Determinants of the t Peptide Involved in Folding, Degradation, and Secretion of Acetylcholinesterase*

The C-terminal 40-residue t peptide of acetylcholinesterase (AChE) forms an amphiphilic α helix with a cluster of seven aromatic residues. It allows oligomerization and induces a partial degradation of AChE subunits through the endoplasmic reticulum-associated degradation pathway. We show that the t peptide induces the misfolding of a fraction of AChE subunits, even when mutations disorganized the cluster of aromatic residues or when these residues were replaced by leucines, indicating that this effect is due to hydrophobic residues. Mutations in the aromatic-rich region affected the cellular fate of AChE in a similar manner, with or without mutations that prevented dimerization. Degradation was decreased and secretion was increased when aromatic residues were replaced by leucines, and the opposite occurred when the amphiphilic α helix was disorganized. The last two residues (Asp-Leu) somewhat resembled an endoplasmic reticulum retention signal and caused a partial retention but only in mutants possessing aromatic residues in their t peptide. Our results suggested that several “signals” in the catalytic domain and in the t peptide act cooperatively for AChE quality control.

The C-terminal 40-residue t peptide of acetylcholinesterase (AChE) forms an amphiphilic ␣ helix with a cluster of seven aromatic residues. It allows oligomerization and induces a partial degradation of AChE subunits through the endoplasmic reticulum-associated degradation pathway. We show that the t peptide induces the misfolding of a fraction of AChE subunits, even when mutations disorganized the cluster of aromatic residues or when these residues were replaced by leucines, indicating that this effect is due to hydrophobic residues. Mutations in the aromatic-rich region affected the cellular fate of AChE in a similar manner, with or without mutations that prevented dimerization. Degradation was decreased and secretion was increased when aromatic residues were replaced by leucines, and the opposite occurred when the amphiphilic ␣ helix was disorganized. The last two residues (Asp-Leu) somewhat resembled an endoplasmic reticulum retention signal and caused a partial retention but only in mutants possessing aromatic residues in their t peptide. Our results suggested that several "signals" in the catalytic domain and in the t peptide act cooperatively for AChE quality control.
The major function of acetylcholinesterase (AChE) 1 is to regulate the cholinergic transmission by cleaving the neurotransmitter acetylcholine. In mammals, alternative splicing of a single gene gives rise to different variants (types R, H, and T), which possess the same catalytic activity but different tissuespecific distributions (1)(2)(3). The catalytic subunits of type T (AChE T ), which are predominant in the nervous system and muscles, are characterized by a C-terminal peptide of 40 amino acids called the t peptide (4).
The t peptide enables AChE T subunits to assemble into dimers and tetramers and to associate with structural proteins possessing a proline-rich attachment domain (PRAD), the collagen ColQ, and the transmembrane protein PRiMA (prolinerich membrane anchor) (5)(6)(7). These proteins anchor PRADlinked tetramers of cholinesterase in the basal lamina of neuromuscular junctions and in neuronal membranes, respectively. The t peptide behaves as an autonomous interaction domain in its association with a PRAD and therefore has been called the tryptophan amphiphilic tetramerization domain (WAT) (8). Its homo-and hetero-oligomerization properties depend on two major features, which are conserved in all vertebrates: 1) a series of seven aromatic residues (Trp 10 , Phe 14 , Trp 17 , Tyr 20 , Trp 24 , Phe 28 , and Tyr 31 ) and 2) a cysteine near its C terminus (Cys 37 ) (9). Spectroscopic and crystallographic studies showed that the t peptide is organized as an amphiphilic ␣-helix in which all of the aromatic residues are grouped on one side (10). 2 Cys 37 may form disulfide bridges between two t peptides in homodimers and homotetramers or between two t peptides and the two PRAD cysteines in the heteromeric PRAD-linked tetramers (10 -16).
The formation of AChE T dimers depends on contact between subunits at the level of two ␣-helices from each catalytic domain (␣ 7,8 and ␣ 10 ), forming the "four-helix bundle" (17), and on the formation of a disulfide bridge between the C-terminal cysteines of two t peptides (18), but it does not require the aromatic residues of the t peptide. It has been shown that rat AChE T subunits, which carry a mutation in the ␣ 10 helix (F535A, 9 amino acids upstream of the t peptide), are deficient in dimerization despite the presence of Cys 37 (18). In contrast, the formation of homomeric or PRAD-linked tetramers depends on the cluster of aromatic residues. It is facilitated by intercatenary disulfide bonds but also occurs in their absence (9).
The presence of a t peptide induces an intracellular degradation of AChE T subunits, which is strongly increased with the F535A mutation (AChE*), unless they assemble with a PRADcontaining N-terminal fragment of ColQ (Q N ), forming PRADlinked complexes. This degradation process follows the ER-associated degradation pathway. It can be blocked by proteasome inhibition and is reduced by inhibiting ER-mannosidase I, indicating that sugar chains are involved in its mechanism (19).
Furthermore, the t peptide seems to play a role in delaying protein secretion. Indeed, we recently showed that deletion of the last two residues increased the secretion of Torpedo AChE T , suggesting that its C terminus behaves as a weak retention signal, perhaps facilitating the assembly of oligomers. This may be due to the similarity of the C-terminal tetrapeptide (CAEL) with the classical endoplasmic reticulum retention signal, KDEL (9,20).
In this study, we designed mutations to assess the importance of the cluster of aromatic residues in the ␣-helical structure and of the aromatic versus hydrophobic nature of these residues in rat AChE T subunits. We investigated the synthesis, secretion, degradation, and interaction with a PRAD for wild type and F535A mutants with and without the C-terminal Cys 37 . We showed that distinct features of the t peptide determine these different processes.

EXPERIMENTAL PROCEDURES
Mutagenesis and Transfection-cDNAs encoding the AChE T subunit of rat AChE and an N-terminal fragment of ColQ (Q N ) were inserted in the pEFBos expression vector (21). The primary sequence of the Q N protein is 5Ј-ESTFLDKAFSLQAALLPMEHKKRSVNKCCLLTPPPP-PMFPPPFFTETNILQEVDLNNLPLEIKPTEPS-3Ј (6) (the signal peptide is not shown; the PRAD and the two cysteines are underlined are in boldface).
Site-directed mutagenesis was realized with the method of Kunkel (22). The C2 mutant was derived by deletion of most of the C-terminal peptide from the H variant of AChE, leaving a cysteine at position 6 downstream of the common catalytic domain (23). Plasmids were transfected in COS cells with the DEAE-dextran method, as described previously (24), usually with 4 g of DNA/100-mm dish. The cellular and secreted activities were generally measured 3 or 4 days after transfection.
Titration of AChE Active Sites by DEPQ-Samples of 50 l containing 15-30 mOD units (1/1000 optical density unit) of cellular or secreted AChE activity were treated with 10, 20, and 30 l of 0.125 nM DEPQ for 1 h at room temperature. The remaining AChE activity was then determined with the Ellman colorimetric method (25).
Soman, Cycloheximide, and Brefeldin A-To study the neosynthesis of AChE, steady-state cultures of transfected cells (3 or 4 days after transfection) were treated with the membrane-permeant inhibitor soman (pinacolylmethylphosphonofluoridate) 5 ϫ 10 Ϫ7 M for 30 min, which irreversibly inhibited the cellular enzyme. After extensive washing, the recovery of AChE activity at 37°C was determined by collecting cells at various times. The secretion of active AChE was also monitored, and aliquots of the culture medium were taken and replaced with fresh medium.
To investigate AChE degradation, cycloheximide (200 g/ml) was added to the culture medium of transfected cells to block protein synthesis and brefeldin A (7 g/ml) was added to block secretion.
Acetylcholinesterase Extraction and Quantification-Intracellular and membrane-bound enzyme was extracted for 30 min at 20°C in a TMg buffer (Tris-HCl 50 mM, pH 7.4, 1% Triton X-100, 10 mM MgCl 2 ) followed by centrifugation for 30 min at 13,000 rpm at 4°C. The medium containing the secreted enzyme was also centrifuged to remove cell debris. Enzyme activity was quantified at 414 nm with the colorimetric method of Ellman using a Labsystem Multiskan RC automatic plate reader.
Metabolic Labeling and Immunoprecipitation-After 3 days of transfection, cells were rinsed and incubated for 45 min in a medium without methionine followed by a 30-min labeling with [ 35 S]methionine and cysteine (Redivue, Amersham Biosciences). After rinsing, the cells were chased in a complete medium, the medium was collected, and the cells were extracted at different times. The cell extract and the medium were incubated overnight with a polyclonal antibody raised against AChE (A63) (28) and immunoadsorbed at 4°C on protein G-Sepharose 4B beads (Sigma). The radiolabeled enzyme was detached from the beads in a buffer containing 1% SDS and 0.5 M ␤-mercaptoethanol and loaded on 7% polyacrylamide gels containing SDS for electrophoresis. After migration, the gels were dried and radioactive AChE protein was revealed with a Fuji image analyzer.
Prediction of Secondary Structure-The secondary structure of mutated t peptides was predicted according to Rost (29) in the PredictProtein site (maple.bioc.columbia.edu/predictprotein/).

RESULTS
Mutations in the t Peptide-We introduced mutations in the t peptides of rat AChE T and of the dimerization-defective mu-tant carrying the F535A mutation in the ␣ 10 helix (AChE*), as illustrated in Fig. 1A. We modified the cluster of aromatic residues of the t peptide in various ways. The segment containing these residues was deleted in the Del 10 -31 and Stp10 mutants, the seven aromatic residues were replaced by leucines in the 7L mutant, and two phenylalanine residues (Phe 14 and Phe 28 ) were replaced by tryptophans in the 5W mutant. In the "scrambled" (Scr) mutant, the positions of the seven aromatic residues were exchanged with those of other residues so that they could be dispersed around the helix (Fig. 1B). In the Scr mutant, the composition of the t peptide and the positions of acidic and basic residues were not modified to maintain potential salt bridges, which may stabilize the helical structure of its oligomeric associations. The 10 -31 region is predicted to form an ␣-helix in the case of the 7L, 5W, and Scr mutants as in the wild type t peptide (see "Experimental Procedures"). In this paper, WT refers to the wild type aromatic region of the t peptide combined or not with the F535A mutation in the catalytic domain (AChE or AChE*) and with mutations in the C-terminal tetrapeptide, which contained a cysteine or a serine at position 37 (indicated by indices C or S, for example: 5W C , 5W S ). In the stop39 mutant, we removed the last two residues (Asp-Leu) so that the C terminus would be Ser-Ser. We also replaced the last four residues by the canonical ER retention signal KDEL. For comparison, we studied mutants lacking the t peptide consisting of the catalytic domain only (C1) or of the catalytic domain followed by a short peptide containing a cysteine (C2). We found that C1 formed exclusively monomers, whereas C2 was largely dimerized.
All of the mutants produced active AChE. We compared the activities of these mutants by titrating their active sites with an irreversible inhibitor, DEPQ. We found that the catalytic turnover per site was identical for cellular and secreted enzymes and was not affected by the presence of the F535A mutation, by the presence or absence of the t peptides, or by the different mutations that were introduced in the t peptide (data not shown).
Dimerization Requires Cys 37 , Whereas Tetramerization with or without Q N Mainly Depends on the Aromatic Cluster- Fig.  2A shows the electrophoretic patterns obtained in non-denaturing electrophoresis for the different mutants of rat AChE T subunits expressed in transfected COS cells with or without Q N . As previously described (6, 30), cellular extracts from cells expressing wild type AChE T subunits contained monomers (T 1 ), dimers (T 2 ), and a small proportion of tetramers (T 4 ), whereas the culture medium contained comparable proportions of monomers, dimers, and tetramers. When the cells co-expressed AChE T subunits with the Q N protein, both cell extract and medium contained Q N -linked tetramers (T 4 -Q N ).
The molecular forms of AChE produced with the 5W C mutant were very similar to those obtained with the wild type, although the formation of homomeric tetramers (T 4 ) was slightly reduced. When this mutant was co-expressed with Q N , the formation of T 4 -Q N complexes was reduced. The Scr C mutant produced monomers and dimers but no tetramers, either homomeric or associated with Q N . This mutant was secreted at a very low level, either without or with Q N . Cells expressing the 7L C mutant also produced monomers and dimers but secreted mostly dimers. It did not form any tetramer, but a slowly migrating component (perhaps hexamers) appeared in the medium (Fig. 2, A and B). The Scr and 7L mutants illustrate the necessity of an aromatic cluster for tetramerization.
Without Cys 37 , dimerization was abolished in all of the cases. The WT S subunits (which only differed from the wild type by the C37S mutation) mainly produced monomers (T 1 ) together with lower levels of tetramers (T 4 ) and T 4 -Q N complexes than the wild type. The Scr S and 7L S mutants only produced monomers. It is noteworthy that the 5W S mutant did not form homomeric tetramers or Q N -linked tetramers, in contrast to the 5W C mutant. This shows that intercatenary disulfide bonds contribute to the stability of both homomeric and Q N -associated tetramers.
Tetramerization was also abolished in the case of the dimerization-deficient AChE* T subunits containing the Scr and 7L mutations with or without Cys 37 . Transfected cells co-expressing AChE*-5W C with Q N secreted T 4 -Q N complexes but at a lower level than with the wild type, and these complexes were not detectable in cellular extracts. The AChE*-5W S mutant, similar to AChE-5W S , did not form such heteromeric complexes.
In Fig. 2, A and B, the relative intensities of AChE bands in the medium reflect the relative levels of activity of the different mutants, as documented below. For example, the Del and 7L mutants were more actively secreted than the WT, 5W, and particularly Scr mutants. Fig. 2B shows the influence of a charged detergent, DOC, on the migration of AChE molecular forms in the presence of the non-ionic detergent Triton X-100. The migrations were normalized to those of the Del S and Del C mutants, which do not possess the cluster of hydrophobic residues and constitute non-amphiphilic standards. The migration of amphiphilic molecules such as the wild type monomers and dimers was slower in the presence of Triton X-100 alone compared with Triton X-100 plus DOC. Only those produced by the Scr C mutant retained a fast migration under these conditions. Fig. 2C shows the ratio of migration in the presence and absence of DOC as an index of amphiphilicity. We obtained similar values for cellular and secreted molecular species. Dimers were systematically less accelerated by DOC than monomers, as expected if their aromatic clusters partially had occluded each other, and tetramers were non-amphiphilic (data not shown). The wild type and 5W mutants presented similar values, the 7L mutants were significantly less sensitive to the detergents, and the Scr mutants were essentially not affected. Thus, the amphiphilic character depends on the clustering of hydrophobic residues. It is interesting that leucines provide a weaker amphiphilic character than tryptophans, which most authors classify as less hydrophobic.

The Amphiphilicity of Monomers and Dimers Depends on a Cluster of Hydrophobic Residues in the t Peptide-
Influence of the Aromatic-rich Segment on Cellular and Secreted AChE Activities- Fig. 3 shows the levels of AChE activity obtained in cellular extracts and in the medium for the different mutants expressed in COS cells normalized to those obtained for the wild type AChE T subunit. (respectively noted AChE* and AChE). This mutation, located in the C-terminal ␣ 10 helix of the catalytic domain, compromises dimerization through the four-helix bundle. In mutant C1, the entire t peptide was deleted, and in mutant C2, it was replaced by the first seven residues from the C-terminal peptide of the AChE H splice variant containing a cysteine (see "Experimental Procedures"). In this paper, WT refers to the wild type aromatic-rich region. B and C, wheel diagrams of the 9 -32 segment of the wild type and Scr mutant. The preceding mutations were combined with mutations in the C-terminal tetrapeptide (underlined), replacement of Cys 37 by a serine as noted by an index (e.g. WT C or WT S ), deletion of the last two residues with the C37S mutation (e.g. WT SS ), and replacement of the last four residues by the ER retention signal KDEL (e.g. WT KDEL ).
When Cys 37 was present, the cellular enzymatic activity was increased to 130 and 160% for Scr C and 5W C , respectively, whereas the corresponding secreted activity was reduced to 30 and 50%. On the contrary, the cellular activity was reduced to 40% or less for 7L C , whereas the corresponding secreted activity was increased to 150%. Secretion was increased even higher FIG. 2. Effect of mutations in the aromatic-rich region on oligomerization of AChE T and AChE* T subunits in the absence and in the presence of Q N . Samples from cellular extracts of transfected cells and from the culture medium were subjected to non-denaturing electrophoresis, and the gels were stained for AChE activity. Small arrows indicate the origin of migration. The bands corresponding to monomers (T 1 ), dimers (T 2 ), tetramers, and Q N -associated tetramers (T 4 Ϯ Q N ) are indicated. A, migration patterns in the presence of Triton X-100 and the anionic detergent DOC. From top to bottom, mutants containing the wild type catalytic domain (AChE) with and without Cys 37 and the dimerization-deficient catalytic domain (AChE*) with and without Cys 37 . Note that only the mutants possessing the WT or 5W t peptide formed T 4 -Q N complexes and that mutants lacking Cys 37 did not form dimers. B, effect of the detergents (Triton X-100 with or without DOC) on the migrations of oligomers formed by wild type AChE T subunits and mutants containing Cys 37 . Only the Scr molecular forms were not retarded in the absence of DOC. C, ratio of migrations in the presence and absence of DOC taken as an index of amphiphilicity. The migrations were normalized to those obtained for deleted C1 monomers in the same gels. The values obtained for monomers with or without Cys 37 were not significantly different and were averaged. The histograms represent the means Ϯ S.E. Note that the monomers are more amphiphilic than dimers and that tetramers are non-amphiphilic (data not shown), indicating a progressive occlusion of the hydrophobic cluster of aromatic residues in the t peptides with oligomerization. The wild type and 5W mutants presented the same amphiphilic character, the 7L mutant was significantly less amphiphilic, and the Scr mutant was non-amphiphilic, similar to the Del mutant (data not shown).
for the Del C mutant (280%) and reached the same level as that for the truncated mutants, which lacked the t peptide, either without a C-terminal cysteine, (C1: 220%) or with cysteine near the end of the catalytic domain, allowing dimerization (C2: 270%). The cellular activities of these mutants were either slightly increased (110% for Del C ) or decreased (70% for C2). In the absence of cysteine Cys 37 , both cellular and secreted activities were lower, suggesting an increased degradation, but the effects of the mutations were qualitatively similar.
Thus, secretion was strongly increased when the aromatic sector was removed (Del, C1, or C2) and to a lesser extent when aromatic residues were replaced by leucines (7L). In contrast, secretion was reduced and we observed an increase of the cellular activity when two phenylalanines were replaced by tryptophans (5W). Also, this effect was stronger when the clustering of aromatic residues was suppressed by changing their positions (Scr).
The cellular activity obtained with the dimerization-deficient mutant AChE* carrying a wild type t peptide with or without Cys 37 was Ͻ40% wild type, and secretion was almost abolished compared with that of the C2* mutant, indicating a strong degradation as previously reported (18). Despite the fact that the Scr mutation did not increase the cellular activity, possibly because of intracellular degradation, the effects of the different mutations in the t peptide were remarkably similar to those observed without the F535A mutation. It is particularly noteworthy that the dimerization-deficient mutants AChE*-7L C and AChE*-7L S were significantly secreted, in contrast to AChE*-WT C and AChE*-WT S .
Cells expressing the wild type AChE produced and secreted somewhat more activity than cells expressing the C37S mutant; however, Fig. 4 shows that the ratio of secreted to cellular activities were the same, suggesting that the cysteine exerts no retention effect, in agreement with the fact that monomers possessing the CSDL C terminus were actively secreted along with disulfide-linked dimers. In the case of the dimerizationdeficient mutants (AChE*), the ratio of secreted to cellular activities was clearly lower with a cysteine for WT, Scr, and 5W The mutants contained a wild type or a dimerization-deficient catalytic domain noted, respectively, as AChE (plain bars) and AChE* (hatched bars) and possessed Cys 37 or not (C37S) as indicated. The cellular and secreted activities were measured after 3 days of transfection. Because of variation in transfection efficiencies, the values obtained in each experiment were normalized to those obtained for the wild type (AChE-WT C ), which was always used as a control taken at 100%. The values indicated are the means Ϯ S.E. of at least three totally independent transfection experiments. Note that secretion was increased by the deletion of the aromatic-rich region (C1, C2, stop10, or Del) and, to a lesser degree, by the 7L mutation. Because the synthesis of AChE protein was the same for all of the mutants as shown in Fig. 6A and all of the mutants possessed the same catalytic activity (data not shown), the differences observed reflect the production of the active enzyme. mutants, but it was equal for 7L C and 7L S as well as for Del C and Del S .
Effect of the C-terminal Extremity of the t Peptide on Secretion-We compared mutants in which the cysteine was replaced by a serine (SSDL) with mutants lacking the last two residues (stop39, ending with Ser-Ser) and with mutants in which the last four residues were replaced by the classical ER retention signal, KDEL. These C-terminal endings were combined with the different mutations of the aromatic-rich region as described in Fig. 1A.
The KDEL tetrapeptide increased the cellular activity but only moderately for 5W with or without mutation F535A (Fig.  5A). It reduced the secreted activity in all of the mutants but only marginally for 7L (Fig. 5B). The stop39 mutation increased the secretion of WT and Scr but had no clear effect on 7L, 5W, or Del. This mutation did not affect the cellular activity of WT, 7L, or Del and decreased it very weakly for 5W and quite significantly for Scr. The ratio of secreted to cellular activities provided a more convenient index of these effects (Fig. 5C). This ratio was decreased by KDEL in all cases but less markedly for 7L. The stop39 mutation increased it for WT, Scr, and 5W but not for Del and 7L.
Synthesis of AChE Protein and Production of AChE Activity-Metabolic labeling experiments showed that all of the mutant proteins were synthesized at the same rate, as expected, because the vectors and the coding sequences were identical with the exception of a few codons in the region encoding the short non-catalytic C-terminal t peptide. In effect, we found that, after incorporation of radioactive amino acids for a short period, the amount of labeled AChE protein immunoprecipitated by the specific antibody A63 was the same for all mutants. This is illustrated in Fig. 6A for WT S , Del S , WT S *, and Del S *, showing that it is not influenced by the F535A mutation or by the presence of the aromatic-rich region of the t peptide. The relative intensities of the bands obtained after 20 or 40 min were the same within 5%. This incorporation was not modified after irreversible inhibition of preexisting cellular AChE by soman, a cell-permeant organophosphate inhibitor (data not shown).
We also studied the recovery of AChE activity after its inhibition by soman. Within two hours after washing the inhibitor, the cell activity increased linearly, recovering ϳ25% original cell content per hour in the case of WT S . Fig. 6B shows that the presence of a t peptide decreased the rate of production of newly synthesized active AChE compared with mutants that contained no t peptide (C 1 , C 1 *) or a t peptide from which the aromatic region was deleted (Del). In contrast, the F535A mutation in the dimerization zone of the catalytic domain had no influence. Since all AChE mutant proteins were synthesized at the same rate and all mutant enzymes possessed the same catalytic turnover, the fact that the presence of a complete t peptide reduced the production of AChE activity implies that some of the synthesized AChE polypeptides remain inactive.
We then compared the effects of the mutations that we introduced in the aromatic-rich region of the t peptide (Fig. 6C). The production of AChE activity was reduced to similar degrees for the WT, Scr, 5W, and 7L peptides compared with the Del mutant, which lacks this 10 -31 region. The 7L mutant, in which all of the aromatic residues were replaced by leucines, systematically produced the lowest activity of all of the mutants.
Because AChE-WT S subunits assemble as T 4 -Q N tetramers in which the t peptides are occluded, we examined the effect of co-expression with Q N . We found that this did not change the rate of recovery of AChE activity (data not shown).
Secretion of AChE Protein and Activity-We also monitored the secretion of a newly synthesized enzyme after treatment of transfected COS cells with soman (Fig. 6D). Although the cellular activity increased linearly after the removal of the inhibitor, secretion resumed after a lag period of ϳ3 h, corresponding to the time required for the transport of secreted proteins from the ER to the medium. Secretion then resumed and essentially recovered its original level after ϳ5 h. The highest secretion was observed for the Del mutant followed by 7L and then 5W and wild type with Scr being the least secreted, in agreement with the relative amounts of activities in the medium (Fig. 3).
Degradation of AChE Protein and Activity-We analyzed the degradation of AChE in transfected cells, which had approximately reached a steady state 4 days after transfection. After metabolic labeling for 30 min., we followed the evolution of AChE* protein during a chase period of several hours both in the cells and in the medium (Fig. 7A). All of the mutants progressively disappeared from the cells. The WT and Scr mutants did not appear in the medium, and the 5W mutant was barely detectable. In contrast, the 7L and Del mutants were clearly secreted, in agreement with the histograms of Fig. 3.
To analyze the intracellular degradation of AChE activity as well as protein, synthesis was blocked by cycloheximide and secretion was suppressed by brefeldin A. These experiments were performed with mutants lacking Cys 37 with or without the F535A mutation. Fig. 7, B and C, shows that AChE activity was degraded faster for all of the mutants that contained the F535A mutation (AChE*) compared with corresponding mutants without this mutation (AChE). Fig. 7D shows that the decrease of radioactively labeled protein (AChE*) was similar to that of enzymatic activity under the same conditions. In all of the cases, the Del mutant appeared more stable than the wild type and the 7L mutant was intermediate. The Scr and 5W mutants were similar to the wild type or were less stable. We also obtained similar results for mutants possessing a C-terminal KDEL tetrapeptide without brefeldin A (data not shown). DISCUSSION Previous studies established that the t peptide of AChE represents an autonomous interaction domain that allows the formation of homo-oligomers as well as hetero-oligomers with the anchoring proteins ColQ and PRiMA, which both possess a PRAD. The t peptide contains 40 amino acids and forms an amphiphilic ␣-helix with a cluster of seven aromatic residues. Besides its role in oligomerization, the t peptide can target AChE T subunits for degradation through the ER-associated degradation pathway rather than secretion, particularly when the catalytic domain contains the F535A mutation in its dimerization zone.
In this study, we investigated the features of the t peptide that determine the cellular fate of rat AChE toward degradation or secretion. For this purpose, we modified the aromaticrich region of the t peptide of AChE T subunits in various ways, disorganizing the aromatic cluster (Scr), replacing phenylalanines by the more bulky and less hydrophobic tryptophans (5W), replacing aromatic by the aliphatic hydrophobic leucines (7L), or deleting the entire aromatic-rich region 10 -31 (Del). We also modified the last four residues, which contain the Cys 37 and two C-terminal residues resembling those of the ER retention signal, KDEL.
Amphiphilic Properties and Oligomerization-Mutations in the aromatic-rich region of Torpedo AChE previously showed that the clustering of aromatic residues explains the amphiphilic properties of AChE T monomers and dimers (10). In this study, amphiphilicity was conserved when the two phenylalanines were replaced by tryptophans (5W), even though these residues are less hydrophobic, or when the seven aromatic residues of the t peptide were replaced by leucines (7L). In these mutants, dimers appeared less amphiphilic than monomers, indicating a partial occlusion of the hydrophobic clusters. Amphiphilicity was suppressed when the aromatic-rich region was deleted (Del and Stop10 mutants) but also when the cluster of aromatic residues was disrupted by displacement of these aromatic residues around the predicted helix without changing the amino acid composition (Scr).
In agreement with a previous analysis of oligomeric associations of Torpedo AChE T subunits, we found that aromatic residues, clustered on one side of an amphiphilic ␣-helix, are necessary for the assembly of homomeric or PRAD-associated tetramers, whereas the presence of a cysteine (Cys 37 ) is sufficient for the formation of stable dimers (9). Accordingly, the 5W mutants retained the capacity to form homomeric or PRADlinked tetramers but not the Del, Scr, or 7L mutants.
We particularly focused our attention on the synthesis, degradation, and secretion of the various mutants. Very clearly, FIG. 6. Mutations of the aromatic-rich region do not modify the rate of biosynthesis of the AChE protein but modify the rate of production and secretion of AChE activity. A, COS cells were transfected with the same amounts of cDNA expressing AChE-WT, AChE-Del, AChE*-WT, and AChE*-Del (all with the C37S mutation). They were labeled with [ 35 S]methionine and cysteine for 20 min, and cell extracts were collected immediately without chase. In the autoradiogram, immunoprecipitated AChE subunits appear as triplets because of heterogeneity in their glycosylation (18). Note that Del mutants lacking the aromatic-rich region of the t peptide migrate faster than WT mutants containing the full-length t peptide. B, effect of the F535A mutation and of the aromatic-rich region of the t peptide on the rate of neosynthesis of active AChE. Three days after transfection with equal amounts of cDNA, transfected cells were treated with 5 ϫ 10 Ϫ7 M soman, an irreversible organophosphate inhibitor of AChE and washed extensively and the recovery of AChE activity was monitored as a function of time. The rate of recovery was faster when the entire t peptide or its aromatic-rich region was deleted (•, AChE-C1; E, AChE*-C1; OE, AChE-Del S ) than when the t peptide was present (Ⅺ, AChE-WT S ; f, AChE*-WT S ). The cellular activity is normalized to that obtained in cells expressing the Del S mutant at the steady state. The F535A mutation had no significant effect. C, the rate of recovery was reduced compared with AChE-Del S for mutants of the aromatic-rich region (in this experiment, all of the mutants contained the C37S mutation). The symbols are as follows: OE, Del; •, WT; E: 7L; Ⅺ, Scr; ࡗ, 5W). D, recovery of secretion for mutants differing in their aromatic-rich region after soman treatment. The symbols are as in C.
the effects of mutations in the aromatic-rich region were qualitatively the same with or without the C-terminal cysteine (Cys 37 or C37S) and with or without the F535A mutation, which perturbs dimerization (AChE or AChE*). Thus, the observed effects were not related to dimerization.
Synthesis of Active AChE-Titration experiments with an organophosphate reagent of the active site (DEPQ) showed that mutations in the t peptide do not affect the catalytic turnover of the enzyme, as expected, because the catalytic domain and the t peptide are functionally independent. In addition, the F535A mutation, which affects dimerization of the catalytic domain, had no influence on its catalytic turnover. Therefore, the catalytic activities produced by the different mutants were directly proportional to the numbers of active AChE subunits.
We found that mutations or deletions in the t peptide with or without the F535A mutation do not affect the rate of synthesis of AChE proteins in COS cells, in agreement with the fact that the mutations were restricted to a very small part of the protein in the non-catalytic C-terminal region.
However, the production of AChE activity and thus active AChE subunits was sensitive to the presence of a t peptide. After irreversible inhibition of the preexisting enzyme, the synthesis of newly synthesized active AChE was not affected by the F535A mutation but was increased by deletion of the t peptide or of its aromatic-rich region (Del). Co-expression with Q N did not increase the rate of production of active wild type enzyme despite the formation of the T 4 -Q N complexes in which the t peptides are engaged in a tight interaction with the PRAD in T 4 -Q N complexes (11) in such a way that their aromatic-rich regions are occluded. The lack of effect of Q N on the production of active AChE suggests that complete folding precedes the assembly of the complex, although this association has also been shown to occur in the ER (9).
The presence of a t peptide therefore prevents a fraction of AChE polypeptides from acquiring their active conformation, possibly because its hydrophobic aromatic-rich region tends to become inserted in the catalytic core of the protein, perhaps at the molten globule stage, leading to an inactive conformation of the active site. If polypeptides lacking the t peptide are entirely folded into active catalytic domains, a proportion of ϳ40% appears to become misfolded in the presence of a wild type t peptide. The Scr, 5W, or 7L mutants also reduced the production of active enzyme. This effect was strongest with the 7L mutant in which all seven aromatic residues were replaced by leucines and weaker when two phenylalanines were replaced by tryptophans (5W), which are less hydrophobic. Therefore, misfolding seems to result from the hydrophobicity of residues in the t peptide and does not depend on their aromatic character or on their clustering in an amphiphilic ␣-helix.
Degradation-Previous studies showed that degradation was increased by the F535A mutation and by the presence of aromatic residues in the t peptide (19). Although degradation was faster for the dimerization deficient mutant (AChE*) than for the wild type catalytic domain (AChE), the influence of mutations in the aromatic-rich region was similar in both cases. We obtained similar degradation half-lives by studying the decrease of cellular AChE activity or of labeled AChE protein when secretion was blocked either by a KDEL retention signal or by brefeldin A (Fig. 7, C and D). This finding suggested that inactive and active AChE molecules were degraded FIG. 7. Effect of mutations in the aromatic-rich region on degradation. A, the newly synthesized protein was labeled by incorporation of [ 35 S]methionine and cysteine for 30 min, and radioactive AChE* subunits containing the indicated mutations in the t peptide (with Cys 37 ) were analyzed by autoradiography during a chase period of 8 h in the cells and in the medium. Note that the wild type and Scr mutant were not detectably secreted, whereas the 7L and Del mutants were found in the medium. In B, C, and D, transfected COS at the steady state were incubated with cycloheximide and brefeldin A to block protein synthesis and secretion, and the decrease of AChE activity was followed for 20 h for mutants of the t peptide lacking Cys 37 . B, wild type catalytic domain (AChE). C, catalytic domain presenting the F535A mutation (AChE*). D, radioactive AChE* protein was followed in cellular extracts after metabolic labeling. The symbols are as follows: OE, Del; •, WT; E, 7L; Ⅺ, Scr; ࡗ, 5W. The data are shown on the same semi-logarithmic scale. Note that the decrease of AChE activity was faster for AChE* than for the corresponding AChE mutants.
in the same manner and that brefeldin A did not perturb the degradation process. In particular, the 7L mutant, which produced the highest proportion of misfolded polypeptides, was degraded less rapidly than the wild type and the Scr or 5W mutants but more rapidly than the Del mutant. Thus, degradation is triggered by the presence of hydrophobic residues but much more efficiently by aromatic residues than by leucines. It does not depend on the organization of an amphiphilic ␣-helix as shown by the rapid degradation of the Scr mutant. It is interesting to emphasize the cooperativity between the F535A mutation in the catalytic domain and the functionally distinct t peptide for inducing degradation.
Effect of the C-terminal Tetrapeptide-We found that the C-terminal residues of the t peptide with or without the cysteine (CSDL or SSDL) do not influence the acquisition of AChE activity. Cys 37 does not prevent secretion of monomers. The ratio of secreted to cellular activity was only reduced when the presence of this cysteine was combined with the 5W mutation or with the F535A mutation in the catalytic domain and a t peptide containing aromatic residues (WT, Scr, or 5W). This finding indicates a cooperativity among various "signals" in different parts of the protein.
The role of the C-terminal tetrapeptide of AChE as a retention signal is not clear, and retention by an appended KDEL motif was reversed by dimerization through Cys 37 (20). We showed that the removal of the last two residues (Glu-Leu) of Torpedo AChE T subunits decreased the cellular activity and concomitantly increased the secreted activity ϳ4-fold (9). To examine a possible intracellular retention of monomeric rat AChE T subunits, we compared the influence of the C-terminal tetrapeptide SSDL with that of the canonical ER retention signal, KDEL, and we studied the effect of deleting the last two residues (Asp-Leu). The KDEL tetrapeptide did exert a retention effect, because it nearly blocked secretion and largely increased the cellular activity. It did not change the half-life of cellular AChE compared with that observed in brefeldin Atreated cells. However, retention was much less marked in the 7L mutant, which remained nearly secreted as with the SSDL terminus, indicating that the action of a KDEL "signal" may be strongly modulated by upstream elements. It is possible that the C-terminal leucine is partially occluded because of interactions with the other leucines. Removal of the Asp-Leu residues from SSDL clearly increased the secreted activity of the Scr mutant, but had no effect on secretion of the 7L and Del mutants. In addition, we observed no corresponding increase of their cellular activity, except for Scr. Therefore, it is not possible to consider that an SSDL tetrapeptide systematically acts as a retention signal but these observations confirm that it may exert an effect that is modulated by mutations in the aromaticrich region.
Effect of the Aromatic-rich Region on Secretion-We studied the rate of recovery of AChE activity and the rate of secretion after irreversible inhibition of the cellular AChE. Secretion resumed after a lag period of ϳ3 h and recovered its initial rate after 5 h.
For the Del and 7L mutants, the rate of secretion into the medium is nearly equal to the rate of synthesis of AChE activity in the cells observed after the removal of the inhibitor, indicating that most of the newly synthesized enzyme is secreted. In contrast, the ratio of secretion to synthesis is ϳ40 -50% for WT and 5W and Ͻ25% for Scr, indicating that the presence of aromatic residues reduces the efficiency of secretion, especially when the helical cluster is disorganized. How-ever, the strongly hydrophobic leucine residues do not divert the active 7L molecules from secretion. Therefore, secretion is not limited by the presence of an amphiphilic helix but rather by exposed aromatic residues.
Thus, it is possible to identify the features of the t peptide that affect the folding, degradation, and secretion of AChE subunits. Interference with a correct folding of the catalytic domain depends primarily on hydrophobicity, not on the aromatic nature of residues, or on their organization in a helical cluster. Degradation is stronger with aromatic residues than leucines and again is not sensitive to their ␣-helical organization. The influence of aromatic residues reinforces the strong effect due to the mutation F535A in the dimerization zone of the catalytic domain, although this is apparently not due to the capacity of catalytic domains for dimerization. Secretion efficiency was less reduced by leucines than by aromatic residues, and this effect was much stronger when the ␣-helical clustering of these residues was disorganized. The C-terminal Asp-Leu residue exerts a moderate retention effect, which is much weaker than that of a KDEL signal and only occurs in the presence of aromatic residues.
The characteristics of the t peptide that control folding are quite different from those that control degradation or secretion. AChE and its t peptide thus offer an extremely interesting model for analyzing the mechanisms of cellular trafficking of a secreted protein, with or without oligomerization.