Aczonin, a 550-kD putative scaffolding protein of presynaptic active zones, shares homology regions with Rim and Bassoon and binds profilin - PubMed (original) (raw)

Aczonin, a 550-kD putative scaffolding protein of presynaptic active zones, shares homology regions with Rim and Bassoon and binds profilin

X Wang et al. J Cell Biol. 1999.

Abstract

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 Cys(4) 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.

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Figures

Figure 1

Figure 1

Sequence alignment of mouse aczonin (mACZ), chicken aczonin (cACZ), and rat Bassoon (Bsn). The long splicing variants are shown (mouse, L; chicken, XL), and the position at mouse codon 4829 is indicated by an asterisk where the QQLRIQP sequence can instead be followed by the short SKRRK COOH terminus. Overlining beginning at mouse codon 430 marks three 10-mer repeat units deleted in some mouse cDNAs. The chicken sequence is incomplete for the ∼80 NH2-terminal codons. Upstream of the putative start codon, the mouse cDNA contig continues for 304 nucleotides of GC-rich sequence with no in-frame stop codon. Additional rescreenings did not yield sequences reaching further upstream. Between aczonin and Bassoon, the first nine codons are synonymous, whereas the upstream cDNA sequences are completely dissimilar, also suggesting that the codon assumed here as methionine 1 is the true start codon. Specific sequence motifs (see Fig. 2) are framed by arrowheads above the mouse sequence (except the zinc finger and polyproline motifs that are self-evident) and designated at the right margin. The rat Bassoon sequence is taken from tom Dieck et al. 1998. EMBL/GenBank/DDBJ sequence database accession numbers are Y19185-6 (mouse aczonin-L and S), Y19187 (chicken aczonin-XL), and Y19188 (partial human aczonin; data not shown).

Figure 2

Figure 2

Regional organization of aczonin and partial homology to Bassoon and Rim. For aczonin, wide bars indicate sequence regions with high similarity between chicken and mouse, and narrow bars indicate sequences with low interspecies conservation. Triangles mark regions with 10-mer repeats, and ovoids mark the region with 22-mer repeats in the chicken sequence (actual repeat units are shorter and more numerous than these symbols). Black boxes represent zinc finger (Zn), polyproline (PP), PDZ, and C2 modules as indicated. Shaded boxes in Bassoon and Rim indicate additional sequence regions with similarity to aczonin. Cross-hatched boxes indicate a sequence region of particularly high conservation between mouse aczonin, chicken aczonin, and Bassoon. Regions of sequence similarity are connected by dashed lines. In aczonin, a vertical dashed line near the NH2 terminus indicates the end of the chicken sequence, and two vertical lines between the C2 modules indicate the sites of differential splicing.

Figure 3

Figure 3

Tissue specificity of aczonin mRNA and protein expression. (A) Chicken aczonin mRNA (10 μg poly(A)+ RNA per lane). (B) Human aczonin mRNA (2 μg poly(A)+ RNA per lane). (C) Mouse aczonin protein (80 μg of tissue homogenate protein per lane). Tissue abbreviations are: A, adrenal gland; AC, adrenal cortex; AM, adrenal medulla; B, brain; BS, brain stem; C, cerebellum; FB, forebrain; H, heart; I, small intestine; K, kidney; Li, liver; Lu, lung; M, muscle; O, ovary; Pa, pancreas; Pl, placenta; Sp, spleen; St, stomach; Te, testis; Tm, thymus; and Tr, thyroid. Smears are attributed to partial degradation of these very long mRNA and protein molecules. Long exposures are shown to illustrate tissue specificity.

Figure 4

Figure 4

Immunohisto-chemical localization of aczonin in rat brain. Light microscopic inspection of the cerebellar cortex (A) shows finely punctate staining of the molecular layer (m), and ring-shaped or patchy immunopositive structures in the granule cell layer (g), whereas the medulla (md) is immunonegative. p indicates Purkinje cell layer. Electron microscopy shows that immunoperoxidase reaction product is restricted to the presynaptic active zones (B) of an asymmetric synapse with a dendritic spine in the molecular layer of the dentate gyrus or (C) of a mossy fiber terminal in a cerebellar glomerulus. In B, note that aczonin immunoreactivity is focused to the two junctional zones of the perforated synaptic specialization. Bar: 115 μm (A); 0.33 μm (B); or 1 μm (C).

Figure 5

Figure 5

Distribution of aczonin in subcellular fractionation. (A) Mouse brain homogenate was subjected to 120,000 g fractionation (S, supernatant; P, pellet) in a detergent-free homogenization buffer (HB) containing 150 mM NaCl as described in Materials and Methods. The pellet fraction P was resuspended in the homogenization buffer (HB) or in various extraction buffers (1 M NaCl in homogenization buffer; 1% Triton X-100 in homogenization buffer without NaCl; 100 mM Na2CO3, pH 11.5; 6 M guanidinium chloride) and recentrifuged at 120,000 g. Supernatant and pellet fractions after recentrifugation are termed S′ and P′. Equal aliquots of all fractions were analyzed by SDS-PAGE and immunoblotting with aczonin, tubulin, and synaptophysin antibodies. In the experiment shown, extraction was carried out at 4°C for 20 min. The same distribution was obtained when extraction was performed at room temperature for 30 min. In additional experiments not shown, aczonin could be partially extracted from the pellet by 8 M urea, but not by 3% NP-40. (B) Synaptic vesicles were purified from rat brain according to Hell et al. 1988: H, homogenate; S1 and P1, 47,000 g supernatant and pellet derived from H; S2 and P2, 120,000 g supernatant and pellet derived from S1; supernatant S3, fluffy layer L3, cushion C3, and pellet P3 from the 260,000 g spin of S2; P3′, resuspended and cleared P3 before controlled-pore glass chromatography; PIP and PIIP, pools from breakthrough peak and vesicle peak of the controlled-pore glass chromatography. 30 μg protein was applied per lane and analyzed by immunoblotting as indicated.

Figure 6

Figure 6

In neuronal cell lines, aczonin is associated with endomembranes. In PC12 cells as shown, double-immunofluorescence demonstrates colocalization with the Golgi complex marker, mannosidase II.

Figure 7

Figure 7

Aczonin binds profilin. Recombinant profilins I and II covalently coupled to Sepharose precipitate aczonin from mouse brain lysate (S, supernatant; P, pellet). Profilin binding is blocked by preincubation of the profilin resin with polyproline (PP), but not by polyalanine (PA). Immobilized BSA as a negative control does not precipitate aczonin. See Results for additional control experiments not shown.

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