C-less Permease Research Articles

The role of helix VIII in the lactose permease of Escherichia coli: I. Cys-scanning mutagenesis.

Using a functional lactose permease mutant devoid of Cys residues (C-less permease), each amino acid residue in transmembrane domain VIII and flanking hydrophilic loops (from Gln 256 to Lys 289) was replaced individually with Cys. Of the 34 single-Cys mutants, 26 accumulate lactose to > 70% of the steady state observed with C-less permease, and an additional 7 mutants (Gly 262-->Cys, Gly 268-->Cys, Asn 272-->Cys, Pro 280-->Cys, Asn 284-->Cys, Gly 287-->Cys, and Gly 288-->Cys) exhibit lower but significant levels of accumulation (30-50% of C-less). As expected (Ujwal ML, Sahin-Tóth M, Persson B, Kaback HR, 1994, Mol Membr Biol 1:9-16), Cys replacement for Glu 269 abolishes lactose transport. Immunoblot analysis reveals that the mutants are inserted into the membrane at concentrations comparable to C-less permease, with the exceptions of mutants Pro 280-->Cys, Gly 287-->Cys, and Lys 289-->Cys, which are expressed at reduced levels. The transport activity of the mutants is inhibited by N-ethylmaleimide (NEM) in a highly specific manner. Most of the mutants are insensitive, but Cys replacements render the permease sensitive to inactivation by NEM at positions that cluster in manner indicating that they are on one face of an alpha-helix (Gly 262-->Cys, Val 264-->Cys, Thr 265-->Cys, Gly 268-->Cys. Asn 272-->Cys, Ala 273-->Cys, Met 276-->Cys, Phe 277-->Cys, and Ala 279-->Cys). The results indicate that transmembrane domain VIII is in alpha-helical conformation and demonstrate that, although only a single residue in this region of the permease is essential for activity (Glu 269), one face of the helix plays an important role in the transport mechanism. More direct evidence for the latter conclusion is provided in the companion paper (Frillingos S. Kaback HR, 1997, Protein Sci 6:438-443) by using site-directed sulfhydryl modification of the Cys-replacement mutants in situ.

Cysteine-scanning mutagenesis of helix II and flanking hydrophilic domains in the lactose permease of Escherichia coli.

Using a functional lactose permease mutant devoid of Cys residues (C-less permease), each amino acid residue in putative transmembrane helix II and flanking hydrophilic loops (from Leu34 to Lys74) was replaced individually with Cys. Of the 41 single-Cys mutants, 28 accumulate lactose to > 70% of the steady state observed with C-less permease, and an additional 10 mutants exhibit lower but significant levels of accumulation (25-60% of C-less). His35-->Cys permease exhibits very low activity (ca. 20% of C-less), while Gly64-->Cys or Asp68-->Cys permease is unable to accumulate lactose. However, His35 can be replaced with Arg without effect on transport activity [Padan, E., Sarkar, H.K., et al. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 6765-6768]. In addition, even though mutant Gly64-->Cys or Glu68-->Cys is inactive both in the C-less background and in the wild-type, neither Gly64 [Jung, K., Jung, H., et al. (1995) Biochemistry 34, 1030-1039] nor Glu68 [Jessen-Marshall, A.E., & Brooker, R.J. (1996) J. Biol. Chem. 271, 1400-1404] is essential for active lactose transport. Immunoloblot analysis reveals that all of the mutants except His35-->Cys permease are inserted into the membrane at concentrations comparable to that of C-less permease. The transport activity of the single-Cys mutants is altered by N-ethylmaleimide (NEM) treatment in a highly specific manner. Most of the mutants are insensitive, but Cys replacements render the permease sensitive to NEM inactivation at positions that cluster in a manner indicating that they are on one face of an alpha-helix (Thr45-->Cys, Gly46-->Cys, Phe49-->Cys, Ser53-->Cys, Ser56-->Cys, Gln60-->Cys, and Ser67-->Cys). Interestingly, the same face contains positions where Cys substitution itself leads to low transport activity (Ile52-->Cys, Leu57-->Cys, Gln60-->Cys, and Gly64-->Cys). The results demonstrate that although no residue per se in this region of the permease is irreplaceable, the surface of one face of helix II is important for active lactose transport.

Cysteine-Scanning Mutagenesis of Transmembrane Domain XII and the Flanking Periplasmic Loop in the Lactose Permease of Escherichia coli

Using a functional lactose permease mutant devoid of Cys residues (C-less permease), each amino acid residue in transmembrane domain XII and the periplasmic loop between putative helices XI and XII (loop XI/XII) was replaced individually with Cys. Out of 34 mutants, 31 exhibit 60-100% or more of C-less activity, mutants Gly377-->Cys and Leu385-->Cys exhibit lower rates of transport but accumulate lactose about 60-70% as well as C-less, and mutant Leu400-->Cys exhibits < 20% of C-less activity. Immunoblots reveal that all of the mutant proteins are present in the membrane in amounts comparable to that of C-less with the exception of mutants Gly377-->Cys and Leu385-->Cys which are expressed about 40% as well as C-less and mutant Leu400-->Cys which is hardly detectable. When transferred to the wild-type background, however, mutant Leu400-->Cys is expressed normally and exhibits highly significant transport activity. Finally, each active Cys-replacement mutant was assayed for sensitivity to N-ethylmaleimide, and with three exceptions, the mutants are essentially unaffected by the alkylating agent. Mutants Val367-->Cys, Gly370-->Cys, and Tyr373-->Cys which are predicted to be immediately distal to helix XI in loop XI/XII are significantly inactivated. The periodicity observed suggests that the periplasmic end of transmembrane domain XI may extend to position 373. In the following paper [Voss, J., He, M. M., Hubbell, W. L., & Kaback, H. R. (1996) Biochemistry 35, 12915-12918], site-directed spin labeling of single-Cys mutants at positions 387-402 is used to demonstrate that transmembrane domain XII is in an alpha-helical conformation.

Role of glycine residues in the structure and function of lactose permease, an Escherichia coli membrane transport protein.

By using oligonucleotide-directed, site-specific mutagenesis, the role of 34 Gly residues in the lactose permease of Escherichia coli has been studied systematically. Each of 34 out of a total of 36 Gly residues was replaced with Cys in a functional permease mutant devoid of Cys residues (C-less permease), as previous experiments demonstrate that Gly-402 and Gly-404 can be deleted by truncation of the C-terminus with no loss of activity [Roepe, P. D., et al. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 3992; McKenna, E., et al. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 2969]. Out of the 34 Cys-replacement mutants described, 15 transport lactose with high activity, 16 exhibit decreased but significant ability to catalyze lactose accumulation, and 3 (Gly-64-->Cys, Gly-115-->Cys and Gly-147-->Cys) exhibit no activity whatsoever. The inactive mutants were studied in more detail by replacement of Gly with Ala, Val, or Pro. C-less permease with Gly-115-->Ala or Gly-147-->Ala transports lactose almost as well as the control, while mutants with Val or Pro in place of Gly have little or no capacity to accumulate the disaccharide. In contrast, mutants with Ala, Val, or Pro in place of Gly-64 are inactive. Strikingly, however, when the mutations are placed in the wild-type background, Gly-64-->Ala permease transports lactose, beta,D-galactopyranosyl 1-thio-beta,D-galactopyranoside, and methyl 1-thio-beta,D-galactopyranoside 40-60% as well as wild-type permease, while Gly-64-->Val or Gly-64-->Pro permease is inactive toward all of these substrates. The results indicate that although none of the Gly residues in lactose permease is mandatory for activity, the bulk of the side chain at positions 64, 115, and 147, rather than conformational flexibility at these positions, is particularly important.

Design of a membrane protein for site-specific proteolysis: properties of engineered factor Xa protease sites in the lactose permease of Escherichia coli.

Lactose permease is a polytopic membrane transport protein with 12 hydrophobic transmembrane domains connected by hydrophilic loops on the cytoplasmic and periplasmic sides of the membrane. By the use of an active permease mutant devoid of Cys residues (C-less permease), single recognition sites (Ile-Glu-Gly-Arg) for the protease factor Xa (fXa) were engineered into hydrophilic loops 7, 8, and 10 in the C-terminal half of the protein. Mutants carrying single sites inserted at position 255, 259 (loop 7), 283, 286 (loop 8), or 341 (loop 10) exhibit significant lactose accumulation (30-70% of C-less permease) and normal levels of expression in the membrane. However, despite solubilization in dodecyl beta-D-maltoside, none of the mutant permeases is proteolyzed by fXa to a significant extent. Insertion of two recognition sites in tandem at position 255 results in partial cleavage, and remarkably, introduction of three sites in tandem leads to complete proteolysis by fXa. Importantly, mutants with two or three fXa sites at position 255 accumulate lactose to high levels (70% of C-less) and are present in the membrane in amounts comparable to that of C-less permease. The results indicate that hydrophilic loops 7, 8, and 10 are buried in the tertiary structure of the permease where they are inaccessible to protease. Insertion of tandem sites probably facilitates proteolysis by causing loops to become more accessible to the aqueous phase and by increasing the local concentration of protease recognition sites. The approach should be applicable to other polytopic membrane proteins.

The role of transmembrane domain III in the lactose permease of escherichia coli

Deletion of putative transmembrane helix III from the lactose permease of Escherichia coli results in complete loss of transport activity. Similarly, replacement of this region en bloc with 23 contiguous Ala, Leu, or Phe residues abolishes active lactose transport. The observations suggest that helix III may contain functionally important residues; therefore, this region was subjected to Cys-scanning mutagenesis. Using a functional mutant devoid of Cys residues (C-less permease) each residue from Tyr 75 to Leu 99 was individually replaced with Cys. Twenty-one of the 25 mutants accumulate lactose to > 70% of the steady-state exhibited by C-less permease, and an additional 3 mutants transport to lower, but significant levels (40-60% of C-less). Cys replacement for Leu 76 results in low transport activity (18% of C-less). However, when placed in the wild-type background, mutant Leu 76-->Cys exhibits highly significant rates of transport (55% of wild type) and steady-state levels of lactose accumulation (65% of wild type). Immunoblots reveal that the mutants are inserted into the membrane at concentrations comparable to wild type. Studies with N-ethylmaleimide show that mutant Gly 96-->Cys is rapidly inactivated, whereas the other single-Cys mutants are not altered significantly by the alkylating agent. Moreover, the rate of inactivation of Gly 96-->Cys permease is enhanced at least 2-fold in the presence of beta-galactopyranosyl 1-thio-beta, D-galactopyranoside. The observations demonstrate that although no residue per se appears to be essential, structural properties of helix III are important for active lactose transport.

Cysteine scanning mutagenesis of the N‐terminal 32 amino acid residues in the lactose permease of Escherichia coli

Using a functional lactose permease mutant devoid of Cys residues (C-less permease), each amino acid residue in the hydrophilic N-terminus and the first putative transmembrane helix was systematically replaced with Cys (from Tyr-2 to Trp-33). Twenty-three of 32 mutants exhibit high lactose accumulation (70-100% or more of C-less), and an additional 8 mutants accumulate to lower but highly significant levels. Surprisingly, Cys replacement for Gly-24 or Tyr-26 yields fully active permease molecules, and permease with Cys in place of Pro-28 also exhibits significant transport activity, although previous mutagenesis studies on these residues suggested that they may be required for lactose transport. As expected, Cys replacement for Pro-31 completely inactivates, in agreement with previous findings indicating that "helix-breaking" propensity at this position is necessary for full activity (Consler TG, Tsolas O, Kaback HR, 1991, Biochemistry 30:1291-1297). Twenty-nine mutants are present in the membrane in amounts comparable to C-less permease, whereas membrane levels of mutants Tyr-3-->Cys and Phe-12-->Cys are slightly reduced, as judged by immunological techniques. Dramatically, mutant Phe-9-->Cys is hardly detectable when expressed from the lac promoter/operator at a relatively low rate, but is present in the membrane in a stable form when expressed at a high rate from the T7 promoter. Finally, studies with N-ethylmalemide show that 6 Cys-replacement mutants that cluster at the C-terminal end of putative helix I are inactivated significantly.(ABSTRACT TRUNCATED AT 250 WORDS)

Cysteine scanning mutagenesis of putative transmembrane helices IX and X in the lactose permease of Escherichia coli.

Using a functional lactose permease mutant devoid of Cys residues (C-less permease), each amino-acid residue in putative transmembrane helices IX and X and the short intervening loop was systematically replaced with Cys (from Asn-290 to Lys-335). Thirty-four of 46 mutants accumulate lactose to high levels (70-100% or more of C-less), and an additional 7 mutants exhibit lower but highly significant lactose accumulation. As expected (see Kaback, H.R., 1992, Int. Rev. Cytol. 137A, 97-125), Cys substitution for Arg-302, His-322, or Glu-325 results in inactive permease molecules. Although Cys replacement for Lys-319 or Phe-334 also inactivates lactose accumulation, Lys-319 is not essential for active lactose transport (Sahin-Tóth, M., Dunten, R.L., Gonzalez, A., & Kaback, H.R., 1992, Proc. Natl. Acad. Sci. USA 89, 10547-10551), and replacement of Phe-334 with leucine yields permease with considerable activity. All single-Cys mutants except Gly-296 --> Cys are present in the membrane in amounts comparable to C-less permease, as judged by immunological techniques. In contrast, mutant Gly-296 --> Cys is hardly detectable when expressed at a relatively low rate from the lac promoter/operator but present in the membrane in stable form when expressed at a high rate from T7 promoter. Finally, studies with N-ethylmaleimide (NEM) show that only a few mutants are inactivated significantly. Remarkably, the rate of inactivation of Val-315 --> Cys permease is enhanced at least 10-fold in the presence of beta-galactopyranosyl 1-thio-beta-D-galactopyranoside (TDG) or an H+ electrochemical gradient (delta mu-H+). The results demonstrate that only three residues in this region of the permease -Arg-302, His-322, and Glu-325-are essential for active lactose transport. Furthermore, the enhanced reactivity of the Val-315 --> Cys mutant toward NEM in the presence of TDG or delta mu-H+ probably reflects a conformational alteration induced by either substrate binding or delta mu-H+.

Role of the charge pair aspartic acid-237-lysine-358 in the lactose permease of Escherichia coli.

Using a lactose permease mutant devoid of Cys residues (C-less permease), Asp237 and Lys358 were replaced with Cys or other amino acids to pursue the proposal that the two residues form a charge pair [King, S. C., Hansen, C. L., & Wilson, T.H. (1991) Biochim. Biophys. Acta 1062, 177-186]. Individual replacement of Asp237 with Cys, Ala, or Lys or replacement of Lys358 with Cys, Ala, or Asp virtually abolishes active lactose transport. However, simultaneous replacement of both residues with Cys and/or Ala yields permease with high activity. Therefore, neutral amino acid substitutions at either position are detrimental only because they leave the opposing charge unpaired. Strikingly, moreover, when Asp237 is interchanged with Lys358, high activity is observed. The results indicate strongly that Asp237 and Lys358 interact to form a salt bridge and that neither residue nor the salt bridge per se is important for activity. Immunoblots reveal low membrane levels of the active mutants lacking the putative salt bridge, suggesting a role for the salt bridge in either permease folding or stability and raising the possibility that the salt bridge may exist in a folding intermediate but not in the mature protein. Remarkably, however, a mutant with Cys in place of Asp237 is restored to full activity by carboxymethylation which recreates a negative charge at position 237. Pulse-chase analysis and heat-inactivation studies indicate that the stability of the double mutant with Cys at positions 237 and 358 is comparable to C-less. Therefore, the interaction between Asp237 and Lys358 is likely to be important for permease folding and is maintained in the mature protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Functional interactions between putative intramembrane charged residues in the lactose permease of Escherichia coli.

Using a lactose permease mutant devoid of Cys residue ("C-less permease"), we systematically replaced putative intramembrane charged residues with Cys. Individual replacements for Asp-237, Asp-240, Glu-269, Arg-302, Lys-319, His-322, Glu-325, or Lys-358 abolish active lactose transport. When Asp-237 and Lys-358 are simultaneously replaced with Cys and/or Ala, however, high activity is observed. Therefore, when either Asp-237 or Lys-358 is replaced with a neutral residue, leaving an unpaired charge, the permease is inactivated, but neutral replacement of both residues yields active permease [King, S. C., Hansen, C. L. & Wilson, T. H. (1991) Biochim. Biophys. Acta 1062, 177-186]. Remarkably, moreover, when Asp-237 is interchanged with Lys-358, high activity is observed. The observations provide a strong indication that Asp-237 and Lys-358 interact to form a salt bridge. In addition, the data demonstrate that neither residue nor the salt bridge plays an important role in the transport mechanism. Thirteen additional double mutants were constructed in which a negative and a positively charged residue were replaced with Cys. Only Asp-240-->Cys/Lys-319-->Cys exhibits significant activity, accumulating lactose to 25-30% of the steady state observed with C-less permease. Replacing either Asp-240 or Lys-319 individually with Ala also inactivates the permease, but double mutants with neutral substitutions (Cys and/or Ala) at both positions exhibit essentially the same activity as Asp-240-->Cys/Lys-319-->Cys. In marked contrast to Asp-237 and Lys-358, interchanging Asp-240 and Lys-319 abolishes active lactose transport. The results demonstrate that Asp-240 and Lys-319, like Asp-237 and Lys-358, interact functionally and may form a salt bridge. However, the interaction between Asp-240 and Lys-319 is clearly more complex than the interaction between Asp-237 and Lys-358. In any event, the findings suggest that putative transmembrane helix VII lies next to helices X and XI in the tertiary structure of lactose permease.

Construction of a functional lactose permease devoid of cysteine residues

By use of oligonucleotide-directed, site-specific mutagenesis, a lactose (lac) permease molecule was constructed in which all eight cysteinyl residues were simultaneously mutagenized (C-less permease). Cys154 was replaced with valine, and Cys117, -148, -176, -234, -333, -353, and -355 were replaced with serine. Remarkably, C-less permease catalyzes lactose accumulation in the presence of a transmembrane proton electrochemical gradient (interior negative and alkaline). Thus, in intact cells and right-side-out membrane vesicles containing comparable amounts of wild-type and Cys-less permease, the mutant protein catalyzes lactose transport at a maximum velocity and to a steady-state level of accumulation of about 35% and 55%, respectively, of wild-type with a similar apparent Km (ca. 0.3 mM). As anticipated, moreover, active lactose transport via C-less permease is completely resistant to inactivation by N-ethylmaleimide. Finally, C-less permease also catalyzes efflux and equilibrium exchange at about 35% of wild-type activity. The results provide definitive evidence that sulfhydryl groups do not play an essential role in the mechanism of lactose/H+ symport. Potential applications of the C-less mutant to studies of static and dynamic aspects of permease structure/function are discussed.