translocation of Sec-dependent proteins, K17(DE3) bacteria containing plasmid pTLQHis were treated with 2 mM IPTG to induce synthesis of TolQ. Forty minutes following induction, TolQ can be detected on an SDS-polyacrylamide gel by Coomassie stain (Fig. 7, upper panel). At this point, the production of maltose binding protein (MBP) was induced by the addition of maltose to the culture medium. The processing of MBP was monitored by Western blot, and no precursor was detected (Fig. 7, lower panel). Precursor is observed only following treatment with 1 mM azide (Fig. 7, lower panel, lane AZ) which inhibits Sec-dependent translocation. Western blot analysis with antibody to SecA was also performed on th" @default.
• W2095078885 created "2016-06-24" @default.
• W2095078885 creator A5076646260 @default.
• W2095078885 creator A5087461694 @default.
• W2095078885 date "1996-06-01" @default.
• W2095078885 modified "2023-09-28" @default.
• W2095078885 title "Membrane Insertion Characteristics of the Various Transmembrane Domains of the Escherichia coli TolQ Protein" @default.
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