Correspondence to: Manfred W. Kilimann, Institut für Physiologische Chemie, Ruhr-Universität Bochum, D-44780 Bochum, Germany. Tel:49-234-700-7927 Fax:49-234-7094-193 E-mail:manfred.kilimann{at}ruhr-uni-bochum.de.
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Abstract |
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Neurotransmitter exocytosis is restricted to the active zone, a specialized area of the presynaptic plasma membrane. We report the identification and initial characterization of aczonin, a neuron-specific 550-kD protein concentrated at the presynaptic active zone and associated with a detergent-resistant cytoskeletal subcellular fraction. Analysis of the amino acid sequences of chicken and mouse aczonin indicates an organization into multiple domains, including two pairs of Cys4 zinc fingers, a polyproline tract, and a PDZ domain and two C2 domains near the COOH terminus. The second C2 domain is subject to differential splicing. Aczonin binds profilin, an actin-binding protein implicated in actin cytoskeletal dynamics. Large parts of aczonin, including the zinc finger, PDZ, and C2 domains, are homologous to Rim or to Bassoon, two other proteins concentrated in presynaptic active zones. We propose that aczonin is a scaffolding protein involved in the organization of the molecular architecture of synaptic active zones and in the orchestration of neurotransmitter vesicle trafficking.
Key Words: synapse, neurotransmitter exocytosis, membrane traffic, PDZ domain, zinc finger
NEUROTRANSMITTER vesicle exocytosis is confined to synaptic specializations of distinctive architecture. Cell adhesion and extracellular matrix proteins keep the pre- and postsynaptic components in register and at a defined distance. At the postsynaptic side, a characteristic submembranous cytoskeletal structure, the postsynaptic density, is thought to mediate the anchoring and clustering of neurotransmitter receptors. The presynaptic specialization contains a machinery that mediates the rapid yet highly controlled exocytotic and reendocytotic trafficking of neurotransmitter vesicles. Exocytosis is restricted to the active zone, an area of the presynaptic plasma membrane where neurotransmitter vesicles are lined up in close vicinity to the cytoplasmic face of the membrane. Neurotransmitter release is triggered by Ca2+ influx through voltage-gated ion channels, which are also concentrated in the active zone membrane (
Recently, much has been learned about elementary molecular interactions that immediately contributes to the fusion of vesicles with and their reendocytosis from the presynaptic plasma membrane, such as the formation and structure of the fusion core complex or the involvement of dynamin in membrane scission. However, we need to better understand how the many interactions between individual vesicle, plasmalemmal, cytoskeletal, and cytosolic proteins and lipids are integrated into the high degree of spacial and temporal organization that must underlie neurotransmitter vesicle dynamics. Scaffolding proteins specific for active zones may play a role in this. One active zonespecific protein is Rim, a 170-kD protein that carries a PDZ domain and two C2 domains in its COOH-terminal part, and a pair of Cys4 zinc fingers at its NH2 terminus (
Here, we describe the identification and initial characterization of a new active zonespecific protein, aczonin. Aczonin shares extensive regions of homology either with Bassoon or Rim, but also possesses unique sequence regions, including a polyproline stretch. Probably through this polyproline stretch, aczonin binds profilin, a protein involved in actin cytoskeletal dynamics. Aczonin is mainly associated with a detergent-resistant cytoskeletal-like subcellular fraction. We propose that aczonin is a scaffolding protein that interacts with multiple partner molecules and is involved in organizing the interplay between neurotransmitter vesicles, the cytoskeleton, and the plasma membrane at synaptic active zones.
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Materials and Methods |
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cDNA Cloning and Northern Blot Analysis
Immunoscreening of a chicken brain cDNA expression library in gt11 with a rabbit serum against the aqueous Triton X-114 fraction of chicken brain synaptic plasma membranes (
Antibodies
Two sequence regions that have little or no similarity with Bassoon and Rim (codons 485754 [serum 1] and 18082150 [serum 2]) were amplified from mouse brain RNA and cloned into the His-tag vectors pQE31 and pQE30 (Qiagen). Subclone inserts were sequenced to confirm the absence of mutations. His-tag fusion proteins were expressed in bacteria, purified on nickel agarose, and used to immunize rabbits. Sera were affinity-purified with the same fusion proteins coupled to tresyl chlorideactivated Sepharose (Sigma Chemical Co.). Commercial mAbs for mannosidase II (clone 53FC3; BAbCO), Na/K-ATPase ß subunit (Upstate), Rab3 (clones 42.1 and 42.2; Transduction Laboratories, Inc., and Synaptic Systems), Rab5 (Transduction Laboratories, Inc.), transferrin receptor (clone H68.4; Zymed), and -tubulin (Amersham Pharmacia Biotech), and sera for synaptophysin (Biometra) and rabphilin-3A (Synaptic Systems) were purchased from the sources indicated. An affinity-purified Mena antibody (LKE) was donated by Frank Gertler (Massachusetts Institute of Technology, Cambridge, MA), isoform-specific antisera against profilins I and II were gifts of Walter Witke (European Molecular Biology Laboratory, Heidelberg, Germany), and a KDEL receptor mAb was the gift of Wanjin Hong (University of Singapore, Singapore).
Immunoblotting and Subcellular Fractionation
To determine the tissue distribution of aczonin, tissues were homogenized in 0.32 M sucrose, 1 mM EDTA, 10 mM Tris, pH 7.4, 0.5 mM PMSF, 2 µg/ml pepstatin A, 2 µg/ml leupeptin, with a glass-Teflon homogenizer, or for muscle and heart, a turning-knife homogenizer. After spinning for 3 min at 900 g, 80 µg protein of each supernatant was resolved by SDS-PAGE (5% polyacrylamide), transferred to nitrocellulose, and the blot developed with affinity-purified rabbit antiaczonin and the ECL kit (Amersham Pharmacia Biotech).
For 120,000 g fractionation and reextraction experiments, 900 g supernatants of brain homogenates (in 150 mM NaCl, 1 mM EDTA, 10 mM Tris, pH 7.4, 0.5 mM PMSF, 2 µg/ml pepstatin A, 2 µg/ml leupeptin) were subjected to a 120,000 g centrifugation for 30 min at 4°C. Pellets were washed by resuspending in homogenization buffer followed by a second 120,000 g spin, and then resuspended either in homogenization buffer or in various solubilization buffers (see legend to Fig 5), either for 20 min at 4°C or for 30 min at room temperature. The 120,000 g centrifugation was then repeated. Equal aliquots of pellets and supernatants were analyzed by immunoblotting as described above.
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Preparative subcellular fractionation procedures for the purification of synaptic vesicles or synaptic plasma membranes were performed according to
Immunomorphological Analysis
Immunohistochemical procedures for light and electron microscopical analysis of rat brain were as described previously (
Cell culture, immunofluorescence analysis, and brefeldin A treatment were performed according to conventional procedures. PC12 and NS20Y cells were fixed in 4% paraformaldehyde in PBS, and permeabilized with either 0.04% saponin or 0.2% Triton X-100. For double-labeling experiments, cells were incubated simultaneously with both primary antibodies. Antiaczonin was visualized with a biotinylated goat antirabbit secondary antibody (Vector Labs) followed by streptavidin-FITC. Antimannosidase II, anti-KDEL receptor, or antitransferrin receptor marker antibodies were visualized with a Cy3-conjugated goat antimouse antibody (Dianova).
Protein Binding Experiments
Recombinant Protein Constructs.
The mouse Rab3A sequence was taken from
Profilin Binding Experiments.
Mouse brain was homogenized in lysis buffer (20 mM Tris, pH 7.4, 150 mM NaCl, 4 mM MgCl2, 2 mM EDTA, 10 mM NaF, 1 mM Na3VO4, 2 mM PMSF, 2 µg/ml pepstatin A, 2 µg/ml leupeptin, 2 µg/ml aprotinin, 2 mM benzamidine, and 0.2% Triton X-100). The 120,000 g supernatant was adjusted to 4 mg/ml total protein. The pellet was resuspended in 0.1 M Na2CO3 (pH 11.5) for 30 min at room temperature, dialyzed against several changes of lysis buffer with stepwise decreasing pH until pH 7.4, and adjusted to 6 mg/ml. Recombinant profilins or BSA (MBI Fermentas) were coupled to N-hydroxysuccinimideactivated Hi-Trap columns (Amersham Pharmacia Biotech) following the manufacturer's instructions. 20-µl aliquots of protein-coupled resins were preblocked with 3% BSA in PBS, washed with lysis buffer, and incubated with brain lysate for 4 h at 4°C under constant agitation. After spin, pellets were washed six times in lysis buffer and resuspended in SDS sample buffer. 1/20 vol of supernatants and 1/2 vol of pellets were analyzed by SDS-PAGE and immunoblotting. In poly-amino acid blocking experiments, resins were preincubated with 100 µl of 5 mg/ml polyproline (110 kD; Sigma Chemical Co.) or polyalanine (15 kD; Sigma Chemical Co.) in lysis buffer overnight at 4°C and washed once with 1 ml lysis buffer before incubation with lysates. Alternatively, profilins and other His-tagged protein constructs (see Results) were immobilized on nickel agarose (20 µl of resin per sample), incubated with lysates additionally containing 5 mM imidazole, and washed with lysis buffer additionally containing 50 mM imidazole.
Rab3A Binding Experiments.
For Rab3A overlay assay, equal quantities (2 µg) of glutathione S-transferase (GST)1 fusion proteins with similar-sized inserts from aczonin (amino acids 374654 and 8631115), Rim (11399) as positive control, and paralemmin and HSB (S, 1 mM GDP, or without nucleotides. Blots were washed with overlay buffer and processed for immunodetection using His-tag antibody (Qiagen) or Rab3A antibody (Synaptic Systems) and the ECL kit (Amersham Pharmacia Biotech). Recombinant Rab3A was also immobilized on nickel agarose and employed in precipitation experiments with brain lysates as described above for profilin, in the presence of either 0.5 mM GTP
S or 1 mM GDP. Immunoprecipitations with affinity-purified antiaczonin were performed with Pansorbin (Calbiochem) according to conventional procedures using mouse brain lysate prepared as described above, with the addition of 0.5% Triton X-100 and 0.5% BSA, in the presence of either 0.5 mM GTP
S or 1 mM GDP.
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Results |
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Primary Structure of Mouse and Chicken Aczonin: Sequence Organization, Partial Homology with Bassoon and Rim, and Differential Splicing
Aczonin was identified as a new molecular constituent of neuronal synapses by immunoscreening a brain cDNA expression library with antisera raised against synaptic plasma membranes (
Sequence comparison between mouse and chicken reveals an organization into regions of high or low interspecies conservation (Fig 2). In particular, the sequences surrounding the two zinc finger pairs and the polyproline stretch are poorly conserved, suggesting that they mainly serve as spacers. This interpretation is further supported by the finding that the two zinc finger pairs are flanked by blocks of sequence repeats. Upstream of the first zinc finger pair is a series of degenerate proline- and glutamine-rich 10-mer repeats (consensus, KPxPQQPGPx, other residues often small or aliphatic amino acids). These repeats are found in different numbers (1525) in chicken, mouse, and humans. We also isolated mouse cDNA sequence variants differing in the presence or absence of three 10-mer repeat units (Fig 1), which may reflect differential splicing or a genomic polymorphism. Downstream from the second zinc finger pair, the chicken and mouse sequences diverge again for several hundred amino acids, and the mouse sequence is shorter, whereas the chicken sequence has expanded by eight lysine-rich ~22-mer repeats (core motif, VQKED ... SADKI). We suppose that both repeat series served to expand the spacer sequences during evolution, although it remains possible that the 10-mer repeats have an additional function.
Aczonin shares regions of homology with two other proteins concentrated at active zones, Rim and Bassoon (Fig 2). Rim possesses a similar COOH-terminal array of one PDZ and two C2 domains, and both Cys4 zinc finger pairs of aczonin have sequence similarity with the single pair of Cys4 zinc fingers of Rim. Outside these circumscript molecular modules, we do not detect significant sequence similarity between aczonin and Rim, except a motif of 19 amino acids with 68% identity close to the NH2 termini of both proteins (DLSQLSEEERRQIAAVMSR; Fig 1 and Fig 2). Upstream of the PDZ domain, many but not all sequences highly conserved between mouse and chicken aczonin are homologous to Bassoon. This includes the two zinc finger pairs and the extreme NH2 terminus, but not the polyproline stretch and another proline-rich region downstream from it. A highly charged region between amino acids 15501700 of mouse aczonin is particularly conserved between mouse and chicken aczonin and Bassoon. In summary, the aczonin sequence can be structured into Rim-related, Bassoon-related, and aczonin-specific regions or motifs. The zinc finger motifs are similar in all three proteins.
Two sequence regions near the COOH terminus are subject to differential splicing. Splicing at codon 4829 of the mouse sequence can abort the downstream 210 amino acids, including the C2B domain, and replace them by a short SKRRK COOH terminus. Out of nine adult mouse brain cDNAs from this region that were sequenced, eight encode the shorter (aczonin-S) and one the longer COOH terminus (aczonin-L). From chicken, only one cDNA was isolated. It encodes the longer COOH terminus plus 61 additional codons (AHKS ... PEGA) inserted at a position corresponding to mouse codon 4829 (aczonin-XL). Reverse transcription PCR with primers between positions corresponding to codons 4751 and 4946 of the mouse sequence indicated that mRNAs with and without this insert are expressed in similar quantities in chicken brain. In the human aczonin gene (see Discussion), the sequence encoding the short SKRRK COOH terminus is contiguous with the sequences immediately upstream in the cDNA, whereas the alternative COOH-terminal sequences, including the XL insert, are encoded by exons further downstream.
Aczonin Is a Brain-specific Protein
Northern blot analysis of RNAs from various chicken and human tissues showed that aczonin mRNA, which is very large and therefore partially degraded, is most highly expressed in the brain and detectable at low abundance in several endocrine glands but in none of the other tissues analyzed (Fig 3A and Fig B). In both species, testis mRNA gave a unique band pattern, with smaller molecular sizes than in the other tissues. Antisera were raised against recombinant aczonin partial sequences and employed for Western blot analysis of mouse tissue homogenates. A very large protein far above the 206-kD marker, apparently partially degraded, was detected in different brain regions and, after longer exposure, very weakly in stomach but in none of the other tissues analyzed, including adrenal gland, testis, and pancreas (Fig 3 C). In endocrine cells, aczonin protein may be poorly translated from the mRNA or rapidly degraded. Aczonin mRNA and protein are found in similar abundances in forebrain, cerebellum, and brainstem (Fig 3A and Fig C), indicating expression throughout the brain.
Aczonin Is Concentrated at the Active Zones of Synaptic Terminals
Antisera raised against aczonin-specific sequences labeled neuropil-rich areas throughout the rat brain. Cell bodies and myelin-rich areas were spared (Fig 4 A). The examination of the cerebellum and the dentate gyrus by electron microscopy revealed that immunoreactivity concentrates at the presynaptic side of synaptic specializations. Immunoperoxidase reaction product is restricted to a space reaching from the plasma membrane of the active zone into the interior of the synapse by only a few neurotransmitter vesicle diameters (Fig 4B and Fig C). Aczonin immunoreactivity was found in many but not all synapses. In the glomeruli of the cerebellum, all mossy fiber terminals were decorated, whereas only a fraction of synapses between Golgi and granule cells showed reaction product (Fig 4 C). Terminals of parallel fibers, which form contacts with Purkinje cell dendrites in the molecular layer of the cerebellum, were also labeled in many but not all cases. In the dentate gyrus only a subpopulation of granule cell mossy fiber terminals was immunopositive.
Aczonin Is Associated with a Detergent-resistant Cytoskeletal-like Subcellular Fraction in Brain, and with Intracellular Membranes in Neuronal Cell Lines
120,000 g fractionation of brain homogenate showed that ~90% of total aczonin was recovered in the pellet and ~10% in the supernatant (Fig 5 A, fractions P and S). From the pellet, aczonin could not be extracted with 1 M NaCl or with 1% Triton X-100, but could be with 0.1 M sodium carbonate (pH 11.5). In this behavior, aczonin differed from the intrinsic membrane protein, synaptophysin, which was almost completely solubilized by the detergent but not by sodium carbonate, and was similar to the cytoskeletal control protein, tubulin (Fig 5 A). This result suggests that aczonin is associated through polar interactions with a detergent-resistant cytoskeletal-like subcellular fraction.
The distribution of aczonin in a subcellular fractionation course leading to the purification of synaptic vesicles closely follows that of the plasma membrane marker, the Na/K-ATPase ß subunit, and differs from the synaptic vesicle marker, synaptophysin, which partitions markedly also into the light fractions. In the final step of this procedure, controlled-pore glass gel filtration, synaptophysin is enriched in the small-vesicular fraction permeating the gel (fraction PIIP), whereas aczonin is enriched in the PIP exclusion peak, even more so than the plasma membrane marker. Codistribution with a plasma membrane marker was also observed in a fractionation procedure leading to synaptic plasma membranes (data not shown). These observations indicate that aczonin is not firmly associated with free synaptic vesicles, but rather with larger structures that sediment faster than vesicles in centrifugation and are excluded by the controlled-pore glass chromatography matrix, such as large cytoskeletal aggregates, the plasma membrane, or both.
Neuronal cell lines also express aczonin. In these cells, it is associated with endomembrane structures within the cell body. In PC12 neuroendocrine cells, aczonin immunofluorescence is congruent with that of mannosidase II, a marker of the Golgi complex (Fig 6). This aczonin and mannosidase IIpositive membrane structure is fragmented within ~5 min by brefeldin A, but is unaffected by wortmannin (not shown), further supporting its identification as the Golgi complex or a closely apposed structure such as the TGN. In NS20Y neuroblastoma cells, aczonin instead decorates more finely punctate structures that cluster around the nucleus or at the bases of processes but spare the Golgi region. This apparently vesicular compartment is not labeled by KDEL receptor (marker for ERGolgi intermediate compartment and cis-Golgi) or transferrin receptor (recycling endosomes) immunofluorescence (data not shown). These observations suggest that aczonin, although not an intrinsic membrane protein, associates with membranes, membrane proteins, or the membrane-associated cytoskeleton during earlier stages of the secretory pathway, and in this way probably reaches its presynaptic destination in neurons.
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Aczonin Binds Profilin
A polyproline tract is conserved between chicken, mouse, and human aczonin, whereas extensive flanking regions are not or poorly conserved (Fig 1 and Fig 2). Synthetic polyproline (
Profilin exists in two isoforms, profilin I being expressed in many tissues including brain, and profilin II predominating in brain and skeletal muscle (
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In several independent experiments with equal quantities of covalently immobilized proteins as shown in Fig 7, profilins I and II precipitated aczonin with similar efficiencies. In another set of experiments where equal quantities of recombinant profilins were immobilized on nickel agarose, profilin II consistently precipitated more aczonin than profilin I did. The established profilin-binding protein, Mena (
The specificity of profilin binding to aczonin was underpinned by additional controls (data not shown). Two additional His-tagged negative control constructs immobilized on nickel agarose, recombinant Rab3A (see below), and a 91 amino acid sequence from neurobeachin (construct C3; Wang, X., and M.W. Kilimann, unpublished data) did not precipitate either aczonin or Mena. A negative control target protein, neurobeachin, probed for by Western blotting, was not precipitated by the profilin constructs or by the negative control constructs. Profilins I and II bound both aczonin from the soluble fraction of a brain lysate (corresponding to fraction S of Fig 5 A), and aczonin from the sedimentable fraction solubilized by 0.1 M Na2CO3 (fraction S'/Na2CO3 of Fig 5 A) and then back-dialyzed against lysis buffer.
No Detectable Binding of Rab3A to Aczonin
The two zinc finger pairs of aczonin, but not their flanking sequences, have significant sequence similarity to the zinc finger structures of rabphilin-3A and Rim. Sequence regions from these two proteins encompassing the zinc fingers have been shown to bind Rab3A, a small G protein involved in synaptic vesicle trafficking. However, in three different types of experiments we were unable to detect binding of Rab3A and related Rab proteins to aczonin (data not shown). We expressed the two zinc finger pairs from mouse aczonin together with extensive flanking sequences (amino acids 374654 and 8631115, respectively) as GST fusion proteins, and probed for binding to recombinant Rab3A by blot overlay assay. No binding of recombinant Rab3A to these constructs was detected, in contrast to a corresponding Rim sequence (amino acids 11399 fused to GST) employed as a positive control, which displayed pronounced GTP-dependent binding of Rab3A on the same blot. Recombinant His-tagged Rab3A immobilized on nickel agarose also did not precipitate detectable amounts of holo-aczonin from brain lysate; however, as positive controls, Rab3A precipitated rabphilin-3A, and resin-bound recombinant profilin (see above) precipitated aczonin in the same experiment. Finally, immunoprecipitation of aczonin from brain lysate did not bring down Rab3A, Rab3B, Rab3C, or Rab5 in quantities detectable by Western blot analysis.
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Discussion |
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Aczonin is a very large multidomain protein of ~5,000 amino acids which is specifically concentrated at presynaptic active zones and firmly associated with a detergent-resistant, cytoskeletal-like subcellular fraction. It may be a scaffolding protein that structures the presynaptic cortical cytoplasm, interacts with multiple partner molecules, and orchestrates the interplay of neurotransmitter vesicles with the cytoskeleton, the plasma membrane, and probably cytosolic proteins at the active zone. From the aczonin sequence, several motifs stand out that also suggest links to plasmalemmal, vesicular, and cytoskeletal proteins.
The overall pattern of one PDZ and two C2 domains in the COOH-terminal region is similar to Rim. Whereas many PDZ domain proteins have been identified that are involved in the organization of postsynaptic protein arrays (
The two Cys4 zinc finger pairs of aczonin are homologous to each other, to two similar motifs in Bassoon, and to the single Cys4 zinc finger pairs of Rim, rabphilin-3A, and Noc2. According to cysteine residue spacing (CX2CX17CX2CX4CX2CX15CX2C in aczonin) and additional sequence similarity, the zinc finger pairs of these proteins constitute a distinct subfamily of zinc finger motifs, and the three-dimensional structure of its prototype, the rabphilin-3A zinc finger pair, has been solved recently (
Aczonin contains several proline-rich regions that may include targets for the binding of SH3- or WW-domaincontaining proteins, such as the NH2-terminal 1,100 amino acids around and upstream of the zinc fingers or a conserved sequence region at amino acids 23802500. Most strikingly, a polyproline stretch and short flanking sequences in the middle of the aczonin molecule are conserved between chicken, mouse, and humans, whereas several hundred amino acids around them are not or poorly conserved. This suggested an interaction with profilin, and we could indeed demonstrate that both profilin isoforms bind to aczonin and that this binding is blocked by homopolymeric proline but not by polyalanine. Profilin is an actin and phosphoinositide-binding protein expressed in many cell types, including neurons, where it is concentrated in synaptic terminals (
At the gene level, a polyproline tract is encoded by a triplet repeat that has an intrinsic tendency to expand or contract. Therefore, it may be argued that the polyproline tract of aczonin has arisen by serendipity and may be physiologically meaningless at the protein level, although like any polyproline sequence, it binds profilin. However, it is clearly accessible for profilin binding in the context of the complete aczonin molecule, and it is highly conserved in evolution, even between birds and mammals (chicken, 11; mouse, 22; humans, 22 uninterrupted proline residues and additional prolines immediately upstream or downstream), whereas its flanking sequences are not. Leucine residues at or near the ends of proline runs, as in the aczonin sequences, are also found in other profilin-binding proteins (
The zinc finger region of aczonin is flanked by oligopeptide repeats. Dekapeptide repeats upstream of the first zinc finger motif differ in number between chicken, humans, and mouse and even among different mouse cDNAs, whereas 22-mer repeats downstream from the second zinc finger pair are found only in chicken but not in mouse. Therefore, it seems likely that these repeats primarily served as an evolutionary mechanism to rapidly expand spacer sequences around the zinc finger modules. The 10-mer repeat region is similar (45% predicted amino acid sequence identity) to an untranslated repetitive sequence region in the bovine herpesvirus type 1 BICP22 gene (
We have also determined a partial cDNA sequence from the NH2-terminal region of human aczonin (codons 34794). A partial human cDNA sequence representing the 1,213 COOH-terminal amino acids of the short splicing variant of human aczonin has been reported (KIAA0559;
Computer-assisted secondary structure prediction from the aczonin sequence indicates a high potential of flexibility, but also some sequence stretches with high coiled-coil potential that are shared with Bassoon. For example, anchored at the plasma membrane to ion channels or other transmembrane proteins through its PDZ domain, aczonin could potentially reach into the synaptic terminal across several neurotransmitter vesicle diameters. To give an upper-limit estimate, 5,000 amino acids could form an extended -helix 750-nm long, i.e., 15 vesicle diameters. Thus, aczonin may play a role in organizing the supramolecular structure of the cortical cytomatrix at the active zone. It could constitute or be part of the longer strands seen in quick-freeze deep-etch electron microscopy to tether synaptic vesicles to the active zone (
According to immunoblot analysis and immunolight microscopy, aczonin is found throughout the brain. By immunoelectron microscopy of selected brain areas, we detect it very consistently in the mossy fiber terminals of cerebellar glomeruli, but only in a fraction of the terminals of other synapse populations. It remains to be clarified whether aczonin is present in all synapses, albeit at different concentrations, or only in specific populations, and with what functional features of synapses its level of expression and its different splicing variants correlate.
It will be of particular interest to understand the relationship between aczonin and its partial homologues, Bassoon and Rim. It will also be interesting to see whether Piccolo, which resembles both aczonin and Bassoon in molecular size, subcellular distribution, and immunomorphology, is identical to aczonin or whether it constitutes an additional member of this protein family. It is conceivable that these proteins can partially substitute for each other in different types of synapses, that their homologous domains interact with different isoforms or homologues of partner proteins, or that they work hand in hand within the same synapse. For example, aczonin, with a longer reach into the presynaptic cytoplasm than Rim, may usher vesicles towards the plasma membrane without interfering with Rab3 bound to them, and hand them over to Rim immediately before docking.
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Acknowledgements |
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We thank Frank Gertler, Walter Witke, and Wanjin Hong for antibodies.
This work was supported by the Deutsche Forschungsgemeinschaft, the University of Bochum Medical School (FoRUM intramural research funds), and the Fonds der Chemischen Industrie.
Submitted: 2 June 1999
Revised: 5 August 1999
Accepted: 23 August 1999
1.used in this paper: GST, glutathione S-transferase
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References |
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