S. Bon, Laboratoire de Neurobiologie Cellulaire et Moléculaire, CNRS UMR 8544, Ecole Normale Supérieure, 46 rue d'Ulm, 75005 Paris, France. Fax: + 33 1 44 32 38 87, Tel.: + 33 1 44 32 38 91, E-mail: firstname.lastname@example.org
The C-terminal t peptide (40 residues) of vertebrate acetylcholinesterase (AChE) T subunits possesses a series of seven conserved aromatic residues and forms an amphiphilic α-helix; it allows the formation of homo-oligomers (monomers, dimers and tetramers) and heteromeric associations with the anchoring proteins, ColQ and PRiMA, which contain a proline-rich motif (PRAD). We analyzed the influence of mutations in the t peptide of Torpedo AChET on oligomerization and secretion. Charged residues influenced the distribution of homo-oligomers but had little effect on the heteromeric association with QN, a PRAD-containing N-terminal fragment of ColQ. The formation of homo-tetramers and QN-linked tetramers required a central core of four aromatic residues and a peptide segment extending to residue 31; the last nine residues (32–40) were not necessary, although the formation of disulfide bonds by cysteine C37 stabilized T4 and T4–QN tetramers. The last two residues of the t peptide (EL) induced a partial intracellular retention; replacement of the C-terminal CAEL tetrapeptide by KDEL did not prevent tetramerization and heteromeric association with QN, indicating that these associations take place in the endoplasmic reticulum. Mutations that disorganize the α-helical structure of the t peptide were found to enhance degradation. Co-expression with QN generally increased secretion, mostly as T4–QN complexes, but reduced it for some mutants. Thus, mutations in this small, autonomous interaction domain bring information on the features that determine oligomeric associations of AChET subunits and the choice between secretion and degradation.
In vertebrates, the acetylcholinesterase (AChE) gene generates several types of catalytic subunits through alternative splicing in the 3′ region of the transcripts [1–3]. These subunits possess the same common catalytic domain, followed by distinct C-terminal peptides (r, h and t), characterizing the AChER, AChEH and AChET variants [4–6]. In mammals, AChER subunits seem to be expressed mostly during embryogenesis and in the brain after stress [7,8]; they correspond to a soluble, monomeric enzyme species. AChEH subunits possess one or two cysteines and a GPI-addition signal in their C-terminal peptide: they generate GPI-anchored, disulfide-linked dimers, which represent a major fraction of AChE in Torpedo electric organs and muscles, and are expressed on the surface of blood cells in mammals [9–12]. AChET subunits are expressed in muscles and in the nervous system of higher vertebrates and therefore represent the functional cholinesterase species in the cholinergic system [3,13,14].
The C-terminal t peptide confers several characteristic properties to AChET subunits, allowing them to form a series of homo-oligomers (monomers, dimers, tetramers and higher oligomers) when expressed in transfected COS cells [13,15]; some of these molecules are amphiphilic, i.e. interact with detergent micelles [16,17]. AChET subunits also form hetero-oligomers with the collagen, ColQ, or with the transmembrane protein, PRiMA [18,19]; in mammals, these structural proteins anchor the major functional species of cholinesterases in the basal lamina of the neuromuscular junction and in neuronal cell membranes, respectively [20,21]. In the collagen-tailed and hydrophobic-tailed forms, four catalytic AChE subunits are associated, through their C-terminal t peptides, with proline-rich attachment domains (PRAD) localized in the N-terminal regions of ColQ or PRiMA [19,22,23].
The t peptide of AChE consists of 40 residues, with a series of seven strictly conserved aromatic residues, including three evenly spaced tryptophans, as well as acidic and basic residues that are conserved or semiconserved in most vertebrates . This peptide is necessary for the amphiphilic properties which characterize AChET subunits and some of their oligomers (, , ), for the formation of nonamphiphilic homotetramers (), as well as for the heteromeric association of AChET subunits with QN, an N-terminal fragment of collagen ColQ that contains a proline-rich motif, thus producing T4–QN complexes [23,24].
The t peptide constitutes an autonomous interaction domain, called the WAT [tryptophan (W) amphiphilic tetramerization] domain, because it can associate with a PRAD, even in the absence of the catalytic domain; moreover, addition of a t peptide at the C-terminus of foreign proteins, green fluorescent protein and alkaline phosphatase, endowed them with amphiphilic properties and enabled them to form PRAD-associated tetramers . We also found that the simultaneous presence of the t peptide and of mutations at the interface of AChE dimers – the ‘four helix bundle’– prevented the secretion of AChET subunits . We recently showed that the t peptide induces intracellular degradation through the endoplasmic reticulum associated degradation (ERAD)/proteasome pathway, to different extents, depending on the protein to which it is attached, and that aromatic residues are necessary for this effect .
Recent spectroscopic studies showed that the t peptide is organized as an amphiphilic α helix, in which aromatic residues form a hydrophobic sector [29,30]. In addition, an analysis of intercatenary disulfide bonds in the T4–QN complex also demonstrated that the four t peptides are parallel and oriented in the same direction, opposite to that of the PRAD . The structure of a complex formed between synthetic peptides (four t peptides with one PRAD) confirmed this orientation (M. Harel et al., manuscript in preparation).
In the present study, we mutated aromatic and charged residues, suppressed the C-terminal cysteine or introduced cysteines at other positions, and deleted more or less extended C-terminal segments of the t peptide, in Torpedo AChET subunits, to determine the structural basis of the characteristic properties that the t peptide confers to AChET subunits.
Materials and methods
AChE constructs and site-directed mutagenesis
Mutagenesis was performed according to the method of Kunkel et al.. cDNAs encoding wild-type and mutated Torpedo AChET, as well as the previously described Torpedo QN protein , intact or deleted of its PRAD motif (residues 70–86), were inserted into the pEFBos vector. Throughout this article, the residues of the t peptide are numbered from 1 to 40, corresponding to positions 536–575 in the Torpedo AChET subunit, so that the Torpedo mutants are indicated by the modified residues, e.g. W17P.
Transfection of COS cells
COS cells were transfected by the DEAE-dextran method, as described previously , using 4 µg of DNA encoding the AChE catalytic subunit and 4 µg of DNA encoding QN or PRAD-deleted QN, per 100 mm dish. Because Torpedo AChE folds into its active conformation at 27 °C, but not at 37 °C, the cells were incubated for 2 days at 37 °C after transfection, then transferred to 27 °C and maintained at this temperature for 3–4 days, in a medium containing 10% Nuserum (Inotech, Dottikon, Switzerland), which had been pretreated with 10−6m soman to inactivate serum cholinesterases.
To analyze its heteromeric interaction with an associated structural protein, AChET was coexpressed with QN. By using QN, rather than full-length ColQ, we avoid the complexity caused by the formation of the triple helical collagen and by the low salt aggregation of collagen-tailed AChE forms . We added a flag epitope (DYKDDDDK) at the C-terminus of QN, so that complexes containing this protein could be characterized with the anti-flag immunoglobulin, M2 (Kodak), as described previously . The effect of QN on the level of cellular and secreted activity was analysed by comparing the coexpression of AChET with full-length QN and with a PRAD-deleted QN, to compensate for competition between the two transfected vectors.
The cells were extracted at 20 °C with TMg buffer (1% Triton X-100, 50 mm Tris/HCl, pH 7.5, 10 mm MgCl2), and then centrifuged at 10 000 g for 30 min. Media were also centrifuged at 10 000 g for 30 min to remove cell debris before analysis.
AChE activity was determined according to the colorimetric method of Ellman et al.  at room temperature. As the monomeric Torpedo AChE forms produced by some mutants were inactivated by 5,5′-dithiobis(2-nitrobenzoic acid) , the enzyme samples were incubated for variable periods of time, depending on their activity, with a reaction medium containing acetylthiocholine iodide in phosphate buffer, pH 7; 5,5′-dithiobis(2-nitrobenzoic acid) was then added and the absorbance at 414 nm was determined using a Labsystems (Helsinki, Finland) Multiskan RC automatic plate reader. Alkaline phosphatase and β-galactosidase from Escherichia coli were assayed with the chromogenic substrates p-nitrophenyl phosphate and o-nitrophenyl galactoside, respectively.
Sedimentation and electrophoretic analyses
Centrifugation was performed in 5–20% sucrose gradients (50 mm Tris/HCl, pH 7.5, 50 mm MgCl2, either in the presence of 0.2% Brij-97 or in the presence of 0.2% Triton X-100) in a Beckman SW41 rotor, at 36 000 r.p.m., for 18 h at 6 °C. The gradients contained E. coliβ-galactosidase (16 S) and alkaline phosphatase (6.1 S) as internal sedimentation standards . Amphiphilic molecules generally sediment faster in the presence of Triton X-100 than in the presence of Brij-97, providing an indication of their amphiphilic character.
Electrophoresis in nondenaturating polyacrylamide gels was performed as described by Bon et al. , and AChE activity was revealed by the histochemical method of Karnovsky & Roots . In charge shift electrophoresis, the electrophoretic migration of amphiphilic molecules was accelerated in the presence of sodium deoxycholate, when compared to migration in the presence of the neutral detergent, Triton X-100, alone. As an index of the degree of amphiphilicity, we used the ratio between migration in the presence of DOC to migration in Triton X-100 alone, after normalizing these migrations to that of a nonamphiphilic species, the wild-type tetramers or T4–QN.
Both sedimentation and nondenaturing electrophoresis provide semiquantitative information on the interaction of AChE molecules with micelles, and are generally in complete agreement. However, in the present study, we found that some mutations in the t peptide perturb amphiphilic interactions in such a way that sedimentation became essentially identical in the presence of Triton X-100 and Brij-97, while charge shift electrophoresis still showed a marked influence of the detergent: this was the case for dimers of aromatic mutants such as W17H or W17A. In addition, the T4–QN complexes formed by mutants W17F and W17A showed an unusual retardation in sedimentation in the presence of Triton X-100, compared with Brij-97.
Analyses of AChE activity and molecular forms
Figure 1 shows the sequence of the t peptide of Torpedo AChET subunits, and schematically illustrates its proposed α helical structure, its association with the PRAD of ColQ, and the various oligomers of AChET subunits that result from its interactions.
We analyzed how mutations in the t peptide affect the levels of cellular and secreted activity of Torpedo AChE in transfected COS cells. The activities were normalized to those obtained for wild-type AChET in parallel transfections. Immunofluorescence of the protein produced at early stages after transfection indicated that all mutants were expressed in a similar manner. After 2 days at 27 °C, a temperature which allows the correct folding of active Torpedo AChE (see the Materials and methods), the level of cellular activity reached a plateau and the rate of secretion remained constant. Maximal secretion was obtained for a truncated mutant (I3C/stop4), which retained only the first two residues of the t peptide, followed by a cysteine at position 3; this cysteine allowed the formation of dimers, which lacked the aromatic residues and were therefore nonamphiphilic. The secretion of active wild-type AChET subunits was less than 10% of the truncated mutant, showing that a large fraction is degraded intracellularly [27,28].
The molecular forms of AChE were identified by electrophoresis in nondenaturing polyacrylamide gels and their amphiphilic character was evaluated by charge shift electrophoresis in the presence or absence of sodium deoxycholate . As Torpedo AChET monomers are rapidly inactivated under the conditions of electrophoretic migration, the distribution of AChE molecular forms was analyzed by sedimentation in sucrose gradients.
To analyse the capacity of Torpedo AChET subunits to associate with a PRAD, we coexpressed them with protein QN (Fig. 1D). This QN protein organizes wild-type AChET subunits into tetramers (T4–QN) that are nonamphiphilic and efficiently secreted , reaching ≈ 40% of the secretion observed with the truncated I3C/stop4 mutant. The formation of QN-linked oligomers therefore rescued an important fraction of the wild-type catalytic Torpedo AChET subunits from intracellular degradation.
Mutation of charged residues of the t peptide
The t peptide contains seven acidic (D, E) and eight basic (H, K, R) residues, which may form intracatenary salt bridges in the helical conformation (Fig. 1C) and perhaps intercatenary salt bridges in oligomeric assemblies; we mutated these residues to alanines, individually or in groups.
Mutations D4A/E5A, E7A/R8A, K11A, E13A, R16A, K25A, or D29A did not markedly modify the levels of cellular and secreted activities. However, other mutations had a stronger effect, as shown in Fig. 2A. Both cellular and secreted activities were increased by the point mutation, E1A, but decreased by replacement of the first four acidic residues (E1, D4, E5, E7) by alanines. Mutation H15A enhanced the efficiency of secretion, because it decreased the cellular activity but increased the secreted activity.
Like the wild-type AChET subunits, all mutants produced amphiphilic dimers () and nonamphiphilic tetramers (). However, their proportions varied, as illustrated by the sedimentation profiles of four mutants (Fig. 2B). These profiles did not change with time after transfection. They characterize each mutant and are not simply related to the intracellular concentration of the enzyme, as shown by the fact that the D4A/E5A and R16A mutants produced approximately the same levels of cellular activity with the same proportions of molecular forms as the wild type, but differed in the activity and molecular forms of the secreted enzyme. Conversely, the secreted enzyme was quantitatively and qualitatively similar for mutants K11A and D29A, although the patterns of cellular molecular forms were different.
In all cases, coexpression with QN increased the level of secretion and produced T4–QN complexes, as for the wild type.
Mutation of aromatic residues
The three tryptophans (W10, W17, W24) and Y31 were mutated to alanines, and all seven aromatic residues were mutated to prolines. These mutations had little effect on the cellular activity; the secreted activity was reduced by about half by most mutations, but significantly increased by Y31A (data not shown) and Y31P (Fig. 3A). The major molecular forms produced by these mutants were T2 dimers, as illustrated in electrophoretic patterns (Fig. 3D); the production of tetramers was strongly reduced or abolished, again with the exception of Y31 mutants. The amphiphilic character of T2 dimers was retained when individual aromatic residues were replaced with alanines, but it was reduced when the central aromatic residues were mutated to prolines, in a position-dependent manner (Fig. 3B).
The production of T4–QN complexes, resulting in an increased secretion, was not affected by mutation of Y31, but was reduced by mutations of W10 and F28, and was essentially suppressed by mutations of F14, W17, Y20 and W24, either to alanine (not shown) or to proline (Fig. 3A,D). As illustrated in Fig. 3C, we found that when the W10P mutant was coexpressed with the flagged QN protein, the anti-flag M2 immunoglobulin reacted with a fraction of dimers as well as with tetramers, indicating the presence of T2–QN complexes, in addition to T4–QN complexes, in the culture medium.
We replaced the central tryptophan (W17) with a hydrophobic aromatic residue (F), an aliphatic aromatic residue (L), a heterocyclic residue (H), as well as a proline (P) and an alanine (A). As shown in Fig. 4, these mutations did not strongly modify the cellular activity, which remained within the range of 83–127% of the wild type, but reduced or suppressed the formation of homotetramers; the amphiphilic character of the resulting dimers was similar to that of the wild type with F, L or A, it was significantly reduced with H and it was essentially abolished with P.
Figure 4 also shows that there was no interaction with QN in the case of W17L and W17P, a very small production of T4–QN in the case of W17A, and a significant production of this complex in the case of W17F and W17H. For these two mutants, coexpression with QN increased the level of secreted activity to about 60% of that obtained in the wild type; in addition to the T4–QN complex, this coexpression markedly increased the secretion of dimers, particularly for W17H, but unlike those formed with the W10P mutant, these dimers did not seem to be associated with QN, as they did not react with the M2 antibody. Whereas the sedimentation of the wild-type T4–QN complex was absolutely unaffected by the presence of Triton X-100 or Brij-97 in the gradient, the sedimentation of T4–QN complexes formed with W17F and W17A was reproducibly retarded in the presence of Triton X-100 (compared to Brij-97), showing an opposite effect to that normally observed for amphiphilic enzyme species, such as , or (see Fig. 6A); the electrophoretic migration of these complexes was also slower than that of the wild-type complex. This may reveal an interaction with Triton X-100 micelles, but not with Brij-97 micelles, perhaps because of an unusual exposure of the aromatic groups in these complexes.
It is noteworthy that, in contrast to the wild-type and T4–QN, the tetramers formed with the W17F, W17H or W17A mutants were only observed in the medium, but not in the cell extract. This indicates a significant difference in the cellular trafficking of the wild-type and mutant complexes.
Perturbation of the helical organization of an aromatic cluster
To perturb the α helical organization of the aromatic-rich segment of the t peptide, we deleted residues T12 and M21, located, respectively, in its N-terminal region and near its centre (Fig. 1A). Mutation M21W introduced an additional aromatic residue, which might create a steric disturbance in oligomers or in heteromeric complexes with QN.
These mutations had moderate effects on the level of cellular activity, and decreased secretion to ≈ 50% of the wild type. The three mutants produced mostly amphiphilic dimers in the cell extracts; in the case of M21Δ, the medium only contained tetramers (Fig. 5A), in contrast to the wild type, in which these molecular forms are present both in the cell extracts and in the medium.
Figure 5A also shows that coexpression with QN increased secretion for T12Δ and M21W (to about 35% and 50% of the wild type, respectively), but not for M21Δ; T4–QN complexes of T12Δ and M21W were characterized in the medium by reaction with the M2 antibody, but were undetectable or barely detectable in the cell extracts, in contrast to the wild-type T4–QN complex.
Effect of a cysteine at various positions in the t peptide
The formation of intercatenary disulfide bonds between wild-type AChET subunits depends on the free cysteine residue located near the C-terminus of the t peptide, C37. Mutation of this cysteine to a serine reduced both cellular and secreted activities; it suppressed the formation of dimers and reduced cellular and secreted tetramers (Fig. 6A); in the presence of QN, the secretion of T4–QN complexes was reduced to ≈ 75% of that of the wild type. Thus, the presence of an intercatenary disulfide bond appears to be necessary for dimerization, but not for tetramerization, particularly in the presence of QN.
To determine whether cysteines at other positions could allow dimerization and further oligomerization, we replaced residues I3, A6, T12, S19, M21, M22 or H34 with a cysteine, with or without mutation of C37 (C37S). The relative levels of cellular and secreted activities, as well as the distribution of molecular forms, are illustrated in Fig. 6A,B. Unlike C37S, none of these mutants produced monomers without dimers; therefore, when two cysteines were present, they were not engaged in an intracatenary disulfide bond, but could form intercatenary bonds in dimers.
Mutation I3C (with or without C37S) considerably increased the cellular activity, mostly as amphiphilic dimers; secreted activity was also increased, but to a much lesser degree. The presence of the N-terminal cysteine thus appears to facilitate dimerization and to reduce degradation. This mutation also increased the cellular activity obtained with the W17P mutant, without restoring its capacity to interact with QN.
Mutation A6C somewhat decreased the cellular activity, but strongly increased secretion. The A6C mutant mainly produced a nonamphiphilic 14 S species, possibly corresponding to octamers. This unusual oligomer dissociated during storage, particularly in the presence of detergent (Triton X-100), transiently producing amphiphilic tetramers () (which are not usually observed in the wild type) and amphiphilic dimers (). When C37 was absent (A6C/C37S), the 14 S species was observed in the cell extracts, but seemed to be less stable, being almost entirely converted to dimers in the medium.
The T12C mutation introduced a cysteine in the N-terminal part of the aromatic-rich segment (not shown). In the absence of cysteine C37, this allowed the formation of amphiphilic dimers, which were secreted together with nonamphiphilic tetramers and a 14 S species. This species was, in fact, predominant in the secreted enzyme and appeared to be much more stable than that formed with A6C, as it was not dissociated after secretion. In the presence of cysteine C37, the T12C mutant produced mainly nonamphiphilic tetramers, which represented the only secreted form. This suggests that tetramers may be stabilized when disulfide bonds were formed at the two positions, 12 and 37.
A cysteine at position 19, in the aromatic-rich segment but opposite to the aromatic cluster, had very different effects, depending on the presence of cysteine C37. Without C37, mutant S19C/C37S produced very low levels of cellular or secreted activity. In contrast, mutant S19C (containing two cysteines at positions 19 and 37) showed a high level of secretion, mostly as nonamphiphilic tetramers, as observed for T12C.
Mutations M21C and M22C, with or without cysteine C37, had little effect on cellular activity (compared to the wild-type and C37S mutant, respectively), but increased secretion to various degrees. M21C and M21C/C37S secreted both tetramers and nonamphiphilic dimers, while M22C and M22C/C37S secreted mostly tetramers. The dimers formed with a cysteine at position 21 appeared nonamphiphilic, suggesting that the aromatic clusters may be masked by an intercatenary disulfide bond in the aromatic-rich segment.
Finally, the mutants containing a cysteine at position 34 (H34C, H34C/C37S) behaved essentially like the wild type, suggesting that the C-terminal segment of the t peptide is flexible.
Figure 6B shows that the various cysteine mutants formed T4–QN complexes (reacting with the anti-flag M2 immunoglobulin), except M21C/C37S. Thus, mutation M21C suppressed the heteromeric complex when cysteine C37 was absent, but not when it was present: this illustrates the importance of the C-terminal cysteine for the assembly and/or stabilization of the T4–QN complex, in agreement with the formation of intercaternary disulfide bonds between the t peptide and QN cysteines.
Coexpression with QN generally increased secretion when T4–QN complexes were produced, although this effect was marginal or absent for mutants that showed a high level of secretion without QN (A6C, A6C/C37S, S19C). However, coexpression induced a decrease, of ≈ 40%, in the secretion of mutant M21C/C37S, for which complexes could not be detected (not shown). This suggests that QN did interact with the mutant AChET subunits, but induced their degradation rather than the assembly of a stable, secretable hetero-oligomer.
The role of methionine 21
The fact that M21C/C37S did not associate with QN, whereas M22C/C37S formed a T4–QN complex, may be related to the orientation of the two adjacent methionines relative to the aromatic cluster, and to a possible structural role of methionine M21: this residue is conserved in vertebrate t peptides, while M22 is replaced with other residues in some species. To examine this possibility, we mutated M21 and M22 to alanines or serines (with or without C37S). Figure 5B shows that coexpression with QN increased the level of secretion, indicating the formation of T4–QN complexes, for all mutants except M21A/C37S (and M21S/C37S). Thus, the presence of a methionine at position 22 is dispensable, but a methionine at position 21 contributes to the stability of the complex, especially when cysteine 37 is absent.
The C-terminal region of the t peptide: a retention motif?
The last four residues of the t peptide, CAEL, contain the cysteine involved in intercatenary disulfide bonds and also resemble the classical ER-retention signal, KDEL. To determine whether its presence might induce a partial retention of AChET subunits, we introduced various mutations in this motif (Fig. 7A).
It should first be noted that mutation C37S (where the cysteine was removed) did not increase secretion, but rather decreased both cellular and secreted activities; this effect may result from the fact that suppression of the cysteine prevented dimerization and reduced the level of secreted tetramers, as discussed above.
Dimerization was also suppressed when the CAEL motif was replaced with KDEL (TKDEL) because this mutation removed the cysteine. The presence of a C-terminal KDEL tetrapeptide increased the level of cellular activity by about threefold relative to C37S, mostly corresponding to ; this increase in cellular enzyme appeared to facilitate tetramerization, as tetramers () were secreted at a higher level than with C37S (Fig. 7A).
We also deleted the last two residues, EL (stop39): the mutant in which the C-terminal motif was reduced to CA produced dimers and secreted 1.7-times more activity than the wild type. More extensive deletions, which removed the cysteine, suppressed dimerization, so that monomers were predominant in the cells and in the medium, and the levels of activity were reduced in both compartments; the secretion of homotetramers was reduced in stop34, compared to C37S, and tetramerization was abolished in stop32.
Like C37S, the TKDEL, stop39, stop34 and stop32 mutants formed T4–QN complexes, so that their secretion was increased in the presence of QN (Fig. 7B). In contrast, the shorter mutant, stop29, showed no interaction with QN, as indicated by the fact that coexpression did not affect either the secreted activity or the molecular forms, characterized by sedimentation. To determine whether the difference between stop32 and stop29 could be ascribed to one of the three residues D29, Q30 or Y31, we mutated each of them to alanine: the level of T4–QN complexes was similar to that of stop32 for Y31A/stop32 and Q30A/stop32, but considerably reduced for D29A/stop32, suggesting a specific influence of residue D29; the strong effect observed by mutation of this charged residue contrasts with the result obtained when it was mutated in the full-length t peptide (see above).
Progressive deletions from the C-terminus of the I3C mutant
To analyze the effect of C-terminal deletions without preventing dimerization, we used the I3C/C37S mutant, which produced mostly amphiphilic dimers (Fig. 6A) and formed T4–QN complexes when coexpressed with QN(Fig. 8A). As in the wild type, the replacement of the C-terminal tetrapeptide by KDEL increased the cellular activity (mainly ) and reduced its secretion, but did not abolish association with QN, producing T4–QN complexes which were secreted. Deletion of the last nine residues (I3C/stop32) did not abolish association with QN, but deletion of the last 12 residues (I3C/stop29) suppressed it completely. Thus, the presence of a cysteine at position 3 did not modify the requirement of residues 29–31 for interaction with QN.
The effect of deletions on the cellular and secreted activities is illustrated in Fig. 8B. The cellular activity remained approximately constant for all deletions, about 50% of the value observed with the full-length t peptide. Removal of the last two residues (EL) increased secretion, as in the case of the wild type. More extensive deletions in the C-terminal region reduced secretion, compared to that of I3C/C37S/stop39, but deletions within the aromatic region progressively increased it, reaching a plateau when all aromatic residues were removed. The dimers were amphiphilic when they contained at least 29 residues of the t peptide (stop29 and longer), but not if they contained 24 residues or fewer (stop24 and shorter), i.e. when they lacked some of the core aromatic residues.
The t peptide: an elongated amphiphilic α helix with a cluster of aromatic residues
Previous studies suggested that the amphiphilic properties of the t peptide reflect the formation of a cluster of aromatic residues in its α helical conformation . The present mutations confirm the role of aromatic residues, but show that they differ considerably in their importance.
Amphiphilicity was not affected by mutations of charged residues, or by deletions of the C-terminal region which removed up to 11 residues, i.e. maintained all the aromatic residues, except Y31. The amphiphilic character was reduced to various extents when the central residues (F14, W17, Y20, W24, F28) were replaced with prolines, but was not affected by mutation of the first and last residues (W10, Y31). Replacement of the most critical residue, W17, by other, different, amino acids (F, L, A, H, P) showed that amphiphilicity was indifferent to their aromatic nature, that it did not strongly depend on their hydrophobicity, but was very sensitive to the α helical structure. Thus, the amphiphilic properties of the t peptide appear to depend predominantly on the spatial organization of a cluster of hydrophobic residues.
In agreement with a previous study , we obtained no evidence that two cysteines, introduced at several positions in the N- and C-terminal regions of the mutated t peptides, could form an intracaternary disulfide bond. The present results thus confirm that the t peptide forms an elongated amphiphilic helix, rather than folding back on itself as a hairpin in which the N- and C-terminal ends might be joined by a disulfide bond.
Homomeric associations of AChET subunits
In contrast to the wild-type AChET subunits, the C37S mutant did not form stable dimers, showing that an intercaternary disulfide bond is necessary. The position of this bond appeared very flexible, as dimers were formed when cysteines were introduced at various positions along the t peptide (with or without cysteine C37): this did not seem to depend on the orientation of the residue relative to the helical axis, although some positions produced higher proportions of dimers than others. However, although most dimers were amphiphilic, those formed in the presence of a cysteine at position 21 were nonamphiphilic, indicating that, in this case, the hydrophobic patches occluded each other because of the formation of a disulfide bond joining the aromatic clusters near their centers.
Depending on the position of an added cysteine, AChET subunits could form predominantly dimers or tetramers – or even higher oligomers sedimenting at 14 S, possibly octamers. This shows that the interactions between the t peptides may present different geometries. Thus, the t peptides can form different homomeric assemblies, which may be stabilized by intercaternary disulfide bonds and are influenced by the positions of these linkages.
In contrast to dimers, tetramers can be formed in the absence of cysteine in the t peptide [27,36], although at a lower level than for the wild type. Tetramers are generally nonamphiphilic (), but some tetramers may also be amphiphilic (), particularly those resulting from the dissociation of the nonamphiphilic 14 S species. Thus, aromatic clusters may be either occluded or at least partly exposed in tetrameric assemblies, indicating that they correspond to distinct quaternary organizations.
Heteromeric associations with the PRAD-containing protein, QN
The major physiological role of the t peptide is clearly to allow the functional localization of AChET tetramers through their association with PRAD-containing proteins, ColQ and PRiMA. In the present study, we focused our attention on the formation of quaternary associations with an N-terminal fragment of ColQ, QN. This protein assembles with wild-type AChET subunits to form QN-linked tetramers (T4–QN), which are nonamphiphilic. Previous studies showed that in these hetero-oligomers, two catalytic subunits are disulfide-linked with QN, while the other two are disulfide-linked together. However, in the absence of cysteine C37, this association still occurs, indicating that it does not require the formation of intercaternary disulfide bonds.
The complex was formed when the t peptide carried an additional cysteine at positions 3, 6, 12, 19, 22 or 34, with or without the original cysteine C37. It was not formed with a cysteine at position 21, except when C37 was present: a cysteine instead of a methionine at position 21 therefore appears unfavorable. This may be partly because of the formation of nonamphiphilic disulfide-linked dimers in which the aromatic clusters are not available for interaction with the proline-rich domain of QN (PRAD). However, the mutation of methionine 21 to an alanine or a serine also weakened the formation of the complex, which again required the presence of cysteine C37: mutants M21A/C37S and M21S/C37S did not associate with QN. In contrast, similar mutations of methionine M22 did not prevent the formation of T4–QN complexes. This demonstrates that the complex was stabilized by disulfide bonds through cysteine 37, and by the presence of methonine 21, in agreement with strong interactions of this methionine with the PRAD in a complex of isolated peptides (WAT)4 PRAD (M. Harel et al., manuscript in preparation). Nevertheless, the fact that M21 could be replaced with a tryptophan suggests that the complex can accommodate the steric constraint owing to a more bulky residue.
The role of aromatic residues in the formation of the QN-linked complex has been established previously [4,28,37]. Using deletions of single residues, we show here that the structure of the cluster is crucial for this quaternary interaction: it was abolished by deletion of residue 21, in the middle of the aromatic-rich segment. The fact that deletion of residue 12, near the N-terminal end of the aromatic cluster, had no such effect, indicates that the orientation of the aromatic cluster relative to the catalytic domain is not crucial. This is consistent with the notion of a flexible junction between the catalytic domain and the amphiphilic helical region of the t peptide . In fact, addition of a variable number of residues between the catalytic domain and the t peptide did not prevent association with QN (N. Morel & S. Bon, unpublished).
In the present study, we assessed the importance of aromatic residues, individually, by point mutations. We observed that mutation of the central residues (F14, W17, Y20, W24) to proline or alanine had a much stronger effect than mutation of W10 and F28, and that Y31 had no effect. Although not identical, the impacts of these mutations were similar to those observed on the amphiphilic character of AChET subunits. However, mutations of W17 to different amino acids clearly dissociated the two properties, as mutation W17L maintained the amphiphilic character, but totally abolished the association with QN, like mutation W17P. This association was reduced when W17 was replaced with a phenylalanine or a histidine, and even more strongly when it was replaced with an alanine, emphasizing the importance of an aromatic side-chain.
With some mutants, we obtained evidence that QN induced the formation of AChET dimers: coexpression with QN strongly increased the secretion of dimers that were not covalently associated with QN (as in the case of W17F and W17H), or were at least partially disulfide-linked with it (as in the case of W10P). Such dimers may represent an intermediate stage in the assembly of QN-linked tetramers, or result from the dissociation of unstable tetramers.
QN-linked tetramers are usually nonamphiphilic, indicating that the clusters of aromatic residues are occluded when the t peptides are associated with the PRAD, in agreement with their strong involvement in these quaternary interactions and with the crystallographic structure of a complex of synthetic peptides (M. Harel, H. Dvir, S. Bon, W. Q. Liu, M. Vidal, C. Garbay, J. L. Sussman, J. Massoulié & I. Silman, unpublished results). However, the QN-linked tetramers, found with the W17F and W17A mutants, showed some interaction with detergents, suggesting that they were less compact than the wild-type complexes. In fact, the formation of QN-linked complexes in the presence of cysteines within the t peptides reveals a considerable flexibility, because disulfide bonds between these residues do not seem to be compatible with the distances between pairs of homologous residues in a complex of wild-type synthetic peptides .
The heteromeric assocation with QN was not suppressed by removal of the last nine residues (following Y31), showing that it depends primarily on the aromatic-rich segment and does not require the C-terminal part of the t peptide. However, removal of three additional residues (D29, Q30, Y31) abolished the interaction, and point mutations showed that this was mostly caused by the deletion of D29: although mutations of charged residues in the full-length t peptide had little effect on association with QN, this suggests that a salt bridge contributed significantly to its stability when the complex was weakened by a C-terminal deletion.
Similarity between nonamphiphilic homomeric and QN-linked tetramers
We observed that the formation of homomeric tetramers was suppressed by all mutations which affected the heterometic complex, T4–QN, suggesting that both quaternary assemblies depend on the same interactions and possess a similar organization, in agreement with the fact that they are both nonamphiphilic. In fact, except for M21C/C37S, mutations that affected QN-linked tetramers appeared to reduce homomeric tetramers more severely, indicating that tetramers are generally stabilized by the presence of a PRAD. In T4–QN complexes, the four α helical t peptides are organized as a super-helix, forming a hollow tube lined by aromatic side-chains, which is occupied by the PRAD. This central space is unlikely to be filled with water molecules or to remain empty in tetramers; it may be reduced by a change in the pitch of the super-helix.
Homo- and hetero-oligomerization occur in the ER, subcellular trafficking
The presence of an ER-retention motif (KDEL) at the C-terminus of the t peptide blocked secretion, as expected, but did not prevent dimerization when a cysteine was introduced at position 3, in the N-terminal region of the t peptide. The KDEL motif actually increased the formation of homotetramers () in the TKDEL mutant, compared to the C37S mutant which also lacked the C-terminal cysteine and was terminated by the tetrapeptide, SAEL. Similarly, a C-terminal KDEL did not block the formation of heteromeric complexes T4–QN; furthermore, the KDEL motif was sterically masked in the complexes, as the complexes were efficiently secreted. The fact that retention of AChET subunits in the ER did not prevent homomeric or heteromeric associations indicates that they occur in this compartment.
We found that the last two residues of the t peptide, EL, which are also present in the ER-retention tetrapeptide, KDEL, exert a weaker, but significant, retention effect, as their deletion increased secretion. It is possible that these residues help to retain isolated AChET subunits in the ER, and thus facilitate their physiological association with the anchoring proteins, ColQ and PRiMA.
Structural differences certainly explain that complexes formed with the T12Δ and some aromatic mutants followed a different cellular trafficking than the wild type: these mutant complexes were secreted, but not detectable in cellular extracts, showing that they did not accumulate in the secretory compartment, but were either rapidly secreted or degraded. We made a similar observation for homomeric tetramers in the case of mutant M21Δ.
Oligomerization, secretion and degradation
In steady-state cultures, we may assume that all mutants were produced at the same rate, as they only differ in the short C-terminal t peptide, so that the rates of secretion of different mutants represent the difference between the common rate of synthesis and the rate of degradation. In a previous study, we established that the presence of the t peptide induces a partial degradation of AChET subunits through the ERAD pathway and that this effect depends on the presence of aromatic residues . Occlusion of these residues may explain that oligomerization generally facilitates secretion: for example, the relative proportions of monomers, dimers and tetramers in cellular extracts and in the medium indicate that the secretion of wild-type AChET subunits increases with their degree of oligomerization. This explains why the introduction of cysteines at certain positions increased secretion, by facilitating the formation of dimers (I3C), tetramers (M22C) or 14 S oligomers (A6C). Conversely, suppression of cysteine C37 (C37S or deletions) reduced secretion.
Similarly, coexpression with QN generally increased secretion by recruiting AChET subunits into T4–QN hetero-oligomers. However, we observed an opposite effect in the case of a few mutants (S19C, M21C/C37S): this implies that the interaction of AChET subunits with QN produced distorted complexes that were degraded more actively than without QN. Thus, oligomerization does not always protect against degradation, but may actually increase it.
We found that extensive deletions of the aromatic-rich segment increased secretion, indicating a reduced degradation, in agreement with a determining role of aromatic residues in this process [4,28,37]. However, the present analysis of point mutations within this segment shows that degradation is not simply determined by the number of aromatic residues: degradation was increased by a large variety of mutations, including addition of an aromatic residue (M21W), replacement with aromatic or other residues (e.g. W17 to F, L, H, A or P; M21 to A or S) or deletions (T12Δ, M21Δ). This suggests that degradation was increased by disorganization of the aromatic cluster of the wild-type t peptide. Thus, the structure of the wild-type t peptide limits degradation.
Variations in the secretion level showed that degradation was also affected by mutations in the N-terminal and C-terminal region flanking the aromatic-rich segment. Secretion was increased by mutation E1A and decreased by mutation ‘4A’ in the N-terminal region. It was increased by deletion of the last two C-terminal residues (EL), and decreased by mutation of the cysteine (C37S) or by deletion of four to 11 residues from the C-terminus. When secretion was suppressed by addition of a C-terminal ER-retention signal (KDEL), changes in the level of cellular activity reflected variations in degradation: an analysis of such mutants confirmed that deletion in the C-terminal segment increased degradation (stop32-KDEL, stop29-KDEL), relative to a mutant in which only two residues were mutated to introduce the KDEL tetrapeptide (TKDEL). The fact that deletions of C-terminal segments increased degradation may result from a disorganization of the α-helical structure.
Mutations of charged residues showed that the level of degradation is not simply correlated with the capacity of the t peptide for oligomerization. Using progressive deletions in a mutant that possessed an N-terminal cysteine at position 3, allowing an efficient dimerization, we found that the targeting of AChET subunits towards degradation or secretion depends, in a complex manner, on different regions of the t peptide. All of these results show that various elements and features of the t peptide contribute to determine the cellular fate of the protein and deserve further investigations. It is highly probable that processes similar to those demonstrated here, in COS cells, operate in muscle and nerve cells that normally express functional AChE, but this will have to be established.
We thank Ms Claudine Schmitt for technical assistance and M. Noël Perrier for critical reading of the manuscript. This work was supported by grants from the Centre National de la Recherche Scientifique, the Association Française contre les Myopathies, the Direction des Forces et de la Prospective, and the European Community; S. Belbeoc'h was the recipient of a PhD grant from the French Ministry of Research.