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Molecular dissection of the photoreceptor ribbon synapse: physical interaction of Bassoon and RIBEYE is essential for the assembly of the ribbon complex.

tom Dieck S, Altrock WD, Kessels MM, Qualmann B, Regus H, Brauner D, Fejtová A, Bracko O, Gundelfinger ED, Brandstätter JH - J. Cell Biol. (2005)

Bottom Line: Identifiable CAZ proteins segregate into two compartments at the ribbon: a ribbon-associated compartment including Piccolo, RIBEYE, CtBP1/BARS, RIM1, and the motor protein KIF3A, and an active zone compartment including RIM2, Munc13-1, a Ca2+ channel alpha1 subunit, and ERC2/CAST1.A direct interaction between the ribbon-specific protein RIBEYE and Bassoon seems to link the two compartments and is responsible for the physical integrity of the photoreceptor ribbon complex.Finally, we found the RIBEYE homologue CtBP1 at ribbon and conventional synapses, suggesting a novel role for the CtBP/BARS family in the molecular assembly and function of central nervous system synapses.

View Article: PubMed Central - PubMed

Affiliation: Department of Neuroanatomy, Max Planck Institute for Brain Research, D-60528 Frankfurt/Main, Germany.

ABSTRACT
The ribbon complex of retinal photoreceptor synapses represents a specialization of the cytomatrix at the active zone (CAZ) present at conventional synapses. In mice deficient for the CAZ protein Bassoon, ribbons are not anchored to the presynaptic membrane but float freely in the cytoplasm. Exploiting this phenotype, we dissected the molecular structure of the photoreceptor ribbon complex. Identifiable CAZ proteins segregate into two compartments at the ribbon: a ribbon-associated compartment including Piccolo, RIBEYE, CtBP1/BARS, RIM1, and the motor protein KIF3A, and an active zone compartment including RIM2, Munc13-1, a Ca2+ channel alpha1 subunit, and ERC2/CAST1. A direct interaction between the ribbon-specific protein RIBEYE and Bassoon seems to link the two compartments and is responsible for the physical integrity of the photoreceptor ribbon complex. Finally, we found the RIBEYE homologue CtBP1 at ribbon and conventional synapses, suggesting a novel role for the CtBP/BARS family in the molecular assembly and function of central nervous system synapses.

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Biochemical and yeast two-hybrid analysis of the Bassoon interaction with members of the CtBP family. (A) Immunoblots showing that Bassoon complexes containing RIBEYE, Piccolo, and ERC2/CAST1 can be coimmunoprecipitated from wild-type retina extracts with a monoclonal anti-Bassoon antibody. Under the conditions used, no considerable amounts of RIM1/2, Munc13-1, and cadherin are detectable in immunoprecipitates. (B) Yeast two-hybrid analysis shows strong binding of Bassoon constructs harboring aa 1653–2087 (fragments RB29, RB35, RB46) with the RIBEYE B-domain and the RIBEYE homologue CtBP1. Construct RB45 shows only a weak interaction; construct RB44 is self activating. Specificity of the binding is shown by the lack of binding to the RIBEYE A-domain. −, no reporter gene activation; −/+, weak reporter gene activation; +++, strong reporter gene activation. Zn1, Zn2: double zinc fingers; cc1, cc2, cc3: coiled-coil domains; poly-Q: polyglutamine stretch. Numbers indicate Piccolo–Bassoon homology regions 1–10; dashed line indicates the region of Bassoon missing in the mutant protein.
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fig4: Biochemical and yeast two-hybrid analysis of the Bassoon interaction with members of the CtBP family. (A) Immunoblots showing that Bassoon complexes containing RIBEYE, Piccolo, and ERC2/CAST1 can be coimmunoprecipitated from wild-type retina extracts with a monoclonal anti-Bassoon antibody. Under the conditions used, no considerable amounts of RIM1/2, Munc13-1, and cadherin are detectable in immunoprecipitates. (B) Yeast two-hybrid analysis shows strong binding of Bassoon constructs harboring aa 1653–2087 (fragments RB29, RB35, RB46) with the RIBEYE B-domain and the RIBEYE homologue CtBP1. Construct RB45 shows only a weak interaction; construct RB44 is self activating. Specificity of the binding is shown by the lack of binding to the RIBEYE A-domain. −, no reporter gene activation; −/+, weak reporter gene activation; +++, strong reporter gene activation. Zn1, Zn2: double zinc fingers; cc1, cc2, cc3: coiled-coil domains; poly-Q: polyglutamine stretch. Numbers indicate Piccolo–Bassoon homology regions 1–10; dashed line indicates the region of Bassoon missing in the mutant protein.

Mentions: The Bassoon mutant phenotype suggests that interactions of Bassoon with its natural binding partners are involved in the assembly of the ribbon structure. Therefore, we immunoprecipitated and analyzed Bassoon protein complexes from wild-type retina. Our immunoblot analyses revealed that large amounts of RIBEYE, Piccolo, and ERC2/CAST1 were coimmunoprecipitated specifically with Bassoon (Fig. 4 A). Control IgG did not precipitate these proteins (Fig. 4 A). Only very low amounts of RIM1/2 and Munc13-1 coimmunoprecipitated with Bassoon as observed upon long exposition of the blots shown in Fig. 4 A (unpublished data). The cell adhesion molecule cadherin (Fig. 4 A), the Ca2+ channel α1 subunit (unpublished data), and the kinesin motor protein KIF3A (see Fig. 6 F) were not coimmunoprecipitated with Bassoon. There was also no immunoprecipitation of the polyphosphoinositide phosphatase synaptojanin 1 (see Fig. 6 F), which was recently shown to be the protein mutated in the zebrafish nrc mutant that displays a photoreceptor phenotype similar, although not completely identical, to the Bassoon mutant phenotype (Van Epps et al., 2004).


Molecular dissection of the photoreceptor ribbon synapse: physical interaction of Bassoon and RIBEYE is essential for the assembly of the ribbon complex.

tom Dieck S, Altrock WD, Kessels MM, Qualmann B, Regus H, Brauner D, Fejtová A, Bracko O, Gundelfinger ED, Brandstätter JH - J. Cell Biol. (2005)

Biochemical and yeast two-hybrid analysis of the Bassoon interaction with members of the CtBP family. (A) Immunoblots showing that Bassoon complexes containing RIBEYE, Piccolo, and ERC2/CAST1 can be coimmunoprecipitated from wild-type retina extracts with a monoclonal anti-Bassoon antibody. Under the conditions used, no considerable amounts of RIM1/2, Munc13-1, and cadherin are detectable in immunoprecipitates. (B) Yeast two-hybrid analysis shows strong binding of Bassoon constructs harboring aa 1653–2087 (fragments RB29, RB35, RB46) with the RIBEYE B-domain and the RIBEYE homologue CtBP1. Construct RB45 shows only a weak interaction; construct RB44 is self activating. Specificity of the binding is shown by the lack of binding to the RIBEYE A-domain. −, no reporter gene activation; −/+, weak reporter gene activation; +++, strong reporter gene activation. Zn1, Zn2: double zinc fingers; cc1, cc2, cc3: coiled-coil domains; poly-Q: polyglutamine stretch. Numbers indicate Piccolo–Bassoon homology regions 1–10; dashed line indicates the region of Bassoon missing in the mutant protein.
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Related In: Results  -  Collection

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fig4: Biochemical and yeast two-hybrid analysis of the Bassoon interaction with members of the CtBP family. (A) Immunoblots showing that Bassoon complexes containing RIBEYE, Piccolo, and ERC2/CAST1 can be coimmunoprecipitated from wild-type retina extracts with a monoclonal anti-Bassoon antibody. Under the conditions used, no considerable amounts of RIM1/2, Munc13-1, and cadherin are detectable in immunoprecipitates. (B) Yeast two-hybrid analysis shows strong binding of Bassoon constructs harboring aa 1653–2087 (fragments RB29, RB35, RB46) with the RIBEYE B-domain and the RIBEYE homologue CtBP1. Construct RB45 shows only a weak interaction; construct RB44 is self activating. Specificity of the binding is shown by the lack of binding to the RIBEYE A-domain. −, no reporter gene activation; −/+, weak reporter gene activation; +++, strong reporter gene activation. Zn1, Zn2: double zinc fingers; cc1, cc2, cc3: coiled-coil domains; poly-Q: polyglutamine stretch. Numbers indicate Piccolo–Bassoon homology regions 1–10; dashed line indicates the region of Bassoon missing in the mutant protein.
Mentions: The Bassoon mutant phenotype suggests that interactions of Bassoon with its natural binding partners are involved in the assembly of the ribbon structure. Therefore, we immunoprecipitated and analyzed Bassoon protein complexes from wild-type retina. Our immunoblot analyses revealed that large amounts of RIBEYE, Piccolo, and ERC2/CAST1 were coimmunoprecipitated specifically with Bassoon (Fig. 4 A). Control IgG did not precipitate these proteins (Fig. 4 A). Only very low amounts of RIM1/2 and Munc13-1 coimmunoprecipitated with Bassoon as observed upon long exposition of the blots shown in Fig. 4 A (unpublished data). The cell adhesion molecule cadherin (Fig. 4 A), the Ca2+ channel α1 subunit (unpublished data), and the kinesin motor protein KIF3A (see Fig. 6 F) were not coimmunoprecipitated with Bassoon. There was also no immunoprecipitation of the polyphosphoinositide phosphatase synaptojanin 1 (see Fig. 6 F), which was recently shown to be the protein mutated in the zebrafish nrc mutant that displays a photoreceptor phenotype similar, although not completely identical, to the Bassoon mutant phenotype (Van Epps et al., 2004).

Bottom Line: Identifiable CAZ proteins segregate into two compartments at the ribbon: a ribbon-associated compartment including Piccolo, RIBEYE, CtBP1/BARS, RIM1, and the motor protein KIF3A, and an active zone compartment including RIM2, Munc13-1, a Ca2+ channel alpha1 subunit, and ERC2/CAST1.A direct interaction between the ribbon-specific protein RIBEYE and Bassoon seems to link the two compartments and is responsible for the physical integrity of the photoreceptor ribbon complex.Finally, we found the RIBEYE homologue CtBP1 at ribbon and conventional synapses, suggesting a novel role for the CtBP/BARS family in the molecular assembly and function of central nervous system synapses.

View Article: PubMed Central - PubMed

Affiliation: Department of Neuroanatomy, Max Planck Institute for Brain Research, D-60528 Frankfurt/Main, Germany.

ABSTRACT
The ribbon complex of retinal photoreceptor synapses represents a specialization of the cytomatrix at the active zone (CAZ) present at conventional synapses. In mice deficient for the CAZ protein Bassoon, ribbons are not anchored to the presynaptic membrane but float freely in the cytoplasm. Exploiting this phenotype, we dissected the molecular structure of the photoreceptor ribbon complex. Identifiable CAZ proteins segregate into two compartments at the ribbon: a ribbon-associated compartment including Piccolo, RIBEYE, CtBP1/BARS, RIM1, and the motor protein KIF3A, and an active zone compartment including RIM2, Munc13-1, a Ca2+ channel alpha1 subunit, and ERC2/CAST1. A direct interaction between the ribbon-specific protein RIBEYE and Bassoon seems to link the two compartments and is responsible for the physical integrity of the photoreceptor ribbon complex. Finally, we found the RIBEYE homologue CtBP1 at ribbon and conventional synapses, suggesting a novel role for the CtBP/BARS family in the molecular assembly and function of central nervous system synapses.

Show MeSH
Related in: MedlinePlus