AssemblyofAcetylcholinesteraseTetramersbyPeptidic … · 2007-01-26 · permeant inhibitor soman (pinacolyl methylphosphonofluori-date) at 5 10 7 M for 30 min, as described previously

Assembly of Acetylcholinesterase Tetramers by PeptidicMotifs from the Proline-rich Membrane Anchor, PRiMACOMPETITION BETWEEN DEGRADATION AND SECRETION PATHWAYS OFHETEROMERIC COMPLEXES*

Received for publication, July 31, 2006, and in revised form, October 24, 2006 Published, JBC Papers in Press, December 8, 2006, DOI 10.1074/jbc.M607221200

Hiba Noureddine‡, Claudine Schmitt‡, Wangqing Liu§, Christiane Garbay§, Jean Massoulie‡, and Suzanne Bon‡1

From the ‡Laboratoire de Neurobiologie Cellulaire et Moleculaire, CNRS UMR 8544, Ecole Normale Superieure,46 Rue d’Ulm, 75005 Paris and the §Laboratoire de Pharmacochimie Moleculaire et Cellulaire, INSERM U648,UFR Biomedicale des Saints Peres, 45 Rue des Saints Peres, 75006 Paris, France

The membrane-bound form of acetylcholinesterase (AChE)constitutes themajor component of this enzyme in themamma-lian brain. These molecules are hetero-oligomers, composed offour AChE catalytic subunits of type T (AChET), associated witha transmembrane protein of type 1, called PRiMA (proline-richmembrane anchor). PRiMA consists of a signal peptide, anextracellular domain that contains a proline-rich motif (14 pro-lines with an intervening leucine, P4LP10), a transmembranedomain, and a cytoplasmic domain. Expression of AChET sub-units in transfected COS cells with a truncated PRiMA, withoutits transmembrane and cytoplasmic domains (Pstp54 mutant),produced secreted heteromeric complexes (T4-Pstp54), insteadofmembrane-bound tetramers. In this study, we used a series ofdeletions and point mutations to analyze the interactionbetween the extracellular domain of PRiMA and AChET sub-units. We confirmed the importance of the polyprolinestretches and defined a peptidic motif (RP4LP10RL), whichinduces the assembly and secretion of a heteromeric complexwith four AChET subunits, nearly as efficiently as the entireextracellular domain of PRiMA. It is noteworthy that deletion ofthe N-terminal segment preceding the prolines had little effect.Interestingly, short PRiMA mutants, truncated within the pro-line-rich motif, reduced both cellular and secreted AChE activ-ity, suggesting that their interaction with AChET subunitsinduces their intracellular degradation.

In the nervous tissue and muscles of mammals, acetylcho-linesterase (AChE,2 EC 3.1.1.7) controls cholinergic transmis-sion by rapidly hydrolyzing the neurotransmitter acetylcholine

after its release from nerve terminals. The functional localiza-tion of AChE depends on the association of its T splice variantwith structural proteins (1–4). Thus, the physiologically activeAChE species correspond essentially to the collagen-tailedforms at the neuromuscular junctions and tomembrane-boundtetramers in the brain.The AChET splice variant is characterized by its 40-residue

C-terminal peptide (t peptide), which contains a C-terminalcysteine and seven aromatic residues, including three evenlyspaced tryptophans, and can be organized as an amphiphilic�-helix (5). This peptide behaves as an autonomous interactiondomain (the tryptophan (W) amphiphilic tetramerizationdomain (WAT)) (6); it allows oligomerization of AChET sub-units into homomeric dimers (T2) and tetramers (T4), as well asheteromeric associations of tetramers with anchoring proteins(7–9).In the collagen-tailed forms, AChET tetramers are associated

with a specific collagen, calledColQ (7, 10). This interaction hasbeen extensively studied; it is based on a tight interactionbetween four t peptides (6) and a proline-rich motif, calledPRAD (“proline-rich attachment domain”), located in theN-terminal noncollagenous region of ColQ (11). Synthetic tand PRAD peptides (40 and 15 residues, respectively) sponta-neously form a complex, the structure of which has been deter-mined by crystallography; four �-helical t peptides form a stag-gered coiled-coil around the PRAD, organized as an elongatedpolyproline II helix (12). All aromatic residues are orientedtoward the interior of this compact cylindrical complex, and thetryptophans are apposed to the rings of the proline residues.From this interaction, it was possible to deduce models for thequaternary organization of four AChET subunits linked to aColQ chain (12, 13).The existence of anN-glycosylated 20-kDa hydrophobic pro-

tein, associated with membrane-bound AChE tetramers, wasoriginally discovered in 1987 by Gennari et al. (14) and by Ine-strosa et al. (15); this membrane anchor was more recentlycloned and called PRiMA (“proline-rich membrane anchor”),because it contains a proline-rich motif, like ColQ (16). Thissuggests that AChET subunits may associate with PRiMA andwith ColQ in a similar manner. However, there are significantdifferences in the numbers of prolines (8 in ColQ and 14 inPRiMA) and in the numbers and positions of cysteines thatcould form intercatenary disulfide bonds with a cysteine

* This work was supported by grants from the CNRS, the Association Fran-caise Contre les Myopathies, the Direction des Forces et de la Prospective,and the European Community. The costs of publication of this article weredefrayed in part by the payment of page charges. This article must there-fore be hereby marked “advertisement” in accordance with 18 U.S.C. Sec-tion 1734 solely to indicate this fact.

1 To whom correspondence should be addressed. Tel.: 33-1-44-32-38-91; Fax:33-1-44-32-38-87; E-mail: [email protected].

2 The abbreviations used are: AChE, acetylcholinesterase; AChET, splice vari-ant T; PriMA, proline-rich membrane anchor; ColQ, collagen Q; ERAD,endoplasmic reticulum-associated degradation; PRAD, proline-richattachment domain; Pstpn, truncated PRiMA mutant, terminating with astop codon at position n; �N-X-Pstpn, PRiMA mutant with a deletion of theN-terminal fragment, starting with residue X before the prolines and ter-minating with a stop codon at position n; WAT, tryptophan (W) amphiphilictetramerization domain.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 282, NO. 6, pp. 3487–3497, February 9, 2007© 2007 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

FEBRUARY 9, 2007 • VOLUME 282 • NUMBER 6 JOURNAL OF BIOLOGICAL CHEMISTRY 3487

located near the C terminus of each of the four t peptides. Inaddition, PRiMA contains a putative N-glycosylation sitebetween the polyproline stretches and the transmembranedomain (17). Alternative splicing produces two PRiMA vari-ants, which differ in their C-terminal domains as follows: theintracellular domains of themajor variant (PRiMA I) and of theminor variant (PRiMA II) contain 40 and 11 residues, respec-tively (18). The mode of association between PRiMA andAChET subunits is physiologically important because theresulting PRiMA-anchored AChE tetramers (16, 18) representthe major enzyme species in the brain (18), and their level isregulated by exercise in muscles (19).We have undertaken an analysis of the association of AChET

with PRiMA. In this study, we mostly used truncated PRiMAmutants containing only the extracellular domain or fragmentsof this domain, but not the transmembrane and cytoplasmicdomains, thus producing soluble heteromeric complexes withAChET subunits. In previous studies, we have shown that asignificant fraction of AChET subunits is degraded intracel-lularly, through the ERAD process (“endoplasmic reticulum-associated degradation”) (20, 21), and that this is mostlyinduced by exposed aromatic residues (21, 22), in agreementwith the fact that the formation of a complex in which theseresidues are occluded may reduce their degradation andincrease their secretion. In this study, we examine how co-ex-pression with PRiMA mutants affects the trafficking, degrada-tion, and secretion of AChET subunits. We show that sometruncated mutants of PRiMA act as degradation inducers,assembling AChET subunits into complexes that are degradedintracellularly, indicating that they fail to pass the quality con-trol of the secretory pathway. By using deletions andmutations,we analyze the influence of residues flanking the polyprolinestretches and also of the leucine located between the prolines,and we define a peptidic motif (RP4LP10RL), which is nearly asefficient as the complete extracellular domain of PRiMA forrecruitment of AChET subunits into secreted heteromericcomplexes.

EXPERIMENTAL PROCEDURES

Vectors and Site-directed Mutagenesis—The AChET sub-units of rat AChE and intact or mutated mouse PRiMA wereexpressed by inserting the corresponding cDNAs into pEF-Bosvectors (23). Throughout this study, the numbering of PRiMAresidues corresponds to themature protein (Fig. 1A); the extra-cellular domain corresponds to residues 1–53. Mutagenesiswas performed by themethod of Kunkel et al. (24), as describedpreviously (25). For deletions, we used mutagenic oligonucleo-tides of about 30 nucleotides containing 15 nucleotides on eachside of the deleted fragment. Truncated mutants are indicatedby the position of stop codons and by themodified residues; forexample, R36E-Pstp37 indicates a mutant in which a stop codonwas introduced at position 37 and arginine 36 was replaced by aglutamic acid. Fig. 1B shows the structure of mutants used inthis study. We introduced a FLAG epitope (DYKDE) after thecleavage site of the signal peptide in PRiMA, so that it wasrecognized by the anti-FLAG monoclonal antibody M1(Sigma). We also used an N-terminal fragment of TorpedoColQ, with or without its PRAD domain (Fig. 1C).

Transfection in COS Cells—Plasmids were transfected inCOS cells with the DEAE-dextran method, as described previ-ously (9), using 2 �g of vector DNA encoding the catalytic sub-unit AChET and various amounts of vector DNA encodingPRiMAmutants, as specified, per 60-mmdish. For comparison,we also used an N-terminal fragment of Torpedo marmorataColQ (Qstp69). In each series of transfections, we completed theamount of vector encoding PRiMA mutants with a vectorencoding a noninteracting protein, the N-terminal domain ofColQ from which the PRAD interaction motif was deleted(�(28–44)-Qstp69) (11), so that the total amount of vectorremained constant, to avoid changes in the synthetic capacity ofthe 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 toinactivate serum cholinesterases. The medium and the cellswere 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 membrane-permeant inhibitor soman (pinacolyl methylphosphonofluori-date) at 5 � 10�7 M for 30 min, as described previously (22).After extensive washing, the recovery of AChE activity wasdetermined by collecting cells at various times in fresh culturemedium at 37 °C. Secretion of newly synthesized AChE onlyresumed after about 150–180 min, and during that period therecovery of cellular activity reflected a balance between neosyn-thesis and intracellular degradation.Cell Extracts—Intracellular and membrane-bound AChE

was extracted for 15 min at 20 °C in a TMg buffer (1% TritonX-100, 50 mM Tris-HCl, pH 7.5, 10 mM MgCl2) containing 25mM benzamidine, followed by centrifugation for 10 min at13,000 rpm at 4 °C. The culture medium containing thesecreted enzyme was also centrifuged at 13,000 rpm for 10 minto remove cell debris before analysis.Enzyme Assays—AChE activity was determined with the col-

orimetric method of Ellman et al. (26) at room temperature.The reaction wasmonitored at 414 nmwith a LabsystemsMul-tiskan RC automatic plate reader (Helsinki, Finland); the opti-cal density was recorded at 20-s intervals over a period of 10min. Alkaline phosphatase and �-galactosidase from Esche-richia coli were assayed with the chromogenic substratesp-nitrophenyl phosphate and o-nitrophenyl galactoside,respectively.Sedimentation and Electrophoresis Analyses—Centrifuga-

tion in 5–20% sucrose gradients (50 mM Tris-HCl, pH 7.5, 20mMMgCl2, in the presence of 1% Brij-96 or 0.2% Triton X-100)was performed in a Beckman SW41 rotor, at 36,000 rpm, for17 h 30 min at 6 °C. Approximately 40 fractions were collected,and AChE activity was measured with the Ellman colorimetricassay, allowing the determination of the sedimentation coeffi-cients of the different molecular forms, and of their relativeactivities. The gradients containedE. coli�-galactosidase (16 S)and alkaline phosphatase (6.1 S) as internal sedimentationstandards. Amphiphilic molecules are characterized by the factthat they interact with detergent micelles; they generally sedi-ment slower in the presence of Brij-96 than of Triton X-100.

Assembly of AChE Tetramers by PRiMA

3488 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 282 • NUMBER 6 • FEBRUARY 9, 2007

Electrophoresis in nondenaturing polyacrylamide gels wasperformed as described by Bon et al. (27). The gels contained0.25% Triton X-100 with or without 0.05% deoxycholate; elec-trophoresis was performed in a refrigerated apparatus under 40V/cm for 2 h. Enzymatic activity was revealed by the histo-chemical method of Karnovsky and Roots (28). This methodallows a rapid qualitative comparison of up to 20 samples in asingle gel. In charge shift electrophoresis, the electrophoreticmigration of amphiphilic molecules was accelerated in thepresence of sodium deoxycholate, when compared with migra-tion in the presence of the neutral detergent Triton X-100.Effect of Synthetic Peptides on Oligomerization of AChET

Subunits—Synthetic peptides corresponding to the interactionmotif of PRiMA (RP4LP10 and RP4LP10RL) were synthesized by

the Merrifield solid phase methodin an Applied Biosystems 431Aautomated peptide synthesizer,with small scale 9-fluorenylme-thoxycarbonyl (Fmoc) chemistry(29). Protected amino acids werepurchased fromAppliedBiosystems(Foster City, CA) (30). Crude pep-tides were purified by reverse phasehigh pressure liquid chromatogra-phy on a Vydac C18 column (5 �m,250 � 10 mm2), using appropriateacetonitrile gradients containing0.1% trifluoroacetic acid.A 10�2 M solution of peptide in

Tris-HCl, pH 8 (1 M), was addedwith fresh medium to transfectedCOS cells expressing only AChETsubunits, at a final concentration of10�4 M. Peptides were also added tocell homogenates, at a final concen-tration of 10�4 M, and incubatedovernight at 20 or 37 °C. In somesamples, a synthetic t peptide(WAT) was added at 2 � 10�4 M.The oligomeric state of AChE wasthen analyzed by nondenaturingelectrophoresis, after dilution 2-foldin buffer containing 1% TritonX-100.

RESULTS

PRiMA Mutants—Fig. 1A showsthe primary sequence of mousePRiMA and the structure of mutantsused in this study. PRiMA consists ofa signal peptide (residues�35 to�1),an extracellular N-terminal domain(residues 1–53) that includes fourcysteines, a proline-rich motif and aputativeN-glycosylation site, a trans-membrane domain (residues 54–78),and two possible cytoplasmic C-ter-minal domains that define PRiMA I

and PRiMA II. We studied the interaction of AChET subunitswith the extracellular domain of PRiMA, usingmutants lackingthe transmembrane and cytoplasmic domains (Fig. 1B). First,we introduced stop codons at various positions (n), producingproteins Pstpn. Second, we deleted most of the N-terminalregion preceding the prolines, in mutants designed as �N; tomaintain a correct signal peptide cleavage site, we conservedthe first or the last residue of this region (Glu-1 and Arg-20,respectively). We also assessed the possible influence of elec-trostatic effects, bymutating the residue preceding the prolinesby other charged or uncharged residues (Asp, Lys, or Ala).Finally, we analyzed the role of the leucine located within thepolyproline region (Leu-25), by replacing it with a proline, andby adding a leucine at position 31, with or without the original

FIGURE 1. Structure of PRiMA and of mutants used in this study. A, sequence of PRiMA, numbered fromthe first residue of the mature protein. The putative signal peptide is shown in lightface letters (�35 to �1),the extracellular domain in boldface letters (residues 1–53), the transmembrane domain in boldface italics,and the cytoplasmic domain in lightface italics. C-ter. I and C-ter. II represent the cytoplasmic domains of themajor and minor splice variants of PRiMA. In the extracellular domain, the putative N-glycosylation site isindicated by an asterisk and the cysteines are underlined. B, structure of mutants. All mutants possess the signalpeptide (not shown) and fragments of the extracellular domain but not the transmembrane and cytoplasmicdomains. They are designated by the position of introduced stop codons (e.g. Pstp54 contains the entire extra-cellular domain, residues 1–53). The deletion of the region preceding the polyproline stretches is indicated by�. In these mutants, a single residue precedes the prolines, so as to maintain a proper cleavage site after thesignal peptide. This residue is indicated, e.g. arginine (R) in �N-R-Pstp54. Mutants in the proline-rich region areindicated by the distribution of prolines and leucine residues (e.g. P4LP10 in the case of the wild type). Theinteraction motif (RP4LP10RL) corresponds to mutant �N-R-Pstp38. C, structure of the N-terminal fragment ofTorpedo ColQ (Qstp69) (the signal peptide is not shown). The underlined fragment corresponds to the PRADinteraction domain. It was deleted in �[28 – 44]Qstp69.



leucine. Thus the original motif P4LP10 was changed to P15,P10LP4, and P4LP5LP4.Formation and Secretion of Soluble Complexes with Truncated

PRiMAMutants, Lacking the Transmembrane Domain—PRiMAand its different mutants were expressed in COS cells togetherwith AChET subunits (also called T subunits). To evaluate theformation of heteromeric complexes, we analyzed the totalAChE activity of the cellular extracts and of themedium, aswellas their composition in molecular forms, using both sedimen-tation and nondenaturing electrophoresis. It should be notedthat allmolecular forms of AChE, which differ in their degree ofoligomerization, with or without associated proteins, possessthe same catalytic activity per active site, so that the level ofAChE activity reflects the number of AChET subunits, inde-pendently of the composition in molecular forms.Fig. 2 illustrates the molecular forms obtained by expressing

AChET subunits alone, with full-length PRiMA, and with itsextracellular fragment, Pstp54. As a control, we used an N-ter-minal fragment of ColQ that contains the PRAD (residues28–44), without the collagenous and trimerization domains(Qstp69) (Fig. 1C). When expressed alone or in the presence of anoninteracting fragment of ColQ from which the proline-richmotif was deleted (�(28–44)-Qstp69), T subunits producedmostly amphiphilicmonomers (T1) and dimers (T2) (27), with asmall proportion of nonamphiphilic tetramers sedimenting at10.5 S, which are thought to be homomeric (T4), i.e. with noassociated endogenous noncatalytic component (Fig. 2A).Monomers and dimers are not readily resolved in the sedimen-tation profiles, andwe therefore quantified their sum (T1�T2).The proportion of T4 tetramers is markedly higher in themedium than in the cell extract, indicating that they aresecreted more efficiently than monomers and dimers, asobserved previously (9).Co-expression ofT subunitswith full-length PRiMA induced

the formation of membrane-bound T4-PRiMA complexes,expressed at the cell surface as demonstrated by immunofluo-rescence (not shown). These heteromeric complexes wererecovered in the cell extract after solubilization in the presenceof 1% Triton X-100; unlike homomeric tetramers (T4), whichsediment at 10.5 S regardless of the presence or absence ofdetergents, the PRiMA-associated tetramers are amphiphilic,and their sedimentation is influenced by detergents: they sedi-ment at 9.8 S in the presence of Triton X-100 and 9 S in thepresence of Brij-96. This was also shown by the fact that they

FIGURE 2. Molecular forms of AChE produced and secreted by transienttransfection of COS cells with AChET expressed alone, with full-lengthPRiMA, and with truncated PRiMA, reduced to its extracellular domain(Pstp54). A, representative sedimentation profiles, obtained from gradientscontaining 1% Brij-96, normalized to the same total AChE activity, indicatingthe relative proportions of monomers (T1), dimers (T2), and tetramers,

associated or not with PRiMA or Pstp54. Profiles corresponding to cell extractsare shown in the top panel, and to the medium in the middle panel. E, AChETexpressed alone; f, AChET expressed with PRiMA; Œ, AChET expressed withPstp54. In general, the relative proportion in the medium, compared with cellextracts, increases with the degree of oligomerization, indicating that secre-tion is facilitated by oligomerization. Soluble nonamphiphilic tetramers (T4)and heteromeric complexes (T4-Pstp54) sediment around 10.5 S, whereasamphiphilic T4-PRiMA complexes, containing the transmembrane domain ofPRiMA, are retarded by their interaction with the detergent (Brij-96) and sed-iment around 9 S. B, migration of AChE forms from cell extracts and culturemedia in nondenaturing electrophoresis. In the profiles corresponding to co-transfected cells (AChET with PRiMA and AChET with the extracellular domainof PRiMA, Pstp54), the simple arrows indicate nonamphiphilic AChE tetramericforms; the arrow with an asterisk indicates the amphiphilic tetramer associ-ated with a complete PRiMA protein. c indicates cellular extract; m indicatesmedium.



migratedmore slowly thanT4 tetramers in nondenaturing elec-trophoresis (Fig. 2B). In addition, the medium contained anincreased level of soluble molecules sedimenting at 10.5 S, sug-gesting that they correspond to nonamphiphilic tetramers.When AChET subunits were co-expressed with an N-flaggedPRiMA, these secreted tetrameric molecules were recognizedby an anti-FLAG antibody (M1), as shown by retardation oftheir migration in nondenaturing electrophoresis (Fig. 3), dem-onstrating that they contain an N-terminal fragment of PRiMAthat might be produced by proteolysis, either intracellularly orat the cell surface.Co-expression of AChET subunits with the Pstp54 mutant,

corresponding to the extracellular domain of PRiMA, inducedthe formation of a soluble AChE form, sedimenting at 10.5 S,and migrating as an AChE tetramer in nondenaturing elec-trophoresis (Fig. 2, A and B). This form, which could not bedistinguished from homomeric T4 tetramers by sedimenta-tion, corresponds to AChET tetramers associated with thePstp54 protein (T4-Pstp54). The fact that the ratio of T4-Pstp54to T1 � T2 is higher in the medium than in the cell extractshows that they are preferentially secreted, as observed forhomomeric tetramers T4.The formation of soluble T4-Pstp54 complexes demonstrates

that the amphiphilic character of T4-PRiMA complexes is dueto the transmembrane domain of PRiMA and confirms thatAChET subunits interact with a peptidic motif located in theN-terminal extracellular region of PRiMA, in agreement with aprevious study (16).Effect of Progressive C-terminal Deletions in the Extracellular

Domain of PRiMA on the Recruitment of AChET Subunits—Weexamined the interaction between AChET subunits andPRiMA mutants in which stop codons were introducedupstream of position 54: Pstp46, Pstp43, Pstp39, Pstp38, Pstp37,Pstp36, Pstp33, and Pstp31 (see Fig. 1B). We studied the AChE

activity and molecular forms pro-duced in COS cells expressing aconstant amount of AChET sub-units (2 �g of vector DNA per60-mm culture dish), togetherwith varying amounts of eachmutant.The proportion of AChET tet-

ramers increased in the cells and inthe medium with the amount ofassociated noncatalytic protein, asillustrated for Pstp33, Pstp54, andQstp69 in Fig. 4. This proportion wasfound to plateau at similar values forPstp54 and Qstp69 but at a muchlower level for Pstp33.Fig. 5 illustrates the effect of the

Pstpnmutants on the cellular activityand on the rate of secretion, with afixed amount of plasmid encodingthe Pstpn mutants (2 �g of DNAencoding AChET and PRiMAmutant, each, per 60-mm culturedish). The level of cellular activity

FIGURE 3. Secreted AChE tetramers induced by co-transfection of AChETsubunits with PRiMA contain an N-terminal fragment of PRiMA. The elec-trophoretic migration of AChE tetramers secreted by COS cells expressingAChET subunits with an N-FLAG-PRiMA was retarded, in nondenaturing gels,after incubation with the monoclonal anti-FLAG antibody M1. AChE tetram-ers are indicated by arrows and the retarded component by an asterisk.

FIGURE 4. Variation of the proportion of AChE tetramers with the amount of PRiMA mutants. COS cells weretransfected with a fixed amount of DNA encoding AChET subunits (2 �g per 60-mm culture dish) and the indicatedamount of DNA encoding PRiMA mutants, or the N-terminal region of ColQ (Qstp69), complemented with DNAencoding a noninteracting protein, so that the total amount of vector was kept constant to avoid modifications inthe protein synthesis capacity of the cells. The proportion of tetramers, associated or not with Pstpn proteins (T4 �T4-Pstpn) was determined from sedimentation profiles, as in Fig. 2. The figure illustrates the results obtained for Pstp31and Pstp54 mutants, producing high or low proportions, respectively, of T4-Pstpn complexes. The curves obtainedwith all other mutants were very similar in shape, differing mostly by the maximal levels of tetramers; co-transfectionwith 2 �g of DNA/dish encoding the various mutants was used in most subsequent experiments.





varied very little when AChET subunits were co-transfectedwith the various PRiMAmutants. Secretion increased by about30% when AChET subunits were co-transfected with Qstp69,indicating that they were partially rescued from intracellulardegradation by the formation of T4-Qstp69 complexes (notshown), but reduced by about 50% when they were co-trans-fected with Pstp31. The secreted activity gradually increasedwith the longer mutants Pstp33 and Pstp36, reaching the approx-imate 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 truncatedPstpn mutants, as well as with Qstp69, indicating that they inter-acted with all mutants, even the shorter ones, such as Pstp33 orPstp31, which produced only minimal levels of cellular orsecreted T4-Pstpn form. The variations observed in the level ofsecreted activity appeared correlated with the proportion ofheteromeric complex in the medium, as shown in Fig. 5C.Remarkably, this correlation includes all the truncated PRiMAmutants analyzed in this study, and also Qstp69. Fig. 5D showsthat the secretion of T4-Pstpn complexes appears proportionalto their cellular activity, except for T4-Pstp36, which was moreefficiently secreted, and formutants containingAla, Asp, orGluresidues preceding the proline-rich segment, which are lessefficiently secreted.The fact thatAChE activitywas reduced, both in the cells and

in the medium, when AChET subunits were co-expressed withshort truncated PRiMA mutants such as Pstp31 suggests thatthey were more degraded than when expressed alone. This wasverified by following the initial rate of recovery of AChE activ-ity, during the 2 h after irreversible inhibition, i.e. before secre-tion of the newly synthesized enzyme; this rate represents thebalance between neosynthesis and intracellular degradation.The rate of neosynthesis must be identical when AChET sub-units are expressed with a noninteracting protein and with dif-ferent PRiMA mutants. We found that the rates of recoveryvaried in the order AChET � �(28–44)-Qstp69 � AChET �Pstp46 � AChET � Pstp33, indicating that Pstp33, and to a lesserdegree Pstp46, induced some degradation of newly synthesizedactive AChET subunits.The ratio of secreted to the cellular activity of each molec-

ular form of AChE can be considered as an index of its secret-ability; Fig. 6 shows that this ratio remained essentially con-stant for monomers and dimers (T1 � T2), but varied markedlyfor AChE tetramers associated with Pstpn proteins. This indi-cates that the secretion of the complexes depended on thelength of the PRiMA fragment, especially between Pstp31 andPstp36, confirming that complexes formed with the shorter

PRiMA mutants were not efficiently secreted. In addition, thefact that this ratio varied in a nonmonotonic manner as a func-tion of the length of themutants from 36 to 54 suggests that theC-terminal residues of the Pstpn proteinsmay either facilitate orreduce secretion.The Pstp38 mutant seemed to produce a maximum of

secreted heteromeric complexes, whereas Pstp36 appeared opti-

FIGURE 5. Effect of the PRiMA mutants on cellular and secreted AChE activity and molecular forms. COS cells were co-transfected with 2 �g of DNAencoding AChET and 2 �g of DNA encoding PRiMA mutants. A, sequence and numbering of the extracellular domain of PRiMA; the cysteines are underlined andthe putative N-glycosylation site is indicated by an asterisk. B, the cellular and secreted AChE activities corresponding to the different mutants were normalized,for each experiment, to those of AChET subunits expressed alone, taken as 100%. The proportions of the molecular forms were determined from sedimentationprofiles. The data corresponding to C-terminal truncated mutants 1–9 are the means of 4 –7 independent transfection experiments, � S.E.; those for othermutants correspond to a representative experiment. The lightly hatched boxes correspond to the activity of monomers and dimers (T1 � T2), and the darklyhatched boxes to tetramers (homomeric tetramers T4 and heteromeric T4-Pstpn complexes). If homomeric T4 tetramers are present in the same proportion to T1and T2, as in the case of AChET subunits alone, they represent a minor species and can be generally neglected. In mutants 16 –18, the deletion of the N-terminalregion is associated with mutations in the proline-rich segment, as indicated in parentheses. Note that a mutant reduced to the RP4LP10RL motif (expressedas mutant �N-R-Pstp38) induced the production and secretion of essentially the same amount of complex as the complete extracellular domain (Pstp54mutant). C, correlation between the total secreted AChE activity and the proportion of tetramers in the medium. The numbers identify the mutants, asindicated in the left column of B. D, correlation between the percentages of T4 complexes in secreted and cellular AChE.

FIGURE 6. Ratio of secreted to cellular activity of AChE forms. The secretedactivity reflects the rate of secretion, whereas the cellular activity, 3– 4 daysafter transfection, approximately corresponds to a steady state value; there-fore, these activities are not comparable, but their ratio may be considered asan index of the efficiency of secretion for each AChE form. These ratios werearbitrarily normalized to 100 for AChE tetramers produced in co-transfectionwith the complete N-terminal domain of PRiMA, Pstp54. For monomers anddimers (T1� T2), the ratio remained approximately constant, in agreementwith the fact that their structure did not change.



mal for the secretability of such complexes. The differencesobserved between Pstp36, Pstp37, Pstp38, and Pstp39 show that theresidues located immediately downstreamof the prolines (RLL)influence the formation and the secretion of the T4-Pstpn com-plexes. However, replacement of Arg by an alanine or a glu-tamic acid in mutant Pstp37 (R36A-Pstp37 and R36E-Pstp37) orreplacement of RLL by alanines in mutant (RLL/AAA-Pstp39)had essentially no effect on recruitment of AChET subunits,indicating that the charge of these residues is not important.It is interesting that Pstp46 and Pstp54 produced very similar

results, because Pstp54 possesses a putative N-glycosylation siteand a cysteine that are removed in Pstp46, suggesting that thesetwo elements do not interfere, positively or negatively, with theinteraction betweenPRiMAandAChET subunits in transfectedCOS cells. The lack of influence of these elements was con-firmed bymutating Cys-48 to a serine in Pstp54 and by suppress-ing the glycosylation site by replacing Thr-46 by an alanine;these mutations did not affect the production and secretion ofT4-Pstpn complexes (not shown).Influence of the Region Preceding the Polyproline Stretches—To

assess the possible role of the peptidic region that precedes thepolyproline stretches of PRiMA, we deleted it in Pstp46 and Pstp54.Tomaintain a proper cleavage site between the signal peptide andthe mature protein, the prolines stretches were preceded by anarginine (as in the wild type) or by other charged or neutralresidues (Lys, Glu, Asp, and Ala), as shown in Fig. 1B.With an arginine preceding the prolines, we found that dele-

tion of the N-terminal fragment (residues 1–19) from mutantsPstp46 or Pstp54 (�N-R-Pstp46 and �N-R-Pstp54) slightly reducedthe amount of cellular and secreted complexes (Fig. 5B). Thefact that the two deleted mutants produced the same effectconfirms that residues 45–53 do not influence the assembly orthe trafficking of the complexes.We then examined a possible effect of a charged or neutral

residue preceding the prolines, in mutants �N-R-Pstp46, �N-K-Pstp46, �N-A-Pstp46, �N-D-Pstp46, and �N-E-Pstp46 (Fig. 5B andFig. 6). We found that the amount of secreted AChE tetramersis higher with a neutral or acidic residue but that their secret-ability appears lower, as indicated by the ratio of secreted tocellular activities, suggesting that this residue influences theefficiency of secretion (Fig. 5D).

It was surprising that the secretion of AChE complexes waslower with a positively charged residue upstream of the pro-lines, because this residue is an arginine in thewild type PRiMA.However, when we replaced this arginine by a glutamic acid inPstp46, we found no modification of the yield of T4-Pstp46 com-plexes, indicating that this charge effect is negligible when theN-terminal segment (residues 1–19) is present (not shown).Mutations in the Proline-rich Region—PRiMA contains two

groups of successive prolines, separated by a leucine residue(P4LP10). To determine whether this leucine is required for theinteraction with AChET subunits, we mutated it to a proline,starting from the �N-E-Pstp46 mutant. The production andsecretion of AChE tetramers remained unchanged with theresulting mutant, noted �N-(P15)-Pstp46. However, the produc-tion of complexes was significantly reduced by displacement ofthe leucine to a symmetrical position in the proline stretches(P10LP4) and even more by addition of a second leucine

(P4LP5LP4), as shown in Fig. 5B. These mutations had littleeffect on the secretability of the complexes (Fig. 6). Thus, therecruitment of AChET subunits into heteromeric T4-Pstp46complexes is weakened by an interruption of the prolinestretches by a leucine, and this suggests that a succession of 10prolines is sufficient to ensure a maximal efficiency.A Proline-rich Peptidic Motif, Sufficient for Recruitment of

AChET Subunits—In the preceding sections, we showed that itis possible to maintain an efficient recruitment of AChET sub-units into T4-Pstpn hetero-oligomers after deletion of theN-ter-minal region of PRiMAprecedingArg-20 and of theC-terminalregion following Leu-37. Therefore, the 20–37 peptidic motif,which contains the polyproline stretches (RP4LP10RL), may besufficient for this interaction. To verify this conclusion, wecombined N-terminal and C-terminal deletions in the �N-R-Pstp38 mutant. This mutant was co-expressed with AChET sub-units, and compared with Pstp54, �N-R-Pstp38, and Pstp38, allexpressed at 2 �g of vector/dish (Fig. 5). All mutants inducedthe production of T4-Pstpn, confirming the effects observedwith separate deletions of the N-terminal and C-terminalregions. Thus, the 18-residue peptide 20–37, derived fromPRiMA, possesses the capacity to assemble four AChET sub-units into stable T4-Pstpn complexes in a manner similar to thatof the full-length extracellular domain of PRiMA (Pstp54).It is noteworthy that deletion of the N-terminal region, in

�N-R-Pstp54 and �N-R-Pstp38, increased the ratio of secreted tocellular T4-Pstpn by about 25%, compared with the correspond-ing mutants Pstp54 and Pstp38, suggesting that secretion wasfacilitated by the N-terminal deletion. Deletion of the C-termi-nal region, in Pstp38 and �N-R-Pstp38, compared with Pstp54 and�N-R-Pstp54, also appeared to increase secretability.Assembly of AChET Tetramers by Synthetic Peptides—We

synthesized the PRiMA-derived peptides RP4LP10 andRP4LP10RL, to determine whether they could induce theassembly of AChET monomers and dimers into tetramers. In afirst series of experiments, the peptides were added at a finalconcentration of 10�4 M to COS cells expressing only AChETsubunits, during the transfection process and/or in the culturemedium. The cells and themediumwere analyzed by nondena-turing electrophoresis after 3 days, as indicated above. Thisshowed a clear increase in the proportion of AChET tetramers,most probably associated with the peptides (Fig. 7A). Thisresult indicates that the peptides were able to penetrate thebiosynthetic compartments of the living cells.In a second series of experiments, homogenates of COS cells

expressing only AChET subunits were prepared without deter-gent and incubated overnight at 37 °C with the peptides (10�4

M). As shown in Fig. 7B, both peptides induced the assembly oftetramers, under these acellular conditions. When a synthetic tpeptide (WAT), at 2� 10�4 M, was added to the cell homogenatetogether with the PRiMA-derived peptides, it reduced markedlythe recruitment of AChET monomers and dimers into tetramers,indicating a direct interaction between these peptides.

DISCUSSION

The N-terminal Extracellular Domain of PRiMA AssemblesAChET Subunits into Soluble T4-Pstpn Complexes—The assem-bly of AChET tetramers with the transmembrane protein



PRiMA produces membrane-bound complexes, both in thenervous system (14–18, 31) and in muscles (19). In addition,the availability of PRiMA seems to limit the stabilization as wellas the functional localization of AChE in the mammalian brain(18). It is therefore important to analyze the interactionbetween PRiMA and AChET subunits. In this study, weattempted to define the peptidicmotif of PRiMA that is respon-sible for its quaternary association with AChET subunits, byusing deletions upstream, downstream, and within the proline-rich segment.We mostly used truncated PRiMA mutants, lacking the

transmembrane and cytoplasmic domains (Pstpn with a stopcodon at position n), which were co-expressed with AChETsubunits in transiently transfectedCOS cells, producing solublecomplexes (T4-Pstpn), whichwere secreted instead of remainingattached to the cell membrane.Truncated PRiMA Mutants Recruit AChET Subunits into

Heteromeric Complexes, Following Either Secretion or Degrada-tion Pathways—We have shown in a previous study that a sig-nificant fraction of AChET subunits is degraded intracellularlythrough the ERAD process, whereas another fraction issecreted (20, 21). Therefore, the level of intracellular AChEactivity reflects a balance between the rate of synthesis and thecombined rates of degradation and secretion.When COS cells were transfected with a fixed amount of

plasmid encoding AChET subunits, together with variousamounts of plasmid encoding PRiMAmutants, a variable frac-

tion of the subunits was integrated in T4-Pstpn complexes, bothin the cells and in the medium. It was surprising to find thatalthough the proportion of T4-Pstpn complexes varied widely, itappeared to plateau for similar amounts of plasmid DNA, sug-gesting that all mutants presented a similar apparent “affinity”for AChET subunits. This paradox can be resolved by assumingthat even when the observed proportion of complexes was verylow, mutants such as Pstp31 did in fact interact with AChETsubunits, as indicated by amarked reduction in the secretion ofT1�T2, but produced rapidly degraded complexes.With longermutants, the level of secreted T4-Pstpn complex progressivelyincreased. Thus, the variations observed in the amount ofsecreted complex appear to reflect a competition betweensecretion and degradation. Depending on its organization, eachtype ofT4-Pstpn complexwould be orientedwith different prob-abilities toward the secretion pathway or the degradation path-way. This suggests that the rate of secretion depends criticallyon the structure of the complex.It is a classical notion that proteins destined to function as a

multisubunit complex may be retained in the endoplasmicreticulum by the quality control system of the cell, unless theyare appropriately associated with their partners (32). This canbe rationalized by the fact that they expose hydrophobic sur-faces in their unassembled state but not in the complex. Thisseems to be the case for Torpedo AChET subunits, which aresecreted very little when expressed alone in COS cells but canbe secreted when co-expressed with an N-terminal fragment ofColQ (Qstp69) (5). We also observed here that rat AChET sub-units can be rescued byQstp69 fromdegradation through ERAD(20). This is consistent with the fact that their physiologicalfunction depends on association with an anchoring protein,ColQ or PRiMA.The fact that short truncated PRiMA mutants increase the

intracellular degradation of AChET subunits is more unusual.However, Ulloa-Aguirre et al. (33) recently suggested that someproteins induce an inappropriate conformation of normallyexternalized molecules and thus target them toward degrada-tion by the cellular quality control and proposed to call thishypothetical process “protein shipwrecking” (33). Here wepresent evidence that some PRiMAmutantsmay behave in thismanner, and that there is in fact a continuous spectrumbetweenmutants that induce degradation and those that rescueAChET subunits from ERAD and promote their secretion asheteromeric complexes.Because the assembly of a complex between proteins

occludes interaction surfaces, rather than exposing hydropho-bic zones, how could the association with short PRiMAmutants such as Pstp31 induce the degradation of AChET sub-units? Although we have presently no experimental evidence,we may propose a hypothetical explanation. We have demon-strated previously that aromatic (but not hydrophobic) residuesof theC-terminal t peptide of AChE play a crucial role to inducethe degradation of AChET subunits by ERAD (22). It is possiblethat a group of aromatic residues remains exposed in the com-plexes formed with short PRiMA mutants, engaging theirtranslocation into the cytoplasm and degradation by protea-somes, whereas these residues aremasked in complexes formed

FIGURE 7. Synthetic PRiMA peptides induce oligomerization of AChETsubunits in vitro. A, COS cells were transfected with AChET, and the syntheticpeptides RP4LP10 and RP4LP10RL were added with fresh culture medium after2 days. The cells were extracted 24 h later and analyzed by nondenaturingelectrophoresis, as well as the medium. B, peptides were added at a concen-tration of 10�4

M to an homogenate of COS cells expressing only AChET sub-units, and incubated overnight at 37 °C. In some samples, a synthetic t pep-tide (WAT) was added at 2 10�4

M, as indicated. The homogenate was thenanalyzed by nondenaturing electrophoresis in the presence of detergents(see “Experimental Procedures”). The peptides induced the formation of sta-ble AChET tetramers (indicated by arrows); this effect was reduced by additionof the t peptide (WAT).



with longer mutants, as demonstrated by crystallography in acomplex of four t peptides with the PRAD of ColQ (12).Definition of a Sufficient Interaction Motif (PRAD) in

PRiMA—The efficiency of secretion of AChE oligomersincreases with their degree of oligomerization (9). The secret-ability of heteromeric T4-Pstpn complexes, indicated by theratio of secreted to cellular activity, was variable; it is notewor-thy that it was increased by deletion of peptidic regions locatedupstream or downstream of the polyproline stretches. A maxi-mum was obtained by removing all residues following the pro-lines (Pstp36); conversely, it was reduced for mutants in whichthe N-terminal region was deleted (�N) and the proline-richsegment was preceded by residues Ala, Glu, or Asp, as opposedto basic residues Arg or Lys.Most of the N-terminal and C-terminal regions flanking the

polyproline stretches could be deleted without suppressing thecapacity of P mutants to engage AChET subunits into T4-Pstpncomplexes. This capacity in fact appeared maximal when mostof the C-terminal region was deleted, leaving only two residues(RL) after the prolines, in Pstp73. However, it is not certain thatthese residues play a specific role in the association with t pep-tides, because replacement of the three residues following theprolines by alanines had essentially no effect (in RLL/AAA-Pstp39 versus Pstp39). Even though we have no direct evidencethat Pstp54 is N-glycosylated under our experimental condi-tions, we found that the presence of a putative N-glycosylationsite, located at position 44, did not influence the association ofPRiMA mutants with AChET subunits. Similarly, the cysteinelocated at position 48 did not affect the formation of T4-Pstpncomplexes.The PRAD of ColQ is normally disulfide-linked to two of the

t peptides, while the other two are disulfide-linked together (5).However, we found previously that the formation of hetero-meric complexes with the N-terminal region of ColQ couldoccur without the two adjacent cysteines preceding the PRAD,indicating that disulfide bonds between ColQ and two AChETsubunits are not required (5, 11). The present results suggest asimilar conclusion in the case of PRiMA, because the formationof complexes was only slightly reduced (20% or less) by deletionofmost of theN-terminal region,which contains four cysteines,located at positions 6, 13, 17, and 19. This does not rule out thepossibility that some of these cysteines may be involved inintercatenary disulfide bonds between PRiMA andAChET sub-units, as shown in membrane-anchored AChE tetramers frombovine brain (14, 15, 34), and thus stabilize the complex.PRiMA contains two polyproline stretches, separated by a

leucine (P4LP10). We found that this leucine is not required forthe interaction and could be replaced by a proline, resulting in asingle suite of 15 prolines (P15). The secretion of T4-Pstpn com-plexeswas decreased by displacement of the leucine (P10LP4) orinterruption of the proline stretches by a second leucine(P4LP5LP4), and it was almost totally abolished when 5 prolineswere deleted in Pstp31 (P4LP5). Thus, the formation of secretablecomplexes requires the presence of a minimal number of unin-terrupted prolines.Our deletion experiments converged on the idea that the

20–37 segment of PRiMA (RP4LP10RL) may be able to recruitAChET subunits nearly as efficiently as the entire extracellular

domain of PRiMA, and this was confirmed by expressing this18-residue peptide as mutant �N-R-Pstp38. In fact, addition ofthe synthetic peptides RP4LP10 and RP4LP10RL to COS cellsexpressing only AChET subunits induced the recruitment ofmonomers and dimers into stable tetramers. Moreover, weobtained the same result by incubating a cell homogenate withthe peptides, and we observed that the assembly of AChET tet-ramers was partially blocked by addition of a synthetic t peptide(WAT). This proves that the PRiMA-derived and t peptidesspontaneously interact in vitro, as shown previously for aColQ-derived PRAD peptide (12), and opens the way to the forma-tion, crystallization, and structural analysis of the core complexthat forms the basis of the association between PRiMA andAChET. It is noteworthy that the PRADof ColQ is very efficientwith only 8 prolines (P3MFP5). This raises the intriguing possi-bility that the complexes of t peptides with the proline-richmotifs of ColQ and PRiMA might significantly differ in theirquaternary organization. Using short PRiMA-derived peptides,it may also be possible to shed some light on the structuraldifferences between complexes that pass or fail the quality con-trol in the secretory pathway.

Acknowledgments—We thank Dr. Noel Perrier and Dr. CinziaFalasca for helpful suggestions.

REFERENCES1. Sikorav, J. L., Duval, N., Anselmet, A., Bon, S., Krejci, E., Legay, C.,

Osterlund, M., Reimund, B., and Massoulie, J. (1988) EMBO J. 7,2983–2993

2. Taylor, P. (1991) J. Biol. Chem. 266, 4025–40283. Massoulie, J. (2002) Neurosignals 11, 130–1434. Massoulie, J., Bon, S., Perrier, N., and Falasca, C. (2005) Chem. Biol. Interact.

157, 3–145. Bon, S., Dufourcq, J., Leroy, J., Cornut, I., and Massoulie, J. (2004) Eur.

J. Biochem. 271, 33–476. Simon, S., Krejci, E., and Massoulie, J. (1998) EMBO J. 17, 6178–61877. Krejci, E., Coussen, F., Duval, N., Chatel, J. M., Legay, C., Puype, M.,

Vandekerckhove, J., Cartaud, J., Bon, S., and Massoulie, J. (1991) EMBO J.10, 1285–1293

8. Duval, N., Krejci, E., Grassi, J., Coussen, F.,Massoulie, J., and Bon, S. (1992)EMBO J. 11, 3255–3261

9. Bon, S., and Massoulie, J. (1997) J. Biol. Chem. 272, 3007–301510. Krejci, E., Thomine, S., Boschetti, N., Legay, C., Sketelj, J., and Massoulie,

J. (1997) J. Biol. Chem. 272, 22840–2284711. Bon, S., Coussen, F., and Massoulie, J. (1997) J. Biol. Chem. 272,

3016–302112. Dvir, H., Harel, M., Bon, S., Liu, W. Q., Vidal, M., Garbay, C., Sussman,

J. L., Massoulie, J., and Silman, I. (2004) EMBO J. 23, 4394–440513. Zhang, D., and McCammon, J. A. (2005) PLoS Comput. Biol. 1, 484–49114. Gennari, K., Brunner, J., and Brodbeck, U. (1987) J. Neurochem. 49, 12–1815. Inestrosa, N. C., Roberts, W. L., Marshall, T. L., and Rosenberry, T. L.

(1987) J. Biol. Chem. 262, 4441–444416. Perrier, A. L., Massoulie, J., and Krejci, E. (2002) Neuron 33, 275–28517. Boschetti, N., and Brodbeck, U. (1996) FEBS Lett. 380, 133–13618. Perrier, N. A., Kherif, S., Perrier, A. L., Krejci, E., Dumas, S., Mallet, J., and

Massoulie, J. (2003) Eur. J. Neurosci. 18, 1837–184719. Gisiger, V., Belisle, M., and Gardiner, P. F. (1994) Eur. J. Neurosci. 6,

673–68020. Belbeoc’h, S., Massoulie, J., and Bon, S. (2003) EMBO J. 22, 3536–354521. Belbeoc’h, S., Falasca, C., Leroy, J., Ayon, A., Massoulie, J., and Bon, S.

(2004) Eur. J. Biochem. 271, 1476–148722. Falasca, C., Perrier, N. A., Massoulie, J., and Bon, S. (2005) J. Biol. Chem.

280, 878–886



23. Mizushima, S., and Nagata, S. (1990) Nucleic Acids Res. 18, 532224. Kunkel, T. A., Roberts, J. D., and Zakour, R. A. (1987) Methods Enzymol.

154, 367–38225. Morel, N., Leroy, J., Ayon, A., Massoulie, J., and Bon, S. (2001) J. Biol.

Chem. 276, 37379–3738926. Ellman, G. L., Courtney, K. D., Andres, V., and Featherstone, R. M. (1961)

Biochem. Pharmacol. 7, 88–9527. Bon, S., Rosenberry, T. L., and Massoulie, J. (1991) Cell. Mol. Neurobiol.

11, 157–17228. Karnovsky, M. J., and Roots, L. (1964) J. Histochem. Cytochem. 12,

219–222

29. Fields, G. B., and Noble, R. L. (1990) Int. J. Pept. Protein Res. 35, 161–21430. Sigler,G.F., Fuller,W.D.,Chaturvedi,N.C.,Goodman,M., andVerlander,M.

(1983) Biopolymers 22, 2157–216231. Boschetti, N., Liao, J., and Brodbeck, U. (1994) Neurochem. Res. 19,

359–36532. Ellgard, L., and Helenius, A. (2003) Nat. Rev. Mol. Cell Biol. 3, 181–19133. Ulloa-Aguirre, A., Janovick, J. A., Brothers, S. P., and Conn, P. M. (2004)

Traffic 5, 821–83734. Perrier, A. L., Cousin, X., Boschetti, N., Haas, R., Chatel, J. M., Bon, S.,

Roberts,W. L., Pickett, S. R., Massoulie, J., Rosenberry, T. L., and Krejci, E.(2000) J. Biol. Chem. 275, 34260–34265



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