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• W2068611210 abstract "The membrane-bound form of acetylcholinesterase (AChE) constitutes the major component of this enzyme in the mammalian brain. These molecules are hetero-oligomers, composed of four AChE catalytic subunits of type T (AChET), associated with a transmembrane protein of type 1, called PRiMA (proline-rich membrane anchor). PRiMA consists of a signal peptide, an extracellular domain that contains a proline-rich motif (14 prolines with an intervening leucine, P4LP10), a transmembrane domain, and a cytoplasmic domain. Expression of AChET subunits in transfected COS cells with a truncated PRiMA, without its transmembrane and cytoplasmic domains (Pstp54 mutant), produced secreted heteromeric complexes (T4-Pstp54), instead of membrane-bound tetramers. In this study, we used a series of deletions and point mutations to analyze the interaction between the extracellular domain of PRiMA and AChET subunits. We confirmed the importance of the polyproline stretches and defined a peptidic motif (RP4LP10RL), which induces the assembly and secretion of a heteromeric complex with four AChET subunits, nearly as efficiently as the entire extracellular domain of PRiMA. It is noteworthy that deletion of the N-terminal segment preceding the prolines had little effect. Interestingly, short PRiMA mutants, truncated within the proline-rich motif, reduced both cellular and secreted AChE activity, suggesting that their interaction with AChET subunits induces their intracellular degradation. The membrane-bound form of acetylcholinesterase (AChE) constitutes the major component of this enzyme in the mammalian brain. These molecules are hetero-oligomers, composed of four AChE catalytic subunits of type T (AChET), associated with a transmembrane protein of type 1, called PRiMA (proline-rich membrane anchor). PRiMA consists of a signal peptide, an extracellular domain that contains a proline-rich motif (14 prolines with an intervening leucine, P4LP10), a transmembrane domain, and a cytoplasmic domain. Expression of AChET subunits in transfected COS cells with a truncated PRiMA, without its transmembrane and cytoplasmic domains (Pstp54 mutant), produced secreted heteromeric complexes (T4-Pstp54), instead of membrane-bound tetramers. In this study, we used a series of deletions and point mutations to analyze the interaction between the extracellular domain of PRiMA and AChET subunits. We confirmed the importance of the polyproline stretches and defined a peptidic motif (RP4LP10RL), which induces the assembly and secretion of a heteromeric complex with four AChET subunits, nearly as efficiently as the entire extracellular domain of PRiMA. It is noteworthy that deletion of the N-terminal segment preceding the prolines had little effect. Interestingly, short PRiMA mutants, truncated within the proline-rich motif, reduced both cellular and secreted AChE activity, suggesting that their interaction with AChET subunits induces their intracellular degradation. In the nervous tissue and muscles of mammals, acetylcholinesterase (AChE, 2The abbreviations used are: AChE, acetylcholinesterase; AChET, splice variant T; PriMA, proline-rich membrane anchor; ColQ, collagen Q; ERAD, endoplasmic reticulum-associated degradation; PRAD, proline-rich attachment domain; Pstpn, truncated PRiMA mutant, terminating with a stop codon at position n; ΔN-X-Pstpn, PRiMA mutant with a deletion of the N-terminal fragment, starting with residue X before the prolines and terminating with a stop codon at position n; WAT, tryptophan (W) amphiphilic tetramerization domain. EC 3.1.1.7) controls cholinergic transmission by rapidly hydrolyzing the neurotransmitter acetylcholine after its release from nerve terminals. The functional localization of AChE depends on the association of its T splice variant with structural proteins (1Sikorav J.L. Duval N. Anselmet A. Bon S. Krejci E. Legay C. Osterlund M. Reimund B. Massoulié J. EMBO J. 1988; 7: 2983-2993Crossref PubMed Scopus (100) Google Scholar, 2Taylor P. J. Biol. Chem. 1991; 266: 4025-4028Abstract Full Text PDF PubMed Google Scholar, 3Massoulié J. Neurosignals. 2002; 11: 130-143Crossref PubMed Scopus (242) Google Scholar, 4Massoulié J. Bon S. Perrier N. Falasca C. Chem. Biol. Interact. 2005; 157: 3-14Crossref PubMed Scopus (55) Google Scholar). Thus, the physiologically active AChE species correspond essentially to the collagen-tailed forms at the neuromuscular junctions and to membrane-bound tetramers in the brain. The AChET splice variant is characterized by its 40-residue C-terminal peptide (t peptide), which contains a C-terminal cysteine and seven aromatic residues, including three evenly spaced tryptophans, and can be organized as an amphiphilic α-helix (5Bon S. Dufourcq J. Leroy J. Cornut I. Massoulié J. Eur. J. Biochem. 2004; 271: 33-47Crossref PubMed Scopus (22) Google Scholar). This peptide behaves as an autonomous interaction domain (the tryptophan (W) amphiphilic tetramerization domain (WAT)) (6Simon S. Krejci E. Massoulié J. EMBO J. 1998; 17: 6178-6187Crossref PubMed Scopus (76) Google Scholar); it allows oligomerization of AChET subunits into homomeric dimers (T2) and tetramers (T4), as well as heteromeric associations of tetramers with anchoring proteins (7Krejci E. Coussen F. Duval N. Chatel J.M. Legay C. Puype M. Vandekerckhove J. Cartaud J. Bon S. Massoulié J. EMBO J. 1991; 10: 1285-1293Crossref PubMed Scopus (118) Google Scholar, 8Duval N. Krejci E. Grassi J. Coussen F. Massoulié J. Bon S. EMBO J. 1992; 11: 3255-3261Crossref PubMed Scopus (39) Google Scholar, 9Bon S. Massoulié J. J. Biol. Chem. 1997; 272: 3007-3015Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). In the collagen-tailed forms, AChET tetramers are associated with a specific collagen, called ColQ (7Krejci E. Coussen F. Duval N. Chatel J.M. Legay C. Puype M. Vandekerckhove J. Cartaud J. Bon S. Massoulié J. EMBO J. 1991; 10: 1285-1293Crossref PubMed Scopus (118) Google Scholar, 10Krejci E. Thomine S. Boschetti N. Legay C. Sketelj J. Massoulié J. J. Biol. Chem. 1997; 272: 22840-22847Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). This interaction has been extensively studied; it is based on a tight interaction between four t peptides (6Simon S. Krejci E. Massoulié J. EMBO J. 1998; 17: 6178-6187Crossref PubMed Scopus (76) Google Scholar) and a proline-rich motif, called PRAD (“proline-rich attachment domain”), located in the N-terminal noncollagenous region of ColQ (11Bon S. Coussen F. Massoulié J. J. Biol. Chem. 1997; 272: 3016-3021Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar). Synthetic t and PRAD peptides (40 and 15 residues, respectively) spontaneously form a complex, the structure of which has been determined by crystallography; four α-helical t peptides form a staggered coiled-coil around the PRAD, organized as an elongated polyproline II helix (12Dvir H. Harel M. Bon S. Liu W.Q. Vidal M. Garbay C. Sussman J.L. Massoulié J. Silman I. EMBO J. 2004; 23: 4394-4405Crossref PubMed Scopus (93) Google Scholar). All aromatic residues are oriented toward the interior of this compact cylindrical complex, and the tryptophans are apposed to the rings of the proline residues. From this interaction, it was possible to deduce models for the quaternary organization of four AChET subunits linked to a ColQ chain (12Dvir H. Harel M. Bon S. Liu W.Q. Vidal M. Garbay C. Sussman J.L. Massoulié J. Silman I. EMBO J. 2004; 23: 4394-4405Crossref PubMed Scopus (93) Google Scholar, 13Zhang D. McCammon J.A. PLoS Comput. Biol. 2005; 1: 484-491Google Scholar). The existence of an N-glycosylated 20-kDa hydrophobic protein, associated with membrane-bound AChE tetramers, was originally discovered in 1987 by Gennari et al. (14Gennari K. Brunner J. Brodbeck U. J. Neurochem. 1987; 49: 12-18Crossref PubMed Scopus (68) Google Scholar) and by Inestrosa et al. (15Inestrosa N.C. Roberts W.L. Marshall T.L. Rosenberry T.L. J. Biol. Chem. 1987; 262: 4441-4444Abstract Full Text PDF PubMed Google Scholar); this membrane anchor was more recently cloned and called PRiMA (“proline-rich membrane anchor”), because it contains a proline-rich motif, like ColQ (16Perrier A.L. Massoulié J. Krejci E. Neuron. 2002; 33: 275-285Abstract Full Text Full Text PDF PubMed Scopus (217) Google Scholar). This suggests that AChET subunits may associate with PRiMA and with ColQ in a similar manner. However, there are significant differences in the numbers of prolines (8 in ColQ and 14 in PRiMA) and in the numbers and positions of cysteines that could form intercatenary disulfide bonds with a cysteine located near the C terminus of each of the four t peptides. In addition, PRiMA contains a putative N-glycosylation site between the polyproline stretches and the transmembrane domain (17Boschetti N. Brodbeck U. FEBS Lett. 1996; 380: 133-136Crossref PubMed Scopus (22) Google Scholar). Alternative splicing produces two PRiMA variants, which differ in their C-terminal domains as follows: the intracellular domains of the major variant (PRiMA I) and of the minor variant (PRiMA II) contain 40 and 11 residues, respectively (18Perrier N.A. Khérif S. Perrier A.L. Krejci E. Dumas S. Mallet J. Massoulié J. Eur. J. Neurosci. 2003; 18: 1837-1847Crossref PubMed Scopus (37) Google Scholar). The mode of association between PRiMA and AChET subunits is physiologically important because the resulting PRiMA-anchored AChE tetramers (16Perrier A.L. Massoulié J. Krejci E. Neuron. 2002; 33: 275-285Abstract Full Text Full Text PDF PubMed Scopus (217) Google Scholar, 18Perrier N.A. Khérif S. Perrier A.L. Krejci E. Dumas S. Mallet J. Massoulié J. Eur. J. Neurosci. 2003; 18: 1837-1847Crossref PubMed Scopus (37) Google Scholar) represent the major enzyme species in the brain (18Perrier N.A. Khérif S. Perrier A.L. Krejci E. Dumas S. Mallet J. Massoulié J. Eur. J. Neurosci. 2003; 18: 1837-1847Crossref PubMed Scopus (37) Google Scholar), and their level is regulated by exercise in muscles (19Gisiger V. Bélisle M. Gardiner P.F. Eur. J. Neurosci. 1994; 6: 673-680Crossref PubMed Scopus (51) Google Scholar). We have undertaken an analysis of the association of AChET with PRiMA. In this study, we mostly used truncated PRiMA mutants containing only the extracellular domain or fragments of this domain, but not the transmembrane and cytoplasmic domains, thus producing soluble heteromeric complexes with AChET subunits. In previous studies, we have shown that a significant fraction of AChET subunits is degraded intracellularly, through the ERAD process (“endoplasmic reticulum-associated degradation”) (20Belbeoc'h S. Massoulié J. Bon S. EMBO J. 2003; 22: 3536-3545Crossref PubMed Scopus (40) Google Scholar, 21Belbeoc'h S. Falasca C. Leroy J. Ayon A. Massoulié J. Bon S. Eur. J. Biochem. 2004; 271: 1476-1487Crossref PubMed Scopus (32) Google Scholar), and that this is mostly induced by exposed aromatic residues (21Belbeoc'h S. Falasca C. Leroy J. Ayon A. Massoulié J. Bon S. Eur. J. Biochem. 2004; 271: 1476-1487Crossref PubMed Scopus (32) Google Scholar, 22Falasca C. Perrier N.A. Massoulié J. Bon S. J. Biol. Chem. 2005; 280: 878-886Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar), in agreement with the fact that the formation of a complex in which these residues are occluded may reduce their degradation and increase their secretion. In this study, we examine how co-expression with PRiMA mutants affects the trafficking, degradation, and secretion of AChET subunits. We show that some truncated mutants of PRiMA act as degradation inducers, assembling AChET subunits into complexes that are degraded intracellularly, indicating that they fail to pass the quality control of the secretory pathway. By using deletions and mutations, we analyze the influence of residues flanking the polyproline stretches and also of the leucine located between the prolines, and we define a peptidic motif (RP4LP10RL), which is nearly as efficient as the complete extracellular domain of PRiMA for recruitment of AChET subunits into secreted heteromeric complexes. Vectors and Site-directed Mutagenesis—The AChET subunits of rat AChE and intact or mutated mouse PRiMA were expressed by inserting the corresponding cDNAs into pEF-Bos vectors (23Mizushima S. Nagata S. Nucleic Acids Res. 1990; 18: 5322Crossref PubMed Scopus (1499) Google Scholar). Throughout this study, the numbering of PRiMA residues corresponds to the mature protein (Fig. 1A); the extracellular domain corresponds to residues 1–53. Mutagenesis was performed by the method of Kunkel et al. (24Kunkel T.A. Roberts J.D. Zakour R.A. Methods Enzymol. 1987; 154: 367-382Crossref PubMed Scopus (4560) Google Scholar), as described previously (25Morel N. Leroy J. Ayon A. Massoulié J. Bon S. J. Biol. Chem. 2001; 276: 37379-37389Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). For deletions, we used mutagenic oligonucleotides of about 30 nucleotides containing 15 nucleotides on each side of the deleted fragment. Truncated mutants are indicated by the position of stop codons and by the modified residues; for example, R36E-Pstp37 indicates a mutant in which a stop codon was introduced at position 37 and arginine 36 was replaced by a glutamic acid. Fig. 1B shows the structure of mutants used in this study. We introduced a FLAG epitope (DYKDE) after the cleavage site of the signal peptide in PRiMA, so that it was recognized by the anti-FLAG monoclonal antibody M1 (Sigma). We also used an N-terminal fragment of Torpedo ColQ, with or without its PRAD domain (Fig. 1C). Transfection in COS Cells—Plasmids were transfected in COS cells with the DEAE-dextran method, as described previously (9Bon S. Massoulié J. J. Biol. Chem. 1997; 272: 3007-3015Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar), using 2 μg of vector DNA encoding the catalytic subunit AChET and various amounts of vector DNA encoding PRiMA mutants, as specified, per 60-mm dish. For comparison, we also used an N-terminal fragment of Torpedo marmorata ColQ (Qstp69). In each series of transfections, we completed the amount of vector encoding PRiMA mutants with a vector encoding a noninteracting protein, the N-terminal domain of ColQ from which the PRAD interaction motif was deleted (Δ(28–44)-Qstp69) (11Bon S. Coussen F. Massoulié J. J. Biol. Chem. 1997; 272: 3016-3021Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar), so that the total amount of vector remained constant, to avoid changes in the synthetic capacity of the cells. After transfection, COS cells were incubated at 37 °C, in a medium containing 10% NUserum (Inotech, Dottikon, Switzerland), which had been pretreated with 10–5 m soman to inactivate serum cholinesterases. The medium and the cells were collected after 3–4 days. Analysis of AChE Recovery after Irreversible Inhibition—The AChE activity of transfected cells (3 or 4 days after transfection) was irreversibly inhibited by incubation with the membranepermeant inhibitor soman (pinacolyl methylphosphonofluoridate) at 5 × 10–7 m for 30 min, as described previously (22Falasca C. Perrier N.A. Massoulié J. Bon S. J. Biol. Chem. 2005; 280: 878-886Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar). After extensive washing, the recovery of AChE activity was determined by collecting cells at various times in fresh culture medium at 37 °C. Secretion of newly synthesized AChE only resumed after about 150–180 min, and during that period the recovery of cellular activity reflected a balance between neosynthesis and intracellular degradation. Cell Extracts—Intracellular and membrane-bound AChE was extracted for 15 min at 20 °C in a TMg buffer (1% Triton X-100, 50 mm Tris-HCl, pH 7.5, 10 mm MgCl2) containing 25 mm benzamidine, followed by centrifugation for 10 min at 13,000 rpm at 4 °C. The culture medium containing the secreted enzyme was also centrifuged at 13,000 rpm for 10 min to remove cell debris before analysis. Enzyme Assays—AChE activity was determined with the colorimetric method of Ellman et al. (26Ellman G.L. Courtney K.D. Andres V. Featherstone R.M. Biochem. Pharmacol. 1961; 7: 88-95Crossref PubMed Scopus (21719) Google Scholar) at room temperature. The reaction was monitored at 414 nm with a Labsystems Multiskan RC automatic plate reader (Helsinki, Finland); the optical density was recorded at 20-s intervals over a period of 10 min. Alkaline phosphatase and β-galactosidase from Escherichia coli were assayed with the chromogenic substrates p-nitrophenyl phosphate and o-nitrophenyl galactoside, respectively. Sedimentation and Electrophoresis Analyses—Centrifugation in 5–20% sucrose gradients (50 mm Tris-HCl, pH 7.5, 20 mm MgCl2, in the presence of 1% Brij-96 or 0.2% Triton X-100) was performed in a Beckman SW41 rotor, at 36,000 rpm, for 17 h 30 min at 6 °C. Approximately 40 fractions were collected, and AChE activity was measured with the Ellman colorimetric assay, allowing the determination of the sedimentation coefficients of the different molecular forms, and of their relative activities. The gradients contained E. coli β-galactosidase (16 S) and alkaline phosphatase (6.1 S) as internal sedimentation standards. Amphiphilic molecules are characterized by the fact that they interact with detergent micelles; they generally sediment slower in the presence of Brij-96 than of Triton X-100. Electrophoresis in nondenaturing polyacrylamide gels was performed as described by Bon et al. (27Bon S. Rosenberry T.L. Massoulié J. Cell. Mol. Neurobiol. 1991; 11: 157-172Crossref PubMed Scopus (46) Google Scholar). The gels contained 0.25% Triton X-100 with or without 0.05% deoxycholate; electrophoresis was performed in a refrigerated apparatus under 40 V/cm for 2 h. Enzymatic activity was revealed by the histochemical method of Karnovsky and Roots (28Karnovsky M.J. Roots L. J. Histochem. Cytochem. 1964; 12: 219-222Crossref PubMed Scopus (2999) Google Scholar). This method allows a rapid qualitative comparison of up to 20 samples in a single gel. In charge shift electrophoresis, the electrophoretic migration of amphiphilic molecules was accelerated in the presence of sodium deoxycholate, when compared with migration in the presence of the neutral detergent Triton X-100. Effect of Synthetic Peptides on Oligomerization of AChET Subunits—Synthetic peptides corresponding to the interaction motif of PRiMA (RP4LP10 and RP4LP10RL) were synthesized by the Merrifield solid phase method in an Applied Biosystems 431A automated peptide synthesizer, with small scale 9-fluorenylmethoxycarbonyl (Fmoc) chemistry (29Fields G.B. Noble R.L. Int. J. Pept. Protein Res. 1990; 35: 161-214Crossref PubMed Scopus (2336) Google Scholar). Protected amino acids were purchased from Applied Biosystems (Foster City, CA) (30Sigler G.F. Fuller W.D. Chaturvedi N.C. Goodman M. Verlander M. Biopolymers. 1983; 22: 2157-2162Crossref Scopus (83) Google Scholar). Crude peptides were purified by reverse phase high pressure liquid chromatography on a Vydac C18 column (5 μm, 250 × 10 mm2), using appropriate acetonitrile gradients containing 0.1% trifluoroacetic acid. A 10–2 m solution of peptide in Tris-HCl, pH 8 (1 m), was added with fresh medium to transfected COS cells expressing only AChET subunits, at a final concentration of 10–4 m. Peptides were also added to cell homogenates, at a final concentration of 10–4 m, and incubated overnight at 20 or 37 °C. In some samples, a synthetic t peptide (WAT) was added at 2 × 10–4 m. The oligomeric state of AChE was then analyzed by nondenaturing electrophoresis, after dilution 2-fold in buffer containing 1% Triton X-100. PRiMA Mutants—Fig. 1A shows the primary sequence of mouse PRiMA and the structure of mutants used in this study. PRiMA consists of a signal peptide (residues –35 to –1), an extracellular N-terminal domain (residues 1–53) that includes four cysteines, a proline-rich motif and a putative N-glycosylation site, a transmembrane domain (residues 54–78), and two possible cytoplasmic C-terminal domains that define PRiMA I and PRiMA II. We studied the interaction of AChET subunits with the extracellular domain of PRiMA, using mutants lacking the transmembrane and cytoplasmic domains (Fig. 1B). First, we introduced stop codons at various positions (n), producing proteins Pstpn. Second, we deleted most of the N-terminal region preceding the prolines, in mutants designed as ΔN; to maintain a correct signal peptide cleavage site, we conserved the first or the last residue of this region (Glu-1 and Arg-20, respectively). We also assessed the possible influence of electrostatic effects, by mutating the residue preceding the prolines by other charged or uncharged residues (Asp, Lys, or Ala). Finally, we analyzed the role of the leucine located within the polyproline region (Leu-25), by replacing it with a proline, and by adding a leucine at position 31, with or without the original leucine. Thus the original motif P4LP10 was changed to P15, P10LP4, and P4LP5LP4. Formation and Secretion of Soluble Complexes with Truncated PRiMA Mutants, Lacking the Transmembrane Domain—PRiMA and its different mutants were expressed in COS cells together with AChET subunits (also called T subunits). To evaluate the formation of heteromeric complexes, we analyzed the total AChE activity of the cellular extracts and of the medium, as well as their composition in molecular forms, using both sedimentation and nondenaturing electrophoresis. It should be noted that all molecular forms of AChE, which differ in their degree of oligomerization, with or without associated proteins, possess the same catalytic activity per active site, so that the level of AChE activity reflects the number of AChET subunits, independently of the composition in molecular forms. Fig. 2 illustrates the molecular forms obtained by expressing AChET subunits alone, with full-length PRiMA, and with its extracellular fragment, Pstp54. As a control, we used an N-terminal fragment of ColQ that contains the PRAD (residues 28–44), without the collagenous and trimerization domains (Qstp69) (Fig. 1C). When expressed alone or in the presence of a noninteracting fragment of ColQ from which the proline-rich motif was deleted (Δ(28–44)-Qstp69), T subunits produced mostly amphiphilic monomers (T1) and dimers (T2) (27Bon S. Rosenberry T.L. Massoulié J. Cell. Mol. Neurobiol. 1991; 11: 157-172Crossref PubMed Scopus (46) Google Scholar), with a small proportion of nonamphiphilic tetramers sedimenting at 10.5 S, which are thought to be homomeric (T4), i.e. with no associated endogenous noncatalytic component (Fig. 2A). Monomers and dimers are not readily resolved in the sedimentation profiles, and we therefore quantified their sum (T1 + T2). The proportion of T4 tetramers is markedly higher in the medium than in the cell extract, indicating that they are secreted more efficiently than monomers and dimers, as observed previously (9Bon S. Massoulié J. J. Biol. Chem. 1997; 272: 3007-3015Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). Co-expression of T subunits with full-length PRiMA induced the formation of membrane-bound T4-PRiMA complexes, expressed at the cell surface as demonstrated by immunofluorescence (not shown). These heteromeric complexes were recovered in the cell extract after solubilization in the presence of 1% Triton X-100; unlike homomeric tetramers (T4), which sediment at 10.5 S regardless of the presence or absence of detergents, the PRiMA-associated tetramers are amphiphilic, and their sedimentation is influenced by detergents: they sediment at 9.8 S in the presence of Triton X-100 and 9 S in the presence of Brij-96. This was also shown by the fact that they migrated more slowly than T4 tetramers in nondenaturing electrophoresis (Fig. 2B). In addition, the medium contained an increased level of soluble molecules sedimenting at 10.5 S, suggesting that they correspond to nonamphiphilic tetramers. When AChET subunits were co-expressed with an N-flagged PRiMA, these secreted tetrameric molecules were recognized by an anti-FLAG antibody (M1), as shown by retardation of their migration in nondenaturing electrophoresis (Fig. 3), demonstrating that they contain an N-terminal fragment of PRiMA that might be produced by proteolysis, either intracellularly or at the cell surface. Co-expression of AChET subunits with the Pstp54 mutant, corresponding to the extracellular domain of PRiMA, induced the formation of a soluble AChE form, sedimenting at 10.5 S, and migrating as an AChE tetramer in nondenaturing electrophoresis (Fig. 2, A and B). This form, which could not be distinguished from homomeric T4 tetramers by sedimentation, corresponds to AChET tetramers associated with the Pstp54 protein (T4-Pstp54). The fact that the ratio of T4-Pstp54 to T1 + T2 is higher in the medium than in the cell extract shows that they are preferentially secreted, as observed for homomeric tetramers T4. The formation of soluble T4-Pstp54 complexes demonstrates that the amphiphilic character of T4-PRiMA complexes is due to the transmembrane domain of PRiMA and confirms that AChET subunits interact with a peptidic motif located in the N-terminal extracellular region of PRiMA, in agreement with a previous study (16Perrier A.L. Massoulié J. Krejci E. Neuron. 2002; 33: 275-285Abstract Full Text Full Text PDF PubMed Scopus (217) Google Scholar). Effect of Progressive C-terminal Deletions in the Extracellular Domain of PRiMA on the Recruitment of AChET Subunits—We examined the interaction between AChET subunits and PRiMA mutants in which stop codons were introduced upstream of position 54: Pstp46, Pstp43, Pstp39, Pstp38, Pstp37, Pstp36, Pstp33, and Pstp31 (see Fig. 1B). We studied the AChE activity and molecular forms produced in COS cells expressing a constant amount of AChET subunits (2 μg of vector DNA per 60-mm culture dish), together with varying amounts of each mutant. The proportion of AChET tetramers increased in the cells and in the medium with the amount of associated noncatalytic protein, as illustrated for Pstp33, Pstp54, and Qstp69 in Fig. 4. This proportion was found to plateau at similar values for Pstp54 and Qstp69 but at a much lower level for Pstp33. Fig. 5 illustrates the effect of the Pstpn mutants on the cellular activity and on the rate of secretion, with a fixed amount of plasmid encoding the Pstpn mutants (2 μg of DNA encoding AChET and PRiMA mutant, each, per 60-mm culture dish). The level of cellular activity varied very little when AChET subunits were co-transfected with the various PRiMA mutants. Secretion increased by about 30% when AChET subunits were co-transfected with Qstp69, indicating that they were partially rescued from intracellular degradation by the formation of T4-Qstp69 complexes (not shown), but reduced by about 50% when they were co-transfected with Pstp31. The secreted activity gradually increased with the longer mutants Pstp33 and Pstp36, reaching the approximate value obtained with AChET alone for longer constructs. The level of secreted T1 + T2 was markedly reduced when AChET subunits were co-transfected with any of the truncated Pstpn mutants, as well as with Qstp69, indicating that they interacted with all mutants, even the shorter ones, such as Pstp33 or Pstp31, which produced only minimal levels of cellular or secreted T4-Pstpn form. The variations observed in the level of secreted activity appeared correlated with the proportion of heteromeric complex in the medium, as shown in Fig. 5C. Remarkably, this correlation includes all the truncated PRiMA mutants analyzed in this study, and also Qstp69. Fig. 5D shows that the secretion of T4-Pstpn complexes appears proportional to their cellular activity, except for T4-Pstp36, which was more efficiently secreted, and for mutants containing Ala, Asp, or Glu residues preceding the proline-rich segment, which are less efficiently secreted. The fact that AChE activity was reduced, both in the cells and in the medium, when AChET subunits were co-expressed with short truncated PRiMA mutants such as Pstp31 suggests that they were more degraded than when expressed alone. This was verified by following the initial rate of recovery of AChE activity, during the 2 h after irreversible inhibition, i.e. before secretion of the newly synthesized enzyme; this rate represents the balance between neosynthesis and intracellular degradation. The rate of neosynthesis must be identical when AChET subunits are expressed with a noninteracting protein and with different PRiMA mutants. We found that the rates of recovery varied in the order AChET + Δ(28–44)-Qstp69 > AChET + Pstp46 > AChET + Pstp33, indicating that Pstp33, and to a lesser degree Pstp46, induced some degradation of newly synthesized active AChET subunits. The ratio of secreted to the cellular activity of each molecular form of AChE can be considered as an index of its secretability; Fig. 6 shows that this ratio remained essentially constant for monomers and dimers (T1 + T2), but varied markedly for AChE tetramers associated with Pstpn proteins. This indicates that the secretion of the complexes depended on the length of the PRiMA fragment, especially between Pstp31 and Pstp36, confirming that complexes formed with the shorter PRiMA mutants were not efficiently secreted. In addition, the fact that this ratio varied in a nonmonotonic manner as a function of the length of the mutants from 36 to 54 suggests that the C-terminal residues of the Pstpn proteins may either facilitate or reduce secretion. The Pstp38 mutant seemed to produce a maximum of secreted heteromeric complexes, whereas Pstp36 appeared optimal for the secretability of such complexes. The differences observed between Pstp36, Pstp37, Pstp38, and Pstp39 show that the residues located immediately downstream of the prolines (RLL) influence the formation and the secretion of the T4-Pstpn complexes. However, re" @default.
• W2068611210 created "2016-06-24" @default.
• W2068611210 creator A5011388889 @default.
• W2068611210 creator A5018982994 @default.
• W2068611210 creator A5019754471 @default.
• W2068611210 creator A5036207583 @default.
• W2068611210 creator A5045848833 @default.
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• W2068611210 date "2007-02-01" @default.
• W2068611210 modified "2023-10-13" @default.
• W2068611210 title "Assembly of Acetylcholinesterase Tetramers by Peptidic Motifs from the Proline-rich Membrane Anchor, PRiMA" @default.
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