Elements of the C-terminal t peptide of acetylcholinesterase that determine amphiphilicity, homomeric and heteromeric associations, secretion and degradation Ste ´ phanie Belbeoc’h, Cinzia Falasca, Jacqueline Leroy, Annick Ayon, Jean Massoulie ´ and Suzanne Bon Laboratoire de Neurobiologie Cellulaire et Mole ´ culaire, CNRS UMR 8544, Ecole Normale Supe ´ rieure, Paris, France The C-terminal t peptide (40 residues) of vertebrate acetyl- cholinesterase (AChE) T subunits possesses a series of seven conserved aromatic residues and forms an amphiphilic a-helix; it allows the formation of homo-oligomers (mono- mers, 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 AChE T on oligomerization and secretion. Charged residues influenced the distribution of homo-oligomers but had little effect on the heteromeric association with Q N , a PRAD-containing N-terminal fragment of ColQ. The formation of homo- tetramers and Q N -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 T 4 and T 4 –Q N 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 Q N , indicating that these associations take place in the endoplasmic reticulum. Mutations that disorganize the a-helical structure of the t peptide were found to enhance degradation. Co-expression with Q N generally increased secretion, mostly as T 4 –Q N 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 AChE T subunits and the choice between secretion and degradation. Keywords: acetylcholinesterase; degradation; disulfide bonds; oligomerization; secretion. In vertebrates, the acetylcholinesterase (AChE) gene gener- ates 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 AChE R ,AChE H and AChE T variants [4–6]. In mammals, AChE R subunits seem to be expressed mostly during embryogenesis and in the brain after stress [7,8]; they correspond to a soluble, monomeric enzyme species. AChE H 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]. AChE T subunits are expressed in muscles and in the nervous system of higher vertebrates and therefore represent the functional cholinest- erase species in the cholinergic system [3,13,14]. The C-terminal t peptide confers several characteristic properties to AChE T 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]. AChE T 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 [5]. This peptide is necessary for the amphiphilic properties which characterize AChE T subunits and some of their oligomers (T a 1 ,T a 2 ,T a 4 ), for the formation of nonamphiphilic homotetramers (T na 4 ), as well as for the Correspondence to S. Bon, Laboratoire de Neurobiologie Cellulaire et Mole ´ culaire, CNRS UMR 8544, Ecole Normale Supe ´ rieure, 46 rue d’Ulm, 75005 Paris, France. Fax: + 33 1 44 32 38 87, Tel.: + 33 1 44 32 38 91, E-mail: jean.massoulie@biologie.ens.fr Abbreviations: AChE, acetylcholinesterase; AChE H , AChE subunit of type H; AChE R , AChE subunit of type R; AChE T ,AChEsubunit of type T; ERAD, endoplasmic reticulum associated degradation; PRAD, proline-rich attachment domain; r, h, t, alternative C-terminal peptides of AChE; WAT, tryptophan (W) amphiphilic tetramerization domain. Enzymes: acetylcholinesterase (E.C. 3.1.1.7). (Received 8 January 2004, revised 20 February 2004, accepted 24 February 2004) Eur. J. Biochem. 271, 1476–1487 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04052.x heteromeric association of AChE T subunits with Q N ,an N-terminal fragment of collagen ColQ that contains a proline-rich motif, thus producing T 4 –Q N 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 [25]. We also found that the simultaneous presence of the t peptide and of mutations at the interface of AChE dimers – the Ôfour helix bundleÕ [26] – prevented the secretion of AChE T subunits [27]. 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 [28]. Recent spectroscopic studies showed that the t peptide is organized as an amphiphilic a helix, in which aromatic residues form a hydrophobic sector [29,30]. In addition, an analysis of intercatenary disulfide bonds in the T 4 –Q N complex also demonstrated that the four t peptides are parallel and oriented in the same direction, opposite to that of the PRAD [30]. 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 AChE T subunits, to determine the structural basis of the character- istic properties that the t peptide confers to AChE T subunits. Materials and methods AChE constructs and site-directed mutagenesis Mutagenesis was performed according to the method of Kunkel et al. [31]. cDNAs encoding wild-type and mutated Torpedo AChE T , as well as the previously described Torpedo Q N protein [23], 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 AChE T 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 [24], using 4 lg of DNA encoding the AChE catalytic subunit and 4 lg of DNA encoding Q N or PRAD-deleted Q N , 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 °Cafter 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 )6 M soman to inactivate serum cholin- esterases. To analyze its heteromeric interaction with an associated structural protein, AChE T was coexpressed with Q N [23]. By using Q N , 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 [32]. We added a flag epitope (DYKDDDDK) at the C-terminus of Q N ,sothat complexes containing this protein could be characterized with the anti-flag immunoglobulin, M2 (Kodak), as described previously [24]. The effect of Q N on the level of cellular and secreted activity was analysed by comparing the coexpression of AChE T with full-length Q N andwitha PRAD-deleted Q N , to compensate for competition between the two transfected vectors. Cell extracts The cells were extracted at 20 °C with TMg buffer (1% Triton X-100, 50 m M Tris/HCl, pH 7.5, 10 m M MgCl 2 ), 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. Enzyme assays AChE activity was determined according to the colorimetric method of Ellman et al. [33] at room temperature. As the monomeric Torpedo AChE forms produced by some mutants were inactivated by 5,5¢-dithiobis(2-nitrobenzoic acid) [34], 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 b-galactosidase from Escherichia coli were assayed with the chromo- genic substrates p-nitrophenyl phosphate and o-nitrophenyl galactoside, respectively. Sedimentation and electrophoretic analyses Centrifugation was performed in 5–20% sucrose gradients (50 m M Tris/HCl, pH 7.5, 50 m M MgCl 2 , 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. coli b-galactosidase (16 S) and alkaline phosphatase (6.1 S) as internal sedi- mentation standards [24]. 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.[16],andAChE activity was revealed by the histochemical method of Karnovsky & Roots [35]. 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 Ó FEBS 2004 Oligomerization and secretion of the AChE t peptide (Eur. J. Biochem. 271) 1477 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 T na 4 or T 4 –Q N . 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 T 4 –Q N complexes formed by mutants W17F and W17A showed an unusual retardation in sedimentation in the presence of Triton X-100, compared with Brij-97. Results Analyses of AChE activity and molecular forms Figure 1 shows the sequence of the t peptide of Torpedo AChE T subunits, and schematically illustrates its proposed a helical structure, its association with the PRAD of ColQ, and the various oligomers of AChE T 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 AChE T 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 nonamphi- philic. The secretion of active wild-type AChE T 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 [16]. As Torpedo AChE T 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 AChE T subunits to associate with a PRAD, we coexpressed them with protein Q N (Fig. 1D). This Q N protein organizes wild-type AChE T subunits into tetramers (T 4 –Q N ) that are nonamphiphilic and efficiently secreted [24], reaching  40% of the secretion observed with the truncated I3C/stop4 mutant. The forma- tion of Q N -linked oligomers therefore rescued an important fraction of the wild-type catalytic Torpedo AChE T subunits from intracellular degradation. Fig. 1. Structure of the t peptide and oligomeric associations of acetylcholinesterase type T subunits (AChE T ). (A) primary sequence of the t peptide from Torpedo AChE T subunits. The residues of the t peptide, encoded by an alternatively spliced 3¢ exon, are numbered from 1 to 40 and correspond to residues 536–575 of the mature Torpedo AChE T subunit; cysteine C37, which is responsible for intercatenary disulfide bonds, is circled. (B) Side view of the t peptide, with its 1–32 segment organized as an a helix. The con- served aromatic residues are located in the upper sector of the helix. (C) Wheel representation of the entire t peptide, putatively organ- ized as an a helix. Aromatic residues, shown in shaded circles, are located in the upper sector; charged residues are in double circles (white for basic residues, grey for acidic residues) and possible salt bridges are marked by hatched bars; cysteine C37 is in a double, grey circle; arrowheads indicate residues that have been mutated to cysteines. (D) Primary sequence of the proline-rich attachment domain (PRAD) motif from Torpedo ColQ. The PRAD residues are shown in bold text (from cysteines 70 and 71 to phenylalanine 86), and a few adjacent residues are shown in non-bold text. (E) Schematic representation of a complex between four t peptides and a PRAD. The N- and C-terminal extremities (indicated N and C) and arrows show the orientations of the t peptides (black zig- zags) running opposite to the PRAD (grey line); cysteines are indicated by circles, joined by lines representing disulfide bonds. (F) Major types of homomeric and heteromeric associations analyzed in this study: T a 1 ,T a 2 and T a 4 , amphiphilic monomer, dimer and tetramer of AChE T subunits; T na 4 , nonamphiphilic tetr- amer; T 4 –Q N , tetramer associated with the N-terminal Q N fragment of ColQ, containing the PRAD motif. The schemes of heteromeric complexes are derived from recent studies (M. Harel, H. Dvir, S. Bon, W. Q. Liu, M. Vidal, C. Garbay, J. L. Sussman, J. Massoulie ´ & I. Silman, unpublished results) [30]. 1478 S. Belbeoc’h et al. (Eur. J. Biochem. 271) Ó FEBS 2004 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. Likethewild-typeAChE T subunits, all mutants produced amphiphilic dimers (T a 2 ) and nonamphiphilic tetramers (T na 4 ). 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 appro- ximately 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 Q N increased the level of secretion and produced T 4 –Q N complexes, as for the wild type. Mutation of aromatic residues The three tryptophans (W10, W17, W24) and Y31 were mutatedtoalanines,andallsevenaromaticresidueswere 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 Fig. 2. Mutations of charged residues in the t peptide. (A) Acetylcho- linesterase (AChE) activities in cell extracts and secreted into the cul- ture medium are shown for the wild type and four mutants. Grey bars and hatched bars correspond to the AChE activities of mutants expressed without or with Q N , respectively (Materials and methods); the activities are normalized to those obtained for the wild type (100%) both in the cell extracts and in the medium; the standard errors were obtained from five independent experiments. For other individual mutations (K11A, E13A, R16A, K25A and D29A), the cellular activities ranged from 77% to 100%, and the secreted activities between 68% and 136%. (B) Sedimentation patterns of cellular and secreted AChE, in sucrose gradients containing 0.2% Triton X-100. The shaded areas, as well as the total areas under the sedimentation profiles, are proportional to the relative activities of the mutants, so that the surface of each peak represents the actual activity of the corresponding molecular form: monomers (T 1 ), dimers (T 2 )and tetramers (T 4 ). Fig. 3. Mutations of aromatic residues. (A) Secreted activities, nor- malized to that of the wild type, for mutants of aromatic residues to prolines: the bars represent secreted activities obtained when acetyl- cholinesterase type T subunits (AChE T ) were expressed without Q N (greybars)andwithQ N (hatched bars); the indicated values are the means of at least three independent experiments. The cellular activities ranged from 86 to 114% without Q N andfrom65to117%withQ N .(B) Amphiphilic character of dimers produced by each mutant, indicated by charge shift electrophoresis. RDOC/TX is the ratio of electrophoretic migrations in the presence of Triton X-100 with sodium deoxycholate and Triton X-100 alone, normalized to those of a nonamphiphilic species (T na 4 ); the indicated values represent the means of three to six independent experiments. (C) Existence of Q N -linked dimers with mu- tant W10P: electrophoretic patterns, in the presence of Triton X-100 and sodium deoxycholate; the third lane shows that a fraction of dimers and tetramers was retarded by the M2 antibody (x), indicating that they were associated with the Q N -flag protein. Note that the coexpression with Q N increased the secretion of dimers as well as tetramers. (D) AChE molecular forms secreted by mutants of aromatic residues to prolines, expressed with and without Q N ; electrophoretic analysis in the presence of Triton X-100 and deoxycholate. s,AChE T dimers; d, Q N -linked dimers; h, tetramers; j,T 4 –Q N complexes (i.e. tetramer associated with the N-terminal Q N fragment of ColQ, containing the proline-rich attachment domain motif); the origin of migration is shown by a thin line. Ó FEBS 2004 Oligomerization and secretion of the AChE t peptide (Eur. J. Biochem. 271) 1479 forms produced by these mutants were T 2 dimers, as illustrated in electrophoretic patterns (Fig. 3D); the pro- duction of tetramers was strongly reduced or abolished, again with the exception of Y31 mutants. The amphiphilic character of T 2 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 T 4 –Q N 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 Q N protein, the anti-flag M2 immunoglobulin reacted with a fraction of dimers as well as with tetramers, indicating the presence of T 2 –Q N complexes, in addition to T 4 –Q N 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 amphi- philic 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 Q N in the case of W17L and W17P, a very small production of T 4 –Q N 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 Q N increased the level of secreted activity to about 60% of that obtained in the wild type; in addition to the T 4 –Q N 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 Q N ,as they did not react with the M2 antibody. Whereas the sedimentation of the wild-type T 4 –Q N complex was abso- lutely unaffected by the presence of Triton X-100 or Brij-97 in the gradient, the sedimentation of T 4 –Q N 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 T a 1 ,T a 2 or T a 4 (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 T na 4 and T 4 –Q N , 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 a 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 Q N . 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 T a 2 dimers in the cell extracts; in the case of M21D,the medium only contained T na 4 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 Q N increased secretion for T12D and M21W (to about 35% and 50% of the wild type, respectively), but not for M21D; T 4 –Q N complexes of T12D 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 T 4 –Q N complex. Effect of a cysteine at various positions in the t peptide The formation of intercatenary disulfide bonds between wild-type AChE T 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 Q N , the secretion of T 4 –Q N 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 Q N . 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). Fig. 4. Molecular forms produced by W17 mutants; interaction with Q N . Sedimentation patterns of cellular and secreted molecular forms; the areas under the profiles are proportional to the corresponding activities; the top of the wild-type T 4 –Q N (tetramer associated with the N-terminal Q N fragment of ColQ, containing the proline-rich attach- ment domain motif) peak exceeds the frame and is shifted downwards. Molecular forms expressed without Q N (––s––) and with Q N (- - -j ) were analyzed in the presence of Triton X-100; sedimentation was also performed in the presence of Brij-97 (Bj) for molecular forms secreted by mutants W17F, W17H and W17A (ÆÆÆÆÆÆ). Note an unusual retardation by Triton X-100 (Tx) for W17F and W17A. 1480 S. Belbeoc’h et al. (Eur. J. Biochem. 271) Ó FEBS 2004 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 degrada- tion. This mutation also increased the cellular activity obtained with the W17P mutant, without restoring its capacity to interact with Q N . Mutation A6C somewhat decreased the cellular activity, but strongly increased secretion. The A6C mutant mainly produced a nonamphiphilic 14 S species, possibly corres- ponding to octamers. This unusual oligomer dissociated during storage, particularly in the presence of detergent (Triton X-100), transiently producing amphiphilic tetramers (T a 4 ) (which are not usually observed in the wild type) and amphiphilic dimers (T a 2 ). 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 T a 2 dimers in the medium. The T12C mutation introduced a cysteine in the N-terminal part of the aromatic-rich segment (not shown). Fig. 6. Oligomeric forms obtained with cysteines at different positions in the t peptide. (A) Sedimentation patterns of cellular and secreted molecular forms in gradients containing Triton X-100 (––) and Brij-97 (- - -); the shaded areas and the areas under the profiles are propor- tional to the corresponding activities. Note that dimers containing cysteines at position 21 did not sediment faster with Triton X-100 than with Brij-97, in contrast to the amphiphilic dimers (wild type, or with cysteines at positions 3, 6 or 34). Mutants A6C and A6C/C37S pro- duced a 14 S species that was progressively dissociated into amphi- philic tetramers (T a 4 ) and ultimately amphiphilic dimers (T a 2 ), as shown for cell extracts in an upper profile. (B) Interaction of cysteine mutants with Q N , as shown by electrophoretic analysis of secreted molecular forms, in the presence of Triton X-100 and sodium deoxy- cholate(comparewithFig.5A).NotethatmutantM21C(with cysteine C37) produced complexes with Q N , retarded by M2, whereas mutant M21C/C37S (without cysteine C37) did not. Fig. 5. Mutations in the aromatic-rich region; mutations of methionines M21 and M22. (A) Electrophoretic patterns of cellular and secreted molecular forms, in the presence of Triton X-100, with and without deoxycholate (DOC); complexes with Q N that were retarded by M2 are indicated by x; the symbols are as in Fig. 4. The secretion of mutant M21D was not increased by coexpression with Q N , and no complex reacting with M2 was detected. In the case of T12D and M21W, T 4 –Q N (tetramer associated with the N-terminal Q N fragment of ColQ, containing the proline-rich attachment domain motif) com- plexes were secreted, but undetectable or barely detectable in cell extracts. (B) Cellular and secreted activities (represented as in Fig. 2A) for mutants containing cysteine C37 or not (C37S). Coexpression with Q N increased the secretion of mutants of methionine M22 and mutants of methionine M21 which possessed cysteine C37, but not of mutants M21A/C37S and M21S/C37S (data not shown), lacking both M21 and C37. Ó FEBS 2004 Oligomerization and secretion of the AChE t peptide (Eur. J. Biochem. 271) 1481 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 T 4 –Q N 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 T 4 –Q N complex, in agreement with the formation of intercaternary disulfide bonds between the t peptide and Q N cysteines. Coexpression with Q N generally increased secretion when T 4 –Q N complexes were produced, although this effect was marginal or absent for mutants that showed a high level of secretion without Q N (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 Q N did interact with the mutant AChE T 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 Q N , whereas M22C/C37S formed a T 4 –Q N 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 Q N increased the level of secretion, indicating the formation of T 4 –Q N 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 contri- butes 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 AChE T 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. Fig. 7. Effects of the C-terminal cysteine (C37), of C-terminal segments and of a KDEL motif on acetylcholinesterase (AChE) molecular forms. (A) Sedimentation patterns of cellular (upper row) and secreted (lower row) enzyme, in gradients containing Triton X-100. The shaded areas and the areas under the sedimentation profiles are proportional to the activities. In mutant T KDEL , the C-terminal tetrapeptide (CAEL) was replaced with the canonical ER retention motif (KDEL). All mutants lacking a cysteine produced and secreted amphiphilic monomers with variable proportions of nonamphiphilic tetramers; these tetramers were secreted at a higher level for T KDEL than for C37S (SAEL). (B) Cellular and secreted activities obtained with and without Q N .The cellular activities decreased with the extent of C-terminal deletions; coexpression with Q N increased secretion to a level comparable to that of the wild type for mutants C37S, T KDEL , stop34 and stop32, but had essentially no effect for stop29. 1482 S. Belbeoc’h et al. (Eur. J. Biochem. 271) Ó FEBS 2004 Dimerization was also suppressed when the CAEL motif was replaced with KDEL (T KDEL ) 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 T a 1 ;this increase in cellular enzyme appeared to facilitate tetra- merization, as tetramers (T na 4 ) were secreted at a higher levelthanwithC37S(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 T KDEL , stop39, stop34 and stop32 mutants formed T 4 –Q N complexes, so that their secretion was increased in the presence of Q N (Fig. 7B). In contrast, the shorter mutant, stop29, showed no interaction with Q N , as indicated by the fact that coexpression did not affect either the secreted activity or the molecular forms, charac- terized by sedimentation. To determine whether the differ- ence 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 T 4 –Q N 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 T 4 –Q N complexes when coexpressed with Q N (Fig. 8A). As in the wild type, the replacement of the C-terminal tetrapeptide by KDEL increased the cellular activity (mainly T a 2 ) and reduced its secretion, but did not abolish association with Q N , producing T 4 –Q N complexes which were secreted. Deletion of the last nine residues (I3C/stop32) did not abolish association with Q N , 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 Q N . 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 aro- matic 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. Fig. 8. Effects of C-terminal segments and of a KDEL motif, in mutants containing an N-terminal cysteine (I3C). (A) Interaction with Q N , indicated by electrophoretic patterns of cellular (top) and secreted (bottom) molecular forms obtained with and without Q N ,inthe presence of Triton X-100 and sodium deoxycholate. Note that mu- tants I3C/C37S, I3C/KDEL and I3C/stop37 produced homomeric T 2 a dimers (s), homomeric T na 4 tetramers (h)andT 4 –Q N (tetramer associated with the N-terminal Q N fragment of ColQ, containing the proline-rich attachment domain motif) complexes (j), whereas I3C/ stop29 did not. Homomeric tetramers of the I3C/KDEL mutant appeared to be partially retained intracellularly. (B) Effect of C-terminal deletions on cellular and secreted activities; progressive deletions were made from the C-terminus of mutants containing an N-terminal cysteine (I3C) which allows an efficient dimerization; in the two longer mutants, the C-terminal cysteine was replaced with a serine (C37S); mutated residues are underlined in the sequence, shown along the horizontal axis. Cellular (upper frame) and secreted (lower frame) activities, expressed as percentage of the wild type, are shown as a function of the remaining length of the t peptide. Asterisks indicate mutants that produced amphiphilic dimers; mutants stop34 and shorter produced nonamphiphilic dimers. Ó FEBS 2004 Oligomerization and secretion of the AChE t peptide (Eur. J. Biochem. 271) 1483 Discussion The t peptide: an elongated amphiphilic a 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 a helical conformation [30]. 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 a helical structure. Thus, the amphi- philic 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 [30], 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 AChE T subunits In contrast to the wild-type AChE T 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 nonamphi- philic, 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, AChE T subunits could form predominantly dimers or tetramers – or even higher oligomers sedimenting at 14 S, possibly octa- mers. 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 influ- enced 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 (T na 4 ), but some tetramers may also be amphiphilic (T a 4 ), 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, Q N The major physiological role of the t peptide is clearly to allow the functional localization of AChE T 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, Q N . This protein assem- bles with wild-type AChE T subunits to form Q N -linked tetramers (T 4 –Q N ), which are nonamphiphilic. Previous studies showed that in these hetero-oligomers, two catalytic subunits are disulfide-linked with Q N , 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 Q N (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 Q N . In contrast, similar mutations of methionine M22 did not prevent the formation of T 4 –Q N 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 Q N - 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 [30]. In fact, addition of a variable number of residues between the catalytic domain and the t peptide did not prevent association with Q N (N. Morel & S. Bon, unpublished). 1484 S. Belbeoc’h et al. (Eur. J. Biochem. 271) Ó FEBS 2004 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 AChE T 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 Q N , 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 theimportanceofanaromaticside-chain. With some mutants, we obtained evidence that Q N induced the formation of AChE T dimers: coexpression with Q N strongly increased the secretion of dimers that were not covalently associated with Q N (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 Q N -linked tetramers, or result from the dissociation of unstable tetramers. Q N -linked tetramers are usually nonamphiphilic, indica- ting 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 interac- tions 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.Massoulie ´ & I. Silman, unpublished results). However, the Q N -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 Q N -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 [30]. The heteromeric assocation with Q N 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 Q N , 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 Q N -linked tetramers We observed that the formation of homomeric T na 4 tetramers was suppressed by all mutations which affected the heterometic complex, T 4 –Q N , 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 Q N -linked tetramers appeared to reduce homomeric tetramers more severely, indicating that tetramers are generally stabilized by the presence of a PRAD. In T 4 –Q N complexes, the four a 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 T na 4 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 (T na 4 )intheT KDEL 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 hetero- meric complexes T 4 –Q N ; furthermore, the KDEL motif was sterically masked in the complexes, as the complexes were efficiently secreted. The fact that retention of AChE T subunits in the ER did not prevent homomeric or hetero- meric associations indicates that they occur in this com- partment. 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 AChE T 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 T12D andsomearomaticmutantsfollowed 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 M21D. 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 AChE T subunits through the ERAD pathway and that this effect depends on the presence of aromatic residues [28]. Occlusion of these residues may explain that oligomerization generally facili- tates 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 AChE T 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). Ó FEBS 2004 Oligomerization and secretion of the AChE t peptide (Eur. J. Biochem. 271) 1485 [...]... 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... 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 a-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... 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 Ô4AÕ in the N-terminal region It was increased by deletion of the last two C-terminal. .. 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 (T1 2D, M21D) This suggests that degradation was increased by disorganization of the aromatic cluster of the. .. 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... 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),... A., Toker, L & Silman, I (1991) Atomic structure of acetylcholinesterase from Torpedo californica: a prototypic acetylcholine-binding protein Science 253, 872–879 ´ Morel, N., Leroy, J., Ayon, A., Massoulie, J & Bon, S (2001) Acetylcholinesterase H and T dimers are associated through the same contact; mutations at this interface interfere with the C-terminal T peptide, inducing degradation rather than... 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 Acknowledgements We thank Ms Claudine Schmitt for technical assistance and M Noel ¨ Perrier for critical reading of the manuscript This work was supported by grants from the Centre National de... et al (Eur J Biochem 271) Conversely, suppression of cysteine C37 (C37S or deletions) reduced secretion Similarly, coexpression with QN generally increased secretion by recruiting AChET subunits into T4 –QN heterooligomers 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. .. rather than secretion J Biol Chem 276, 37379–37389 ´ 28 Belbeoc’h, S., Massoulie, J & Bon, S (2003) The C-terminal T peptide of acetylcholinesterase enhances degradation of unassembled active subunits through the ERAD pathway EMBO J 22, 3536–3545 29 Cottingham, M.G., Voskuil, J.L.A & Vaux, D.J .T (2003) The intact human acetylcholinesterase C-terminal oligomerization domain is alpha-helical in situ and in . 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. . 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