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

Wang X, Kibschull M, Laue MM, Lichte B, Petrasch-Parwez E, Kilimann MW - J. Cell Biol. (1999)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Institut für Physiologische Chemie, Ruhr-Universität Bochum, D-44780 Bochum, Germany.

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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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.
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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.

Mentions: 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.


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

Wang X, Kibschull M, Laue MM, Lichte B, Petrasch-Parwez E, Kilimann MW - J. Cell Biol. (1999)

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.
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Related In: Results  -  Collection

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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.
Mentions: 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.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Institut für Physiologische Chemie, Ruhr-Universität Bochum, D-44780 Bochum, Germany.

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.

Show MeSH
Related in: MedlinePlus