SNARE Motif-Mediated Sorting of Synaptobrevin by the Endocytic Adaptors Clathrin Assembly Lymphoid Myeloid Leukemia (CALM) and AP180 at Synapses

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SNARE motif-mediated sorting of synaptobrevin by the endocytic adaptors clathrin assembly lymphoid myeloid leukemia (CALM) and AP180 at synapses

Seong Joo Kooa, Stefan Markovicb, Dmytro Puchkova, Carsten C. Mahrenholzc, Figen Beceren-Braund, Tanja Maritzena, Jens Dernedded, Rudolf Volkmerc, Hartmut Oschkinatb, and Volker Hauckea,b,1

aInstitute of Chemistry and Biochemistry, NeuroCure Cluster of Excellence, Freie Universität Berlin, 14195 Berlin, Germany; bLeibniz-Institut für Molekulare Pharmakologie, 13125 Berlin, Germany; cInstitut für Immunologie, Charité Universitätsmedizin Berlin, 10117 Berlin, Germany; and dZentralinstitut für Laboratoriumsmedizin und Pathobiochemie, Charité Universitätsmedizin Berlin, 12200 Berlin, Germany

Edited by Axel T. Brunger, Stanford University, Stanford, CA, and approved July 7, 2011 (received for review May 3, 2011) Neurotransmission depends on the exo-endocytosis of synaptic endocytic protein AP180. Unc-11/AP180 mutants in Caenorhabditis vesicles at active zones. Synaptobrevin 2 [also known as vesicle- elegans mislocalize synaptobrevin (13, 14), whereas more general associated membrane protein 2 (VAMP2)], the most abundant syn- endocytic defects are seen in Lap/AP180-deficient Drosophila aptic vesicle protein and a major soluble NSF attachment protein melanogaster strains (15, 16). Whether these phenotypes reflect receptor (SNARE) component, is required for fast calcium-triggered a direct association between AP180 family members and syn- synaptic vesicle fusion. In contrast to the extensive knowledge aptobrevin is unknown. In mammals, the ANTH domain family about the mechanism of SNARE-mediated exocytosis, little is known consists of two members, clathrin assembly lymphoid myeloid about the endocytic sorting of synaptobrevin 2. Here we show that leukemia (CALM) and AP180. AP180 is exclusively expressed in synaptobrevin 2 sorting involves determinants within its SNARE mo- neurons, where it accumulates at nerve terminals (17). By con- tif that are recognized by the ANTH domains of the endocytic adap- trast, CALM is a ubiquitous protein found in both neurons and tors AP180 and clathrin assembly lymphoid myeloid leukemia glia and in nonneuronal tissues (18). Though both CALM and (CALM). Depletion of CALM or AP180 causes selective surface accu- AP180 have been implicated in regulating neurite outgrowth mulation of synaptobrevin 2 but not vGLUT1 at the neuronal sur- (19), their relationship to SV cycling has not been explored. face. Endocytic sorting of synaptobrevin 2 is mediated by direct Here we demonstrate that endocytic sorting of synaptobrevin 2 interaction of the ANTH domain of the related endocytic adaptors is achieved by direct interaction of the ANTH domain of the CALM and AP180 with the N-terminal half of the SNARE motif cen- endocytic adaptors CALM and AP180 with the N-terminal half of tered around M46, as evidenced by NMR spectroscopy analysis and the SNARE motif. These data suggest a unique mechanism of site-directed mutagenesis. Our data unravel a unique mechanism of SNARE motif-dependent endocytic sorting and identify the SNARE motif-dependent endocytic sorting and identify the ANTH fi fi ANTH domain proteins AP180 and CALM as cargo-speci c domain proteins AP180 and CALM as cargo-speci c adaptors for adaptors for synaptobrevin endocytosis in the central nervous synaptobrevin endocytosis. Defective SNARE endocytosis may also system. Our work further contributes to the notion that cargo- underlie the association of CALM and AP180 with neurodevelop- selective mechanisms operate at synapses to maintain the high mental and cognitive defects or neurodegenerative disorders. fidelity of SV protein sorting and recycling. clathrin-mediated endocytosis | structure Results Depletion of the ANTH Domain Proteins AP180 or CALM Causes eurotransmission in the brain depends on the calcium-trig- Selective Surface Accumulation of Synaptobrevin 2. To explore the fi Ngered fusion and recycling of neurotransmitter- lled synaptic possible role of the related ANTH domain proteins AP180 and vesicles (SVs) with the presynaptic membrane at active zones (1). CALM in SV recycling, we knocked down either or both proteins Following their exocytic insertion into the presynaptic membrane, by RNA interference. CALM- or AP180-specific siRNA effi- fi SV proteins need to be retrieved at a precisely de ned stoichi- ciently down-regulated expression of their corresponding target ometry by endocytosis, a process involving clathrin, adaptors, and protein in transfected HEK293 cells (Fig. 1A) and in primary other endocytic proteins (2). Fast calcium-triggered SV fusion hippocampal neurons in culture (Fig. S1A) (19). Under the critically depends on the SV arginine (R)-soluble NSF attachment conditions used, depletion of CALM or AP180 did not adversely protein receptor (SNARE) synaptobrevin [or vesicle-associated affect electrical excitability of neurons or stimulation-dependent membrane protein (VAMP)], which by forming a complex with exocytosis as probed by exo-endocytic cycling of the styryl dye the plasma membrane glutamine (Q)-SNAREs syntaxin and FM4-64 (Fig. 1 B and C). synaptosomal-associated protein (SNAP)-25 (3) drives neuro- < To assess the role of CALM and AP180 in synaptobrevin 2 en- exocytosis (4, 5). Synapses lacking synaptobrevin 2 display 1% docytosis, we made use of superecliptic pHluorin-tagged synapto- of wild-type release when stimulated by action potential (AP)- brevin 2 (synaptopHluorin). Fluorescence of synaptopHluorin mediated calcium influx (6). Proteomic studies have shown that synaptobrevin 2 is a highly abundant SV protein (7) that is exo- endocytically sorted with very high precision (8). Similar obser- Author contributions: D.P., H.O., and V.H. designed research; S.J.K., S.M., D.P., C.C.M., vations have been made for other SV proteins, including syn- F.B.-B., and T.M. performed research; C.C.M. and R.V. contributed new reagents/analytic aptotagmin and vesicular glutamate transporters (VGLUTs). tools S.J.K., S.M., D.P., F.B.-B., J.D., R.V., H.O., and V.H. analyzed data; and S.J.K., H.O., and How such precise sorting of synaptobrevin 2 is achieved has V.H. wrote the paper. remained enigmatic. Synaptobrevin lacks recognizable linear The authors declare no conflict of interest. sorting motifs (9), and unlike other SNARE proteins does not This article is a PNAS Direct Submission. contain a folded N-terminal domain that serves as a targeting Freely available online through the PNAS open access option. determinant in other VAMP family members (10–12). 1To whom correspondence should be addressed. E-mail: [email protected]. Genetic data have linked synaptobrevin sorting to the function This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. of the AP180 N-terminal homology (ANTH) domain-containing 1073/pnas.1107067108/-/DCSupplemental.

13540–13545 | PNAS | August 16, 2011 | vol. 108 | no. 33 www.pnas.org/cgi/doi/10.1073/pnas.1107067108 Downloaded by guest on October 1, 2021 pHluorin, indicative of its impaired endocytic retrieval. This effect was augmented in neurons depleted of both ANTH domain proteins (Fig. 1D), suggesting that AP180 and CALM serve overlaping functions in synaptobrevin 2 internalization. To test whether loss of CALM or AP180 selectively impairs synaptobrevin 2 endocytosis, or whether other SV proteins are also affected, we assessed the partitioning of pHluorin-tagged vGLUT1, another abundant SV component at excitatory synapses. To our surprise, knockdown of either CALM or AP180 or of both proteins together had no effect on the vesicular-to-surface pool ratio of VGLUT1-pHluorin (Fig. 1E). These effects were corroborated by analyzing the axonal distribution of synaptopHluorin and vGLUT1-pHluorin. A partial dispersion of synaptopHluorin but not of vGLUT1-pHluorin was observed in double-knockdown neurons depleted of both CALM and AP180 (Fig. 1F). Hence, CALM and AP180 exert an overlapping cargo-speciﬁc role in the endocytic retrieval of synaptobrevin 2 from the neuronal surface.

Synaptic Vesicle Size Is Altered in AP180-Depleted Neurons. AP180 (termed Lap in flies) mutants in D. melanogaster display enlarged and heterogenously sized SVs (16). To explore possible alterations in SV size we turned to ultrastructural analysis by electron microscopy. Synapses from transfected neurons were identified by immunogold labeling using antibodies against eGFP cotrans- fected together with specific siRNA targeting AP180 or CALM. Nerve terminals from AP180- or CALM-depleted neurons were morphologically normal (Fig. S2A) and showed no significant change in SV density compared with those from control neurons (Fig. 1H). However, SVs from AP180 knockdown neurons appeared slightly larger and more heterogenous, as evidenced by Fig. 1. Depletion of AP180 and CALM causes surface accumulation of syn- – aptobrevin 2. (A)Efficient siRNA knockdown of AP180 or CALM expression. the increased variance of SV size (Fig. S2 A C). Similar alter- HEK293 cells transiently expressing rat AP180 were transfected with siRNA ations were observed in neurons depleted of both AP180 and specific for rat AP180 (Left) or CALM (Right) and analyzed by immunoblot- CALM, whereas SV size was unchanged in CALM-depleted ting. Expression levels of AP180 or CALM are efficiently down-regulated. (B neurons (Fig. 1G and Fig. S2 B and C). As alterations in SV size and C) SV exocytosis probed by FM4-64 in AP180-, CALM-, or AP180- and have also been observed in Lap/AP180 mutants in D. mela- CALM-depleted neurons. (B) Exocytosis kinetics measured by FM4-64 nogaster (16) and in neurons from synaptobrevin 2 knockout mice unloading. (Upper) Scheme of the protocol used to load and unload FM4-64. (20), these data further support a tight functional link between (Lower) Normalized kinetic traces of FM4-64 dye release from synaptic boutons transfected with control siRNA, siRNA directed against AP180 synaptobrevin 2 and AP180. (AP180 KD), CALM (CALM KD), or siRNAs against both proteins. No significant changes in exocytic release kinetics were observed in neurons depleted Direct Association of the ANTH Domains of AP180 and CALM with the of AP180, CALM, or of both proteins. (C) Relative amount of FM4-64 in- SNARE Motif of Synaptobrevin 2. The observed functional con- ternalized and released upon stimulation (ΔF). Data were normalized to the nection between the ANTH domain proteins CALM and AP180 ΔF of nontransfected terminals and represent mean ± SEM. (D) Vesicular vs. and synaptobrevin 2 suggested that these proteins might also surface pools of synaptobrevin 2-pHluorin assessed by acid-base quenching physically interact. We probed this possibility using immunopre- in hippocampal neurons. Synaptobrevin 2-pHluorin accumulates on the cipitation experiments. Indeed, synaptobrevin 2 coprecipitated neuronal surface following depletion of AP180 (P = 0.0172, n = 15 neurons), with full-length AP180 or its ANTH domain but not with an CALM (P = 0.0022, n = 27 neurons), or both proteins (P < 0.0001, n =28 AP180 deletion mutant lacking the ANTH domain (Fig. 2A). neurons). (E) Same as in D but using vGLUT1-pHluorin as a reporter. (F) fi Dispersion of synaptobrevin 2-pHluorin along the axon of AP180/CALM-de- Thus, the ANTH domain is both required and suf cient for syn- pleted neurons. Selective mislocalization of pHluorin-tagged synaptobrevin aptobrevin 2 association. To delineate the molecular determi- 2, but not vGLUT1, along the axon was observed in neurons depleted of nants within synaptobrevin 2 for AP180-ANTH binding, we AP180 and CALM. (Left)Profiles from linescan analyses of neurons. (Scale generated a series of truncation mutants, in which parts of the bar, 5 μm.) (G and H) Morphometric ultrastructural analysis of cumulative SV cytoplasmic domain were deleted. Though deletion of the N- size frequency (G) and SV density (H) by electron microscopy. The average fi terminal 31 amino acids preceding the SNARE motif had no ef- size of SVs is signi cantly increased in terminals from neurons depleted of fect on synaptobrevin association with AP180, deletion mutants AP180 or of both AP180 and CALM. lacking the N-terminal part of the SNARE motif had lost this ability (Fig. 2B). Thus, complex formation between AP180 and synaptobrevin 2 is mediated by ANTH-mediated recognition of critically depends on pH, and can thus be used to quantitatively

determinants within the SNARE motif. CELL BIOLOGY analyze the partitioning of synaptobrevin 2 between the plasma To corroborate these data and to identify further binding membrane and internal SV-localized pools. Fluorescence changes determinants we turned to cellulose-bound peptide SPOT (peps- were monitored from active synaptic boutons, i.e., displaying ap- can) arrays. To this aim, the amino acid sequence of the synapto- propriate responses to electrical stimulation expressing synaptobrevin 2 cytoplasmic domain was covered by an array of cellulose- pHluorin together with siRNA. Fluorescence quantification after bound 15-mer peptides overlapping in sequence by 14 amino acids acid quenching and ammonium dequenching revealed the relative each. This matrix was incubated with His6-tagged AP180-ANTH ratio of pHluorin molecules present on SVs or stranded on the domain and washed extensively, and bound protein was detected by neuronal cell surface. Depletion of either AP180 or CALM led to anti-His6 antibodies bound to HRP-conjugated secondary anti- asignificant increase in the fraction of surface-stranded synapto- bodies. These pepscan overlay assays identified a series of inter-

Koo et al. PNAS | August 16, 2011 | vol. 108 | no. 33 | 13541 Downloaded by guest on October 1, 2021 acting peptides that cover the N-terminal half of the SNARE motif these experiments, 15N-labeled synaptobrevin 2 was used, which of synaptobrevin 2 (amino acids 33–61; Fig. 2C). showed instructive spectra without ligand. In detergent- and lipid- Given that AP180 and CALM share a structurally conserved free buffer, most resonances appear in the random coil region of 1 ANTH domain, we probed binding of synaptobrevin 2 to CALM. the HN chemical shift range (between 8.0 and 8.5 ppm; Fig. 3 A As expected, the ANTH domain of CALM (CALM-ANTH) and B). Signals from residues 81–96 (21) appear at chemical shifts fused to GST avidly pulled down recombinant synaptobrevin 2 <7.9 ppm, indicating their potential involvement in loops and with an efficiency that even exceeded that of AP180-ANTH (Fig. helical structure. However, their intensity is weak with very small – 2D, Left). Moreover, both His6-CALM-ANTH and His6-AP180- signals for residues 89 91, possibly indicating self-association. ANTH directly associated with the N-terminal half of the SNARE Addition of DPC causes a general increase in signal intensity and motif of synaptobrevin 2 (residues 33–54) in direct binding assays the occurrence of additional, as well as more pronounced, helical using recombinant proteins (Fig. 2D, Right). Avid concentration- segments; these include part of the N-terminal SNARE motif dependent binding of CALM-ANTH to synaptobrevin 2 was also (e.g., amino acids 41–54; Fig. 3 A and C) (21). seen in surface plasmon resonance experiments (Fig. 2F). How- If 15N-labeled synaptobrevin 2 is titrated with a double-molar ever, due to the intrinsic tendency of CALM-ANTH to oligo- excess of CALM-ANTH (Fig. 3 A and B) or AP180-ANTH (Fig. merize at elevated concentrations, a reliable KD value could not S3) in aqueous solution, the cross-peaks of large segments of be determined. The observed preference of synaptobrevin 2 for synaptobrevin 2 diminish (Fig. 3B) due to an intermediate ex- CALM over AP180 (see also below) correlates with the slight change regime, or due to formation of a large complex. Dis- decline of CALM expression during postnatal CNS development appearing signals (Fig. 3A, orange) cluster in the N-terminal half and with a concomitant increase of AP180 and synaptobrevin 2 of the SNARE motif (residues 30–68) and in the membrane- levels (Fig. S1B). One might thus speculate that endocytic recy- proximal part of the cytoplasmic domain (residues 74–96). In line cling of synaptobrevin 2 undergoes a developmental switch from with the results of the pulldown experiments, effects are stronger a CALM-based high-affinity/low-capacity to an AP180-based low- for CALM-ANTH than for AP180-ANTH (Fig. S3), which de- affinity/high-capacity retrieval system. monstrates that both the ANTH domains of CALM and AP180 associate with synaptobrevin 2 via the N-terminal half of the Structural Changes Associated with Binding of Synaptobrevin 2 to SNARE motif comprising residues 30–68, in agreement with the ANTH Domains. The structural basis of ANTH domain-mediated biochemical studies (Fig. 2). recognition of synaptobrevin 2 by CALM and AP180 was moni- To study the formation of protein complexes between syn- tored site-specifically via 1H-15N heteronuclear single quantum aptobrevin 2 and the ANTH domains of CALM or AP180 in coherence (HSQC) in the absence or presence of the membrane- a membrane-mimetic environment, titration experiments were mimicking detergent dodecylphosphocholine (DPC) (21). For carried out in the presence of DPC micelles. Under these con-

Fig. 2. Recognition of the SNARE motif of synaptobrevin 2 by the ANTH domains of AP180 and CALM. (A) AP180 associates with synaptobrevin 2 via its ANTH domain. HEK293 cells coexpressing synaptobrevin 2- FLAG together with empty vector, full-length AP180 (FL), the ANTH domain alone (ANTH), or a mutant lacking the ANTH domain (ΔANTH) were subjected to immunoprecipitation using anti-FLAG antibodies. Sam- ples were analyzed by immunoblotting. Input: 1% of the starting material. Note that the immunoblot rep- resenting input material was exposed longer to allow proper visualization of all bands. (B) Synaptobrevin 2 interacts with AP180 via the N-terminal half of its SNARE motif. HEK293 cells coexpressing AP180 and FLAG-tagged truncation mutants of synaptobrevin 2 were subjected to immunoprecipitation as in A. Com- plex formation was disrupted by deletion of residues from the N-terminal half of the SNARE motif of synaptobrevin 2 (Δ1–50). (C) Delineation of the minimal AP180-ANTH binding site within synaptobrevin 2 using peptide SPOT arrays. Peptides displaying nonspeciﬁc binding are boxed in gray and were excluded from the analysis. Binding peptides are boxed in red and the sequence of these peptides is listed below. (D) Direct binding of synaptobrevin 2 to the ANTH domains of CALM and AP180. (Left) Immobilized GST-ANTH domains derived from CALM or AP180 (10 μg) were

incubated with His6-synaptobrevin 2 (1–96) for 1 h at 4 °C. Following extensive washes, samples were analyzed by SDS/PAGE and immunoblotting. (Right) Interaction between GST-synaptobrevin 2 (33–54) comprising the N-terminal half of its SNARE motif immobilized on

beads and incubated with His6-ANTH domains. Samples were analyzed by SDS/PAGE and immunoblotting. (E) Dose-dependent binding of puriﬁed CALM-ANTH to immobilized synaptobrevin 2 (1–96) analyzed by surface plasmon resonance. Shown are binding curves from a representative experiment after background substraction. RU, resonance units.

13542 | www.pnas.org/cgi/doi/10.1073/pnas.1107067108 Koo et al. Downloaded by guest on October 1, 2021 ditions, most cross-peaks remained visible in the presence of CALM-ANTH was observed for D44A (Fig. 4 A and B and Fig. CALM-ANTH. Furthermore, complex formation between 15N- S4B). Because M46A and D44A differentially affected the rec- labeled synaptobrevin 2 and CALM-ANTH resulted in chemical ognition of synaptobrevin 2 by ANTH domain-containing adap- shift changes of those nuclei that participate in binding. Dra- tors, remained competent for SNARE complex formation (Fig. matic effects were observed for R47 and N49 (pink); strong S4A), and have previously been shown to regulate synaptobrevin 2 effects for L32, A37, Q38, V42, D44, M46, V50, and R56 (red); targeting to synaptic-like microvesicles in PC12 cells (25), these and weakly changing chemical shifts for T35, K52, and members mutants were selected for further analysis. of the segment 80–91 (yellow). Nearly no chemical shift changes To probe the effect of these mutations on the partitioning of were observed for residues 61–77, except for A74 (Fig. 3 A and synaptobrevin 2 between internal and surface pools, we expressed C). No clear decision could be taken for V48. A very similar mutant variants of synaptopHluorin in primary hippocampal pattern of chemical shift changes was elicited by AP180-ANTH neurons. Boutons expressing either of these mutant synapto- (Fig. S3), indicating that both ligands interact with the same set pHluorins (M46A or D44A) exhibited proper responses to elec- of residues in synaptobrevin 2. We conclude that the ANTH trical stimulation (Fig. S5). As expected from the biochemical domains of CALM and AP180 associate with synaptobrevin 2 via analysis, synaptopHluorin carrying the M46A mutation exhibited the N-terminal half of its amphipathic SNARE helix. a dramatic increase in the surface-to-vesicular pool ratio, indi- ANTH domain association also elicited alterations in the or- cative of its failure to become internalized (Fig. 4C). Conversely, ganization of the C-terminal helix and the juxtamembrane linker a statistically signiﬁcant reduction in the surface-to-vesicular pool of synaptobrevin 2 (Fig. 3). Previous work has identiﬁed a cluster was observed for D44A (Fig. 4C), likely as a consequence of of aromatic residues Y88–W90 within this region that might its increased binding to AP180 and CALM (Fig. 4B and Fig. S4B). regulate synaptobrevin 2 function (22–24). Our data suggest that These changes were accompanied by a failure of synaptopHlu- this aromatic cluster may form a stable loop structure. However, orin-M46A to be enriched at presynaptic vesicle clusters (Fig. 4D). because the chemical shift-changes induced by CALM-ANTH Thus, ANTH domain-based endocytic sorting of synaptobrevin is (Fig. 3 A and C) or AP180-ANTH (Fig. S3) in the presence of mediated by the N-terminal half of the SNARE helix centered DPC are weak, we consider it unlikely that the aromatic cluster around M46. directly associates with CALM or AP180. Discussion SNARE Motif-Dependent Endocytic Sorting of Synaptobrevin 2. Fi- We identify here the molecular mechanism by which the SV R- nally, we functionally probed the physiological relevance of ANTH SNARE protein synaptobrevin 2 is endocytosed at the neuronal domain recognition of SNARE motif-based sorting determinants surface. Combined cell biological and structural biochemical anal- within synaptobrevin 2 for its exo-endocytic cycling. To this aim, yses indicate that the N-terminal half of the SNARE motif of syn- we generated site-directed mutants of synaptobrevin 2 harboring aptobrevin 2 serves as a sorting determinant for recognition by the mutations within the N-terminal half of the SNARE helix. Based related ANTH domain-containing endocytic adaptors AP180 and on the NMR spectroscopy analysis, we targeted D44, M46, R47, CALM. First, knockdown of AP180 or CALM, or of both proteins and N49 for mutagenesis. M46 constitutes a central part of the together, causes a substantial increase in the fraction of surface- hydrophic face of the N-terminal half of the amphipathic SNARE stranded synaptobrevin 2 along the axon. Second, synaptobrevin via helix (3). Substitution of M46A completely abrogated the ability of determinants within the N-terminal half of its SNARE motif synaptobrevin 2 to associate with GST-CALM-ANTH (Fig. 4 A around M46 is directly bound to and recognized by the ANTH and B), whereas mutation of R47 or N49 to alanines greatly di- domain of CALM or AP180, as shown by biochemical assays and minished binding (Fig. 4A). By contrast, increased binding to NMR spectroscopy analysis. Third, mutations within synaptobrevin

Fig. 3. NMR spectroscopic analyses of structural changes associated with binding synaptobrevin 2 to CALM-ANTH. (A–C) Sequences of synaptobrevin 2 indicating residues in- ﬂuenced by CALM-ANTH binding as detected in 1H-15N- HSQC spectra of solutions in aqueous buffer (Upper sequence and spectrum in B) and in the presence of DPC (Lower sequence and spectrum in C). Sec- ondary structures are indicated above and below the sequences. In the Upper sequence, all residues are shown in orange whose signals disappear upon addition

of CALM-ANTH (cf. also data for CELL BIOLOGY AP180-ANTH in Fig. S3). The chemical shift changes observed upon addition of CALM-ANTH in buffer containing DPC were classiﬁed into three categories (strong: purple; medium: red; weak: yellow) and displayed in the Lower sequence in A.(D) Structure of synaptobrevin 2 (PDB ID code 2KOG) showing residues 30–92 and the residues affected by CALM-ANTH binding in DPC buffer according to the color code in A.

Koo et al. PNAS | August 16, 2011 | vol. 108 | no. 33 | 13543 Downloaded by guest on October 1, 2021 2 that affect the ability of the protein to interact with CALM or AP180 lead to corresponding changes in the efficiency with which it is endocytosed from the neuronal surface. Thus, endocytic recycling of synaptobrevin 2 is achieved by ANTH domain-mediated recognition of the N-terminal half of the SNARE motif. This is a unique mechanism for SNARE sorting that is sub- stantially different from the post-Golgi trafficking recently described for the VAMP-family members Vti1b and VAMP7/Ti- VAMP (10–12). In these cases, specific sorting is achieved by the association of the N-terminal Habc domain of Vti1b and the longin domain of VAMP7/Ti-VAMP with their cognate adaptors EpsinR and Hrb, respectively. Synaptobrevin 2 internalization by AP180/ CALM by contrast involves determinants within the SNARE motif that overlap with the binding site for syntaxin/SNAP-25 (3, 4). Hence, we predict that NSF-mediated disassembly of SNARE complexes (2, 4, 5) is a prerequisite for endocytic recycling of synaptobrevin 2 via AP180/CALM, consistent with the low abundance of syntaxin and SNAP-25 in purified SVs (7). The compa- rably weak interaction between synaptobrevin 2 and CALM or AP180 likely is facilitated by avidity effects (26), which could involve local clustering of exocytosed synaptobrevin 2 molecules (27, 28) and/or lateral cross-linking of ANTH adaptor-SNARE complexes via the assembling clathrin scaffold (29, 30). The results reported here further support and extend previous genetic studies that have suggested a role for AP180 family members in synaptobrevin sorting and SV endocytosis in inverte- brates (14–16, 31, 32). Similar to what has been observed in D. melanogaster (16), AP180-depleted neurons display alterations in SV size, a phenotype reminiscent of that seen in neurons derived from synaptobrevin 2 knockout mice (20). Irregularly shaped and sized vesicles and pits have also been observed in CALM-depleted fibroblasts (33). Paired with the observation that synaptobrevin knockout mice suffer from delayed replenishment of SV membranes following hypertonic sucrose stimulation (20), these data indicate a close physical and functional partnership between synaptobrevin and the AP180 family members CALM and AP180. Why two distinct endocytic proteins, CALM and AP180, are used to retrieve synaptobrevin from the neuronal surface in mammals remains to be determined but could be related to the developmental regulation of their expression profiles and concomitant changes in exo-endocytic cycling of SV proteins. A critical hallmark of the SV cycle is the tight coupling between exo- and endocytosis (1). Such coupling ensures a close balance between exo- and endocytosed membrane and is required to maintain SV protein composition over multiple cycles. How such coupling is achieved remains unknown, but may involve adaptor- dependent sorting mechanisms (34, 35) and/or SV protein clustering (27). Our data show that at least in the case of synaptobrevin, the ANTH domain-containing adaptors AP180 and CALM operate in a cargo-specific manner to selectively retrieve surface- stranded brevin molecules, whereas sorting of other SV cargo such as vGLUT1 procedes unperturbed. These observations favor a scenario in which coordinated activity of cargo-specific adaptors maintains SV composition. Such cargo-specific SV sorting adaptors include AP180 and CALM (this study), stonin 2 (36), and perhaps endophilin (37), among other to-be-identified proteins. Finally, it is tempting to speculate that the function of AP180 and CALM in retrieving surface-stranded synaptobrevin from the neuronal surface may be related to their role in neurode- Fig. 4. SNARE motif-dependent endocytic sorting of synaptobrevin 2. (A) Mutational analysis of the ANTH domain-binding interface within the SNARE motif of synaptobrevin 2. GST-CALM-ANTH was immobilized on beads and incubated with lysates from HEK293 cells expressing synaptobrevin 2-FLAG carrying the indicated mutations. Samples were analyzed by cumulate on the neuronal surface (P < 0.0001, n = 9 neurons), whereas D44A SDS/PAGE and immunoblotting. (B) Quantification of CALM-ANTH binding shows decreased surface pools (P = 0.0073, n = 19 neurons). (D) Axonal dis- to synaptobrevin 2 M46A or D44A as depicted in A. Binding to CALM-ANTH persion of synaptobrevin 2 (M46A). Wild-type and D44A synaptobrevin 2- is abolished significantly by M46A and increased by D44A mutations (P < pHluorin display a pronounced concentration at presynaptic boutons, 0.0001 and P = 0.1415, respectively; n = 3 independent experiments). (C) Exo- whereas M46A is dispersed along the axon. The experiment was carried out endocytic cycling of mutant synaptobrevin 2-pHluorin. M46A is seen to ac- as described in Fig. 1F. (Scale bar, 5μm.)

13544 | www.pnas.org/cgi/doi/10.1073/pnas.1107067108 Koo et al. Downloaded by guest on October 1, 2021 generative disorders, including Alzheimer’s disease (38, 39). pHluorin Imaging. Primary neuronal cultures were prepared from hippocampi Further work is needed to investigate this possibility in detail. of P1–P3 Wistar rats. On 6–8 days in vitro (DIV), neurons were transfected by calcium phosphate transfection (Promega Inc.). On DIV 12–15, neurons Materials and Methods expressing synaptopHluorin or vGLUT1-pHluorin were live-imaged using a charge-coupled device camera (AxioCam; Carl Zeiss, Inc.) on an inverted SI Materials and Methods available online include plasmids, siRNAs, anti- microscope (Axiovert 200M; Carl Zeiss, Inc.) essentially as described pre- bodies, cell culture and transfection, protein puriﬁcation, binding assays, viously (36, 40). See SI Materials and Methods for full details. The fraction of immunoprecipitation, SPOT synthesis and binding studies, surface plasmon surface pool was calculated by the following equation; p = [SpH] / resonance (SPR) measurements, pHluorin imaging, FM4-64 imaging, immu- surface surface ([SpH] + [SpH] )=(F – F )/(F – F )+(F – F ). nostaining of primary neurons, and electron microscopy. surface vesicle physio. acidic physio. acidic basic physio. A total of 200 APs (20 Hz, 100 mA) were used to stimulate the neurons.

Preparation of Detergent Extracts from Mouse Brain. Hippocampi of mice at NMR Spectroscopy. 1H-15NHSQCspectraof15N-labeled synaptobrevin 2 were various ages were isolated and minced by a douncer for 15–20 strokes at 3.9 × g recorded on a Bruker DRX-600 NMR spectrometer in aqueous solution at 18 °C, in lysis buffer [20 mM Hepes (pH 7.4), 100 mM KCl, 2 mM MgCl , 5 mM EDTA, 2 in complex with DPC at 30 °C. Protein concentration was 0.5–1 mM in a buffer 1% Triton X-100, and 1 mM PMSF] supplemented with protease inhibitor consisting of 150 mM NaCl, 5 mM DTT, 1 mM EDTA, 10% D2O, 20 mM Mes at pH mixture (Sigma) and 10 μM ALLN (Calbiochem). After 30 min on ice, the lysate 6.0, and optionally 200 mM DPC. CALM-ANTH or AP180-ANTH was added was centrifuged at 20,800 × g for 10 min at 4 °C. The resulting supernatant stepwise up to twofold stoichiometric excess. Spectra were processed with was collected and its concentration was determined by the Bradford assay. Topspin and analyzed in Sparky version 3.114 (41). Assignments were taken from BMRB entry 16514 (21). Shift tolerances of 0.05 ppm were given for fi Protein Puri cation, Binding Assays, and Immunoprecipitation. Recombinant spectra of DPC-bound synaptobrevin 2. Structures were plotted using University fi fi fi proteins were puri ed using standard protocols. As a nal puri cation step, of California, San Francisco, Chimera (http://www.cgl.ucsf.edu/chimera/)(42). recombinant proteins were subjected to size exclusion chromatography in buffer A [150 mM NaCl, 5 mM DTT, 1 mM EDTA, and Mes (pH 6.0)] for NMR ACKNOWLEDGMENTS. We thank T. Südhof, R. Edwards, and R. Jahn for spectroscopy and in buffer B [20 mM Tris (pH 7.5) and 150 mM NaCl] for SPR plasmids. This work was supported by Deutsche Forschungsgemeinschaft measurements. Binding assays and immunoprecipitation experiments were Grants SFB958/A01, FOR806, and Exc 257-Neurocure and the European Sci- essentially done as described in Diril et al. (36). See SI Materials and Methods ence Foundation. S.J.K. is the recipient of a PhD fellowship from the Max for further details. Delbrück Center for Molecular Medicine.

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