Quaternary Associations of Acetylcholinesterase
II. THE POLYPROLINE ATTACHMENT DOMAIN OF THE COLLAGEN TAIL*

(Received for publication, July 3, 1996, and in revised form, October 23, 1996)

Suzanne Bon , Françoise Coussen and Jean Massoulié Dagger

From the Laboratoire de Neurobiologie Moléculaire et Cellulaire, Unité CNRS 1857, Ecole Normale Supérieure, 46 rue d'Ulm, 75005 Paris, France

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
Acknowledgments
REFERENCES


ABSTRACT

In transfected COS cells, we analyzed the formation of heteromeric associations between rat acetylcholinesterase of type T (AChET) and various constructions derived from the NH2-terminal region of the collagen tail of asymmetric forms, QN. Using a series of deletions and point mutations in QN, we showed that the binding of AChET to QN does not require the cysteines that normally establish intersubunit disulfide bonds with catalytic subunits and that it essentially relies on the presence of stretches of successive prolines, although adjacent residues also contribute to the interaction. We thus defined a <UNL>p</UNL>roline-<UNL>r</UNL>ich <UNL>a</UNL>ttachment <UNL>d</UNL>omain or PRAD, which recruits AChET subunits to form heteromeric associations. Such molecules, consisting of one PRAD associated with a tetramer of AChET, are exported efficiently by the cells. Using the proportion of AChET subunits engaged in heteromeric tetramers, we ranked the interaction efficiency of various constructions. From these experiments we evaluated the contribution of different elements of the PRAD to the quaternary assembly of AChET subunits in the secretory pathway. The PRAD remained functional when reduced to six residues followed by a string of 10 prolines (Glu-Ser-Thr-Gly3-Pro10). We then showed that synthetic polyproline itself can associate with AChET subunits, producing well defined tetramers, when added to live transfected cells or even to cell extracts. This is the first example of an in vitro assembly of AChE tetramers from monomers and dimers. These results open the way to a chemical-physical exploration of the formation of these quaternary associations, both in the secretory pathway and in vitro.


INTRODUCTION

As shown in the preceding article (1), coexpression in COS cells offers a convenient method to analyze the formation of heteromeric molecules in which tetramers of acetylcholinesterase (AChE,1 EC 3.1.1.7) are associated with small binding proteins derived from the NH2-terminal region of the collagen component of collagen-tailed forms (for review, see Ref. 2). When expressed alone in COS cells, AChE subunits of type T (AChET) produce mostly monomers and dimers, with smaller proportions of tetramers and higher oligomers. In the presence of the binding proteins QN/stop and QN/HC, these subunits are recruited into tetramers that are associated with the binding domain and are efficiently secreted or attached to the cell surface by a glycolipidic anchor (GPI) (1).

In this study we used a series of deletions and point mutations in the QN sequence in an attempt to define the attachment domain that allows its interaction with rat AChET. We show that the cysteine residues are not required for interaction with AChET, and we narrow down the binding domain essentially to a stretch of successive prolines. Moreover, we show that polyproline itself can combine with AChET subunits, inducing their polymerization, mainly into tetramers, when added to transfected cells or to a cell extract.


EXPERIMENTAL PROCEDURES

Materials

All reagents were purchased from Prolabo (Paris, France) or from Sigma (St. Louis, MO, U. S. A.). PI-PLC from Bacillus thuringiensis was from Immunotech (Marseille, France). Poly-L-proline with a molecular mass of 1,000-10,000 (mean 8 kDa) and >30,000 (mean 40 kDa) was purchased from Sigma.

Site-directed Mutagenesis and Transfection in COS Cells

Expression vectors encoding the binding proteins QN/HC and QN/stop (QN/stop551) were described previously (1, 3). The structure of the proteins is illustrated in Fig. 1. Site-directed mutagenesis was performed with the single strand method (4). In the case of deletions, we used mutagenic 20-mer oligonucleotides consisting of 10 nucleotides complementary to each side of the deleted fragment. In the case of truncations, TGA stop codons were introduced to terminate the polypeptide chain. Transfection and culture of COS cells were performed as described in the preceding paper (1).


Fig. 1. Structure of the binding proteins and assembly a AChET tetramers; schematic representation of various constructions. Panel A, sequence of the chimeric QN/HC protein. The putative signal peptide is shown in thin letters, the QN domain in bold letters, and the HC domain in bold italics. These two domains are linked by two additional residues (GI), in thin italics, corresponding to a BamHI site introduced during the construction (3). The residues are numbered according to the original sequences of the Torpedo collagen subunit tQ1 for QN (12) and of the Torpedo AChEH subunit for HC (13). Mutations are indicated by letters below the sequence. Residues that were modified en bloc are shown in boxes. Deletions are indicated by lines and arrows above the sequence. Panel B, structure of QN/HC and QN/stop constructions. The sequence of the PRAD is indicated, and it is shown as dark boxes. The NH2- and COOH-flanking parts of the PRAD in QN are gray. The signal peptide and the HC domain are light. Deletions are shown as thin lines. The boxes corresponding to the signal peptide and to the HC region (GPI-addition signal) are not drawn to scale. The + and - signs indicate the capacity of each construct to assemble and bind a tetramer of AChET subunits.
[View Larger Version of this Image (41K GIF file)]


Analysis of AChE Forms

PI-PLC treatments, sedimentation and electrophoretic analyses, were performed as described (1).


RESULTS

Definition of the Proline-rich Attachment Domain (PRAD)

Deletions in the COOH-terminal Part of the QN Domain

We examined the effect of deletions in the COOH-terminal part of the QN domain on the binding of AChET subunits. For this purpose we used two strategies: introduction of stop codons at various positions in the Q sequence, producing QN/stop proteins of various lengths; or deletions of various extents in the chimeric QN/HC protein (Fig. 1).

When coexpressed with rat AChET subunits, QN molecules truncated at position 87 (QN/stop87) induced a significant increase in the proportion of cellular G4na form and more dramatically, a large increase in the released activity, where G4na became predominant over G2a and G1a (not shown). Thus, QN/stop87 was able to induce the formation of heteromeric tetramers, like the QN/stop551 protein, containing the complete QN domain, which we analyzed in the preceding paper (1). In contrast, introduction of a stop codon at position 76 totally abolished this capacity. In the case of QN/stop76, the pattern of AChE forms was identical to that of AChET subunits alone, as illustrated in nondenaturing electrophoresis (Fig. 2). We performed similar experiments in cotransfections with Torpedo AChET subunits; the cells released essentially no activity when Torpedo AChET was expressed alone or with QN/stop76, but coexpression with QN/stop87 induced the secretion of tetramers in the culture medium (not shown).


Fig. 2. Interaction of QN/stop76 and QN/stop87 with rat AChET. Cellular extracts and culture media of cells expressing rat AChET together with QN/stop76 or QN/stop87 were analyzed by nondenaturing electrophoresis, as indicated. The patterns obtained with QN/stop76 were identical to those obtained with AChET alone; QN/stop87 induced a secretion of G4na instead of G1a and G2a.
[View Larger Version of this Image (41K GIF file)]


Deletions in the QN/HC molecule are schematically shown in Fig. 1B. Deletion of COOH-terminal segments of the QN domain, extending up to positions 100 or 85 (QNDelta 100-110/HC, QNDelta 85-110/HC) did not abolish the production of a GPI-G4a form, as shown by the sedimentation profiles of the cellular enzyme and its sensitivity to PI-PLC (not shown). However, QNDelta 85-110/HC somewhat weakened this interaction (see below). In contrast, larger deletions extending into the proline-rich sequence that follows the two vicinal cysteines of QN (QNDelta 81-110/HC and QNDelta 76-110/HC) abolished the binding of AChET.

Together these deletion experiments showed that a large part of the COOH-terminal sequence of QN may be removed without compromising the binding of AChET subunits from rat or from Torpedo. They defined a boundary of the binding domain, between positions 81 (essentially no binding) and 87 (similar to the wild type).

NH2-terminal and COOH-terminal Deletions in the QN Domain

To assess the possible role of the peptidic sequence that precedes the pair of cysteines Cys70-Cys71 in the QN domain, we removed residues 46-69 in deletion Delta 1, leaving only the three residues that immediately follow the putative cleavage site of the signal peptide. In deletion Delta 2, we deleted residues 90-110 in the COOH-terminal part of QN. We introduced these deletions, separately and in combination, in the QN/HC and QN/stop proteins. All of these deleted molecules were able to combine with the rat AChET subunit. For example, QN/stop551 proteins carrying one or both deletions induced the secretion of G4na rather than G1a or G2a (not shown). In the case of QN/HC, the production of GPI-anchored G4a and the release of G4na were similar for the three deleted constructs and for the complete protein, as shown by nondenaturing electrophoresis (Fig. 3) and by sedimentation analyses (not shown). Surprisingly, the Delta 1 deletion actually increased the interaction with AChET, at nonsaturating doses of DNA encoding QN/HC or QN/stop (see below).


Fig. 3. Interaction of deleted QN/HC constructions with rat AChET. Extracts from cells expressing rat AChET together with QNDelta [46-69]/HC (Delta 1), QNDelta [90-533]/HC (Delta 2), and QNDelta [46-69]Delta [90-533]/HC (Delta 1Delta 2) were analyzed by nondenaturing electrophoresis. GPI-anchored G4a forms were produced in all cases, as shown in cell extracts by treatment with PI-PLC. The arrows indicate the conversion of slow migrating to faster migrating G4 molecules. The production of GPI-anchored AChE was accompanied by a marked decrease of the secreted G1a and G2a forms.
[View Larger Version of this Image (32K GIF file)]


These observations establish that at least part of the sequence located between residues 70 and 86 plays a critical role in the interaction of QN with the AChET catalytic subunits. The most prominent feature of this short peptidic sequence is the presence of two stretches of five and three prolines, separated by two residues, Met80-Phe81.

Are Disulfide Bonds Necessary for Association of QN with AChET Subunits?

The QN domain contains a pair of vicinal cysteines (Cys70-Cys71), which form disulfide bonds with cysteines located at position -4 of the COOH terminus of two AChET subunits, whereas the same cysteine residues form a disulfide bond between the other two subunits of the catalytic tetramer (11). To examine whether the formation of disulfide bonds between QN and AChET subunits was a necessary requirement for the assembly or stability of the hetero-oligomeric structure, we constructed mutants that lacked the vicinal cysteines. The double mutation C70G/C71S was introduced in the entire Q subunit and in the QN/HC chimeric protein. In both cases, the mutated proteins behaved exactly like those containing the pair of cysteines, producing hetero-oligomers with rat AChET subunits: collagen-tailed forms in the case of Q, GPI-anchored and released tetramers in the case of QN/HC (not shown).

Mutations in the Proline-rich Region; Comparisons of the Efficiency of Interaction with AChET; Construction of a Minimal Binding Domain

The different QN/HC constructions, when used at a nonsaturating dose of DNA (1 µg/dish), varied markedly in the yield of heteromeric tetramers (cellular G4a and lytic secreted G4na). We could thus distinguish four groups, in the following order: inactive (QNDelta 76-110/HC, QNDelta 81-110/HC), very weak (QN[Gly77-Pro80-Pro81-Gly83]/HC, QN[Gly77-Gly83]/HC, QN[Gly77]/HC), medium (QNDelta 85-110/HC, QN[Gly77-Pro80-Pro81]/HC, QN[Pro80-Pro81-Gly83]/HC), and good (QNDelta 100-110/HC, QN[Pro80-Pro81]/HC, QN[Gly83]/HC, QN/HC).

It was possible to obtain a more quantitative comparison, using the proportion of heteromeric AChE to total activity as an index of the efficiency of interaction, if we assume that the different constructs were expressed at the same level. (Note that they all contained the same signal peptide and cleavage site.) We thus show a ladder of the different QN/stop constructions, according to the proportion of G4na in the culture medium (Fig. 4). This shows that QN/stop85 was slightly but significantly less efficient than QN/stop87, which was itself less efficient than QN/stop551. The Phe85 and Phe86 residues therefore contribute to the binding of AChET subunits. The effect of the Delta 2(90-533) deletion also suggests that more distal residues participate in this interaction. In the case of the QN/HC constructs, it is possible that the interaction may depend on a sufficient distance between the binding domain and the GPI addition signal.


Fig. 4. Comparison of the efficiency of interaction between different QN/stop constructions and rat AChET. Cells were cotransfected with 5 µg of DNA encoding AChET and 1 µg of DNA encoding various QN/stop constructions (see Fig. 1). The proportion of G4na in the culture medium (R) was evaluated after 2 days from sedimentation profiles. The arrows indicate the effects of mutations, labeled after the original residues, as shown in the sequence. The sequence of the interaction domain (PRAD) and the position of the Delta 1 and Delta 2 deletions are shown in the lower part of the figure.
[View Larger Version of this Image (34K GIF file)]


To assess the possible importance of Met80 and Phe81, located between the two proline stretches, in the interaction with AChET subunits, we mutated these residues into prolines. When coexpressed with rat AChET subunits, the QN[Pro80-Pro81]/HC mutant was able to produce GPI-anchored G4a, as well as released G4na, with a slightly reduced efficiency compared to the wild type QN/HC protein (not shown). Similarly, the QN[Pro80-Pro81]/stop85, QN[Pro80-Pro81]/stop87, and QN[Gly72-Gly73-Gly74-Pro80-Pro81]/stop85 mutants produced secreted G4na less efficiently than the corresponding constructions containing Met80-Phe81 (Fig. 4).

We also examined the importance of the proline stretches by replacing the middle residue of each group by a glycine. We found that whereas the P83G mutation had little effect on the yield of GPI-anchored G4a and released G4na, the P77G mutation reduced it markedly, and the double mutation P77G/P83G further weakened the binding. Introducing prolines at positions 80 and 81 partially compensated the effect of P77G but weakened the interaction in the case of P83G or of the double mutation P77G/P83G.

The two cysteines of the attachment domain are separated from the proline stretches by three residues, Leu72-Leu73-Thr74. Mutation of these residues to glycines in complete or partially deleted QN/HC was found to weaken significantly the interaction with AChET. In agreement with the effects of mutations of the Cys70-Cys71 and Leu72-Leu73-Thr74 residues, a deletion including the two cysteines (Delta 46-71) was as effective as deletion Delta 1(46-69) in which they were maintained, whereas removal of the following Leu72-Leu73 residues significantly reduced the efficiency of interaction (Fig. 4).

These observations indicate that a sufficient stretch of proline residues is essential for the association with AChET but that hydrophobic residues located before, between, and after the two proline stretches (Leu72-Leu73-Thr74, Met80-Phe81, Phe85-Phe86) also participate in this interaction. We constructed a mutant, QN/Delta 46-74Pro80-Pro81/stop85, in which the mature protein only consisted of three residues, EST, followed by a stretch of 10 prolines, Pro10, as in the Pro80-Pro81- mutant. We found that this Glu-Ser-Thr-Pro10 peptide was able to combine with AChET, decreasing the proportion of G2a and G1a and increasing markedly the level of secreted G4na, although with less efficiency than other QN/stop constructs (Fig. 4). This suggested that the polyproline sequence may be sufficient to organize AChET tetramers in the absence of other residues.

Conversely, QN/HC and QN/stop constructs in which residues 70-86 were deleted had no effect on the molecular forms of AChE, as shown by sedimentation analyses or by immunofluorescence (not shown). They did not induce the production of GPI-anchored G4a at the surface of the cells or the secretion of G4na in the medium.

Interaction of AChET Subunits with Synthetic Polyproline

Exogenous, Synthetic Polyproline Can Combine with AChET in Transfected Cells

We added various concentrations of synthetic polyproline of about 8 kDa to the culture medium after transfecting cells with AChET. We observed a dose-dependent decrease of G2a and G1a, both in the cell extract (Fig. 5A) and in the culture medium (Fig. 5B), and a concomitant increase of G4na. These effects could be detected at 5 × 10-8 M polyproline; at higher concentrations G4na became the major secreted form of AChE, as observed with QN/stop constructs. In addition, a minor 16 S component appeared in the cell extract and in the medium at higher concentrations of polyproline.2 The sedimentation coefficient of this form suggests that it may result from the binding of two tetramers to a sufficiently long polyproline chain.


Fig. 5. Interaction of exogenous synthetic polyproline with AChE in transfected cells. Shown are sedimentation profiles of the molecular forms of AChE produced by COS cells expressing rat AChET without polyproline (bullet ) or with polyproline at 5 × 10-8 M (open circle ), 5 × 10-7 M (black-triangle), or 5 × 10-5 M (triangle ) (inset). Panel A, cellular extract; panel B, culture medium. Polyproline induced the production and secretion of a G4na form in a manner that resembles the effect of QN/stop.
[View Larger Version of this Image (23K GIF file)]


Polyproline of higher average molecular mass (40 kDa) also interacted with AChE but induced the formation of ill defined components, sedimenting mainly between 6 and 11 S.

We wondered whether AChET and polyproline could combine spontaneously or whether the cellular biosynthetic machinery was required for this interaction. We found that alpha alpha '-dipyridyl, which inhibits hydroxylation of prolines, did not inhibit the production of G4na by polyproline, even when included at 10-4 M in the culture medium for 3 days after transfection. This concentration reduced the production of total AChE activity to less than 20% of the control but did not alter the proportions of molecular forms (not shown).

Interaction of Polyproline with AChET in Cell Extracts

We also examined whether the interaction could occur in an acellular system. When a cell extract, obtained in the absence of detergent (high speed supernatant of a low salt-soluble extract), was incubated with polyproline at 37 °C for 4 h, we observed an important increase of G4na at the expense of G1a and G2a (Fig. 6A). The effect of polyproline on AChE monomers and dimers in a detergent-free cell extract resembled the effect obtained with living cells, except for the absence of the 16 S component. The 13.7 S form was not observed under these conditions, probably because it disappeared during incubation at 37 °C. The efficiency of interaction was reduced markedly in the presence of Triton X-100 (Fig. 6B). In addition, the production of G4na, in the presence of polyproline, occurred less efficiently or more slowly at lower temperatures, 20 or 4 °C (not shown). The interaction between AChE and polyproline was therefore dose-dependent, temperature-dependent, and sensitive to the presence of detergent.


Fig. 6. Interaction of exogenous synthetic polyproline with AChE in an acellular cell extract. Panel A, sedimentation analysis of a soluble extract from COS cells expressing rat AChET, prepared without detergent and incubated for 4 h at 37 °C without polyproline (bullet ) or with polyproline at 10-7 M (open circle ) or 10-5 M (black-triangle). Panel B, electrophoretic analysis of a detergent-free cell extract, incubated for 3 h at 20 °C without polyproline and with various concentrations of polyproline in the absence or in the presence of 1% Triton X-100, as indicated. The intensity of the G4 band was increased by polyproline but at higher concentration in the presence of detergent.
[View Larger Version of this Image (49K GIF file)]


In contrast with cell extracts, we observed only a minimal effect after incubation of the secreted AChE forms with polyproline (less than 5% of G1a and G2a was converted into G4na), and this could in fact reflect the contribution of enzyme forms released by cell lysis. Experiments in which cellular extracts and media from transfected and control COS cells were combined, so that the composition of the mixtures was identical except for the cellular or secreted origin of the AChE molecules, showed that the difference in their capacity to interact with polyproline is an intrinsic property of these monomers and dimers (not shown).


DISCUSSION

In these experiments, we used a series of deletions and mutations in the NH2-terminal domain of the collagen Q subunit to identify the residues that are involved in the attachment of a tetramer of AChET subunits. These modifications were introduced in the QN/HC and QN/stop proteins and in some cases also in the complete collagenic subunit, Q. Assembly of AChET subunits with Q is less convenient because it produces several collagen-tailed molecules (A12, A8, A4) which aggregate in low salt and cannot be analyzed in nondenaturing electrophoresis. The results were however entirely consistent with those obtained for QN/HC and QN/stop proteins and have not been illustrated here.

Cysteines Are Not Required for Interaction of QN with AChET

By comparing the proportions of heteromeric tetramers we were able to rank the efficiency of interaction of different deleted and mutated constructions and thus evaluate the contribution of various elements of the QN domain. Quite unexpectedly, deletion of the peptide preceding the cysteines (Delta 1) actually increased the interaction significantly. We also showed that the vicinal cysteines of QN, Cys70 and Cys71, are not required, although disulfide bonds probably stabilize the quaternary association with AChET. The fact that Cys70 and Cys71 are dispensable is consistent with our previous observation that intersubunit disulfide bonds may be reduced without disrupting the structure of collagen-tailed AChE forms (5). We also showed that sequences following a polyproline stretch, i.e. beyond position Phe86, could be removed without compromising the binding capacity.

PRAD, a Short Conserved Peptidic Domain; Evaluation of Interaction Efficiency in the Secretory Pathway

These experiments showed that the binding domain could be reduced to a short region of 17 residues, starting with cysteines Cys70-Cys71, which we propose to name the polyproline attachment domain or PRAD. This region is remarkably well conserved from Torpedo to higher vertebrates; there are only two replacements in the rat Q subunit3 and none in the chicken.4 The presence of PRAD is sufficient for interaction with AChET subunits, and conversely its deletion abolishes this interaction completely.

The PRAD contains two conserved stretches of five and three consecutive prolines, separated by two residues, Met80-Phe81, preceded and followed by conserved residues, Leu72-Leu73-Thr74 and Phe85-Phe86. By point mutations and deletions we showed that these hydrophobic residues, as well as more distal residues, participate in the interaction with AChET subunits but are not absolutely required.

By analyzing the ratio R of secreted heterotetramers, G4na, to the total activity in the culture medium for nonsaturating doses of QN/stop constructions, as described in the previous paper (1), we obtained an index of the efficiency of interaction, assuming that the binding proteins were expressed at the same level (Fig. 4). This index provides an evaluation of the influence of elements of the binding domain; for example, the M80P/F81P double mutation reduces the value of R to a similar degree in different contexts (QN/stop87, QN/stop85, or QNGly72-Gly73-Gly74/stop85). It seems therefore possible to quantify the contributions of specific residues to quaternary assembly in the secretory pathway.

Interaction of AChE with Synthetic Polyproline

The critical feature of the binding domain PRAD appeared to be the presence of a sufficiently long series of prolines, since a simple Glu-Ser-Thr-Pro10 peptide remained functional. We showed that, in effect, AChET subunits were able to combine with synthetic polyproline. When added to cultures of COS cells expressing AChET, synthetic polyproline of about 8 kDa was able to mimic the effect of the QN domain, even at a concentration as low as 10-9 M, inducing the formation and the release of tetramers G4na at the expense of monomers and dimers. It also produced a heavier complex of 16 S, in lesser proportion. Polyproline was probably taken up by endosomes so that a fraction was able to reach the Golgi apparatus and the endoplasmic reticulum and thus to interact with AChE during its biosynthesis and maturation. We showed that alpha alpha '-dipyridyl, which blocks proline hydroxylation, had no effect on this interaction either in cells or in vitro, even at concentrations that inhibited AChE biosynthesis by more than 70%.

In fact, the biosynthetic machinery of the cell was not absolutely necessary since this interaction could be reproduced in a soluble extract of transfected COS cells expressing rat AChET. Polyproline induced the formation of tetramers G4na from monomers and dimers of rat AChET and possibly also of dimers from monomers. The effect was dose-dependent and temperature-dependent and was reduced considerably in the presence of Triton X-100. It is likely that interaction of the detergent with the COOH-terminal peptide of AChET, organized as an amphiphilic alpha -helix5 prevents its interaction with polyproline. This is the first time that oligomerization of AChE has been obtained in vitro. In previous studies, molecular forms of AChE were dissociated, e.g. by limited proteolysis, or reversibly aggregated, as in the case of collagen-tailed molecules at low ionic strength in the presence of polyanions (6); but monomers or dimers were never seen to form stable tetramers. There was, however, a clear difference between the oligomerization of AChET subunits obtained in living cells and in acellular extracts, since heavy polymers, sedimenting around 16 S, were obtained only in the cells.

It is worth noting that molecules solubilized from cells were able to interact with polyproline, whereas those secreted in the culture medium were not. This is consistent with the fact that post-translational modifications occur during transport and secretion (1). Cleavage of the COOH-terminal T peptide may affect the ability of monomers to form oligomers. For example, soluble monomers that had been purified from bovine brain were found to lack the COOH-terminal part of the T peptide (7). It should be noted, however, that the secreted G1a and G2a forms obtained in our culture media retained their amphiphilic character, which reveals the presence of the amphiphilic alpha -helix of the T peptide.

Possible Interaction of PRAD with Other Proteins

The proline-rich structure of PRAD is reminiscent of the peptide sequences that bind to SH3 domains (8, 9). By analogy, it is therefore tempting to suggest that PRAD is organized as a polyproline II helix. There are marked differences, however, between these proline-rich domains. First, the PRAD exists in the secretory pathway, establishing interactions between externalized proteins, whereas SH3 binding domains are located in the cytosol; such polyproline sequences have not been found previously in extracellular proteins. Second, polyproline in PRAD does not simply provide a scaffold but may actually mimic the interaction. Third, there is no similarity between SH3 domains and the COOH-terminal T peptides of AChET, which are necessary for attachment to the collagen tail. Finally, the stoichiometry of the binding is different, since PRAD simultaneously binds to four AChET subunits. This in fact raises an interesting question about the symmetry of the heteromeric assembly, since polyproline II has an helical repetition of three residues per turn. In any case, it will be interesting to investigate the possible interaction of PRAD with other proteins, such as the 100-kDa protein that is associated with Torpedo asymmetric forms (10, 11). It will also be interesting to examine the possible existence of PRAD in other modular extracellular molecules apart from the Q collagen.

In conclusion, the present study reveals a new type of interaction that occurs intracellularly and withstands extracellular conditions. Such quaternary interactions are important for anchoring AChE and possibly other enzymes and proteins in extracellular matrices. We show that such interactions can induce molecular assembly both in the secretory pathway of living cells and in vitro.


FOOTNOTES

*   This research was supported by grants from the CNRS, the Direction des Recherches et Etudes Techniques, the Association Française contre les Myopathies, and the Human Capital and Mobility program of the European Community. The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Dagger    To whom correspondence should be addressed. Tel.: 33-1-44-32-3891; Fax: 33-1-44-32-3887; E-mail: jean.massoulie{at}biologie.ens.fr.
1    The abbreviations used are: AChE, acetylcholinesterase; AChET, AChE catalytic subunit of type T; AChEH, AChE subunit of type H; Q, collagenic subunit; QN, Q subunit NH2-terminal domain; HC, AChEH subunit COOH-terminal domain; GPI, glycophosphatidylinositol; PI-PLC, phosphatidylinositol-phospholipase C; PRAD, proline-rich attachment domain.
2    This 16 S component must not be confused with the A12 collagen-tailed form.
3    E. Krejci, S. Thomine, C. Legay, J. Sketelj, and J. Massoulié, manuscript in preparation.
4    W. R. Randall, personal communication.
5    S. Bon, I. Cornut, J. Dufourcq, J. Grassi, and J. Massoulié, manuscript in preparation.

Acknowledgments

We thank Anne Le Goff and Rizwana Nawaz for expert technical assistance and Jacqueline Pons for typing the manuscript.


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