Identification of CAST and molecular cloning of its cDNA
To isolate proteins concentrated in the synaptic junction, proteins were extracted from the P2 and PSD fractions of rat brain by a urea-based buffer, and each extract was then subjected to Mono Q column chromatography. Each fraction was subjected to SDS-PAGE, followed by protein staining with silver. When each protein band was compared between the P2 and PSD fractions, 20 protein bands were more concentrated in the PSD than in the P2 fraction (unpublished data). Some of these protein bands are shown in . The corresponding bands for the 20 proteins were cut out from the gels and analyzed by MALDI-TOF mass spectrometry. When a database was searched, most spectra had significant matches to known proteins, such as bassoon (tom Dieck et al., 1998
), synamon/shank 1A (Naisbitt et al., 1999
; Yao et al., 1999
), and synapse-associated protein (SAP) 102 (Muller et al., 1996
; unpublished data). These proteins are enriched in the PSD or presynaptic plasma membrane fraction, indicating that this approach is effective to identify new proteins enriched in the synaptic junction.
Figure 1. Mono Q column chromatographies of the P2 and PSD fractions. Proteins were extracted from the P2 and PSD fractions and each extract was subjected to Mono Q column chromatography. An aliquot (20 μl) of each eluted fraction was subjected to 7.5% (more ...)
Two spectra for ~500- and ~120-kD bands (p500 and p120) had no significant matches in the database. Thus, we determined the partial aa sequences of p500 and p120. Computer homology search revealed that the two peptides derived from p500 were contained within the aa sequence of piccolo (Fenster et al., 2000
). The four peptides derived from p120 were contained in the primary sequence deduced from a human cDNA (KIAA0378). However, this cDNA appeared to lack its NH2
-terminal portion and its function has been unknown. We obtained the full-length cDNA of p120, which encoded the protein consisting of 957 aa with a calculated molecular weight of 110,616 ( A). We named this protein CAST. CAST had no transmembrane segment but had four coiled-coil domains. The COOH-terminal three aa (IWA) was a putative consensus motif for binding to PDZ domains (Songyang et al., 1997
). To confirm whether this clone encoded the full-length cDNA, we constructed an expression vector with the cDNA and expressed the protein by an in vitro translation system. Western blot analysis using a polyclonal antibody (Ab) (anti–CAST-1 Ab) indicated that the expressed protein showed mobility similar to that of native CAST on SDS-PAGE ( B). Thus, we concluded that this clone encoded the full-length cDNA of CAST.
Figure 2. Full-length sequence of CAST. (A) Deduced aa sequence of CAST. Underlines indicate the aa sequences of the four peptide peaks. Boxes indicate coiled-coil domains. The putative consensus motif for binding to PDZ domains is shown in bold letters. These (more ...)
Tissue and subcellular distribution of CAST
Western blot analysis showed that the anti–CAST-1 Ab recognized a protein band of ~120 kD in rat brain, but not in other rat tissues including heart, spleen, lung, muscle, kidney, and testis ( A). This result indicates that CAST is mainly expressed in the brain. Subcellular distribution analysis in rat brain showed that CAST was concentrated in the PSD fraction ( B). The subcellular distribution pattern of CAST was similar to that of the NMDA receptor. In addition, CAST was resistant to solubilization by CHAPS, a zwitter ionic detergent, and NP-40 and Triton X-100, nonionic detergents, although CAST was solubilized by SDS and deoxycholate ( C). These results indicate that CAST is a synaptic protein and tightly associated with the cytoskeletal structure.
Figure 3. Tissue and subcellular distribution of CAST and its solubilization property by various detergents. (A) Tissue distribution. The homogenates of various rat tissues (20 μg of protein each) were analyzed by Western blotting using the anti–CAST-1 (more ...)
Localization of CAST at the CAZ
We then examined the spatial distribution of CAST in a sagittal section of adult mouse brain using another anti-CAST polyclonal Ab (anti–CAST-2 Ab). A widespread immunoreactivity of CAST was detected throughout the central nervous system, including the hippocampus, the cortex, the cerebellum, and the olfactory bulb ( A). Consistently, Western blot analysis revealed that CAST was expressed in the various rat brain regions, such as the hippocampus, the cortex, the cerebellum, the amygdala, and the olfactory bulb (unpublished data). We next examined the precise localization of CAST in the hippocampus by immunohistochemistry. The staining pattern of CAST was compared with that of synaptotagmin I, a synaptic vesicle protein (Matthew et al., 1981
). In the CA3 region of the hippocampus, CAST showed the most intense signal in the stratum lucidum where the synapses are formed between the mossy fiber terminals and the dendrites of pyramidal cells ( B). The staining pattern of CAST was similar to that of synaptotagmin I. In primary cultured rat hippocampal neurons, CAST was also colocalized with bassoon and PSD-95/SAP90 ( C). Immunoelectron microscopic analysis of mouse brain showed that the immunoreactivity of CAST was detected in the presynaptic nerve terminals in the stratum lucidum of the CA3 region (). The CAST signal was concentrated at the CAZ. These results indicate that CAST is localized at the CAZ.
Figure 4. Synaptic localization of CAST. (A) Gross distribution of CAST in mouse brain. (B) CA3 region of mouse hippocampus. The sections were doubly stained using the anti–CAST-2 and anti–synaptotagmin I Abs. (a) Low magnification. (b) High magnification. (more ...)
Figure 5. Localization of CAST at the CAZ in mouse hippocampus. The sections were reacted with the anti–CAST-2 Ab, incubated with immunogold particles (1.4 nm) conjugated with goat IgG against rabbit IgG, and silver enhanced, followed by electron microscopic (more ...)
A ternary complex of CAST with RIM1 and Munc13-1
To explore a function of CAST at the CAZ, we examined the binding of CAST to other CAZ proteins. For this purpose, we first immunoprecipitated CAST by its Ab from the extract of the P2 fraction of rat brain. Among the proteins examined, RIM1 and bassoon were coimmunoprecipitated with CAST ( A, a). Neither Munc13-1 nor PSD-95/SAP90 was coimmunoprecipitated with CAST. Our anti-RIM1 Ab could not be used for immunoprecipitation, but our antibassoon Ab could. CAST and RIM1 were coimmunoprecipitated with bassoon by the antibassoon Ab ( A, b). Furthermore, Munc13-1 was coimmunoprecipitated with bassoon. Because it has been shown that Munc13-1 directly binds RIM1 (Betz et al., 2001
), the RIM1–Munc13-1 complex might be coimmunoprecipitated with bassoon. These results suggest that CAST is associated with other CAZ proteins, including at least RIM1, bassoon, and Munc13-1, although it is unknown why Munc13-1 was not coimmunoprecipitated with CAST by its Ab.
Figure 6. Binding of CAST, RIM1, bassoon, and Munc13-1. (A) Coimmunoprecipitation of other CAZ proteins with CAST. The extract of the P2 fraction was subjected to immunoprecipitation by the anti–CAST-1 or antibassoon Ab. The immunoprecipitate was analyzed (more ...)
We next confirmed the binding of CAST, RIM1, bassoon, and Munc13-1 by the cosedimentation assay. The extract of the P2 fraction was incubated with the Myc–CAST-coupled or Myc–RIM1-coupled affinity beads. Native RIM1 and bassoon bound to the Myc–CAST-coupled affinity beads and native CAST and bassoon bound to the Myc–RIM1-coupled affinity beads ( B). Moreover, native Munc13-1 bound not only to the Myc–RIM1-coupled affinity beads but also to the CAST-coupled affinity beads ( B).
Because it was not clear from these results whether the binding of CAST to other CAZ proteins is direct or indirect, we examined whether CAST forms a complex directly with RIM1 and Munc13-1. We could not examine whether CAST forms a complex with bassoon, because bassoon is a very large protein and transfection of its cDNA has not been done (tom Dieck et al., 1998
). We transfected each expression plasmid of CAST, RIM1, or Munc13-1 into HEK293 cells, extracted each protein, and mixed them in various combinations, followed by immunoprecipitation using the anti-GFP Ab for CAST or the anti-HA Ab for RIM1. RIM1 was coimmunoprecipitated with CAST by the anti-GFP Ab for CAST ( C). Munc13-1 was not, however, coimmunoprecipitated with CAST in the presence or absence of RIM1. Conversely, CAST and/or Munc13-1 were coimmunoprecipitated with RIM1 by the anti-HA Ab. These results, together with the earlier observation that Munc13-1 directly binds RIM1 (Betz et al., 2001
), indicate that CAST forms a ternary complex with at least RIM1 and Munc13-1 by directly binding RIM1 and indirectly binding Munc13-1. It is currently unclear why Munc13-1 is not coimmunoprecipitated with CAST by its Ab, but the immunoprecipitation of CAST might affect the binding of RIM1 and Munc13-1, which is in part consistent with the result in A, a. In addition, bassoon appears to be associated with the ternary complex of CAST, RIM1, and Munc13-1, but it remains to be clarified how bassoon interacts with this complex.
We finally confirmed the direct binding of CAST and RIM1 in a heterologous expression system. EGFP–CAST-1 (full length) or Myc–RIM1 (full length) was expressed in HEK293 cells. EGFP–CAST-1 formed large aggregates ( A, a) and was recovered in the Triton X-100–insoluble fraction ( B, a). In contrast, Myc–RIM1 was mainly distributed in the nucleus ( A, a) and recovered in the Triton X-100–soluble fraction ( B, b). EGFP was distributed throughout the cytoplasm ( A, a). When both EGFP–CAST-1 and Myc–RIM1 were expressed, CAST formed large aggregates again and RIM1 was colocalized with CAST at the aggregates ( A, b). Moreover, RIM1, as well as CAST, was recovered in the Triton X-100–insoluble fraction ( B, c). In contrast, PSD-95/SAP90, which contains three PDZ domains, was not colocalized with CAST ( A, c). These results indicate that CAST forms aggregates and recruits RIM1 to the Triton X-100–insoluble structure and provide another line of evidence for the direct binding of CAST and RIM1.
Figure 7. Aggregation of CAST and recruitment of RIM1 to the aggregates. (A) Colocalization of RIM1 with CAST at the aggregates of CAST. HEK293 cells were transfected with pEGFP–CAST-1 alone, pCMV-Tag3–RIM1 alone, or both. At 36–48 h after (more ...)
Because CAST has a putative COOH-terminal consensus motif for binding to PDZ domains () (Songyang et al., 1997
) and RIM1 has one PDZ domain (Wang et al., 1997
), we examined the direct binding of CAST and RIM1 through the COOH-terminal consensus motif and the PDZ domain by the pull-down assay. The extract of HEK293 cells expressing Myc–RIM1 was incubated with glutathione-Sepharose beads containing various GST fusion proteins of CAST ( A, a). Myc–RIM1 stoichiometrically bound to GST–CAST-4 containing the COOH-terminal consensus motif, but not to other GST fusion proteins ( A, b and c), indicating that the COOH-terminal consensus motif of CAST was essential for its binding to RIM1. We then confirmed that CAST binds to the PDZ domain of RIM1. The extract of HEK293 cells expressing the Myc-tagged COOH terminus of CAST (Myc–CAST-4) was incubated with glutathione-Sepharose beads containing various GST fusion proteins of the PDZ domains of RIM1 and PSD-95/SAP90. Myc–CAST-4 bound to the GST fusion protein containing the PDZ domain of RIM1, but not to GST alone or the GST fusion proteins containing each of the three PDZ domains of PSD-95/SAP90 ( B). Finally, we confirmed the specific binding of the COOH-terminal consensus motif of CAST and the PDZ domain of RIM1 by the immunoprecipitation assay ( C). These results indicate that the binding of CAST and RIM1 is mediated through the COOH-terminal consensus motif and the PDZ domain.
Figure 8. Direct binding of CAST and RIM1. (A) The RIM1-binding site of CAST. (a) GST constructs of CAST. CC, coiled-coil domain. (b) Direct binding of RIM1 to the COOH terminus of CAST. The GST fusion proteins containing various regions of CAST in panel a as well (more ...)
Mechanism of the localization of CAST and RIM1 in neurons
We then examined the mechanism of the localization of CAST and RIM1 in primary cultured rat hippocampal neurons by expressing their various mutants. The various CAST mutants are schematically shown in A, and their activities for their colocalization with bassoon (see below) and binding to RIM1 (unpublished data) are also summarized in the figure. The RIM1-binding activity of the CAST mutants was estimated by the pull-down assay (see Materials and methods). We first expressed both EGFP–CAST-1 and Myc–RIM1 in cultured neurons. Both proteins were colocalized at the synaptic boutons as estimated by the localization of synaptophysin, a well-known synaptic protein (Wiedenmann and Franke, 1985
; B, a). Bassoon was also colocalized there (unpublished data), consistent with the earlier observation that it is synaptically localized in cultured neurons (tom Dieck et al., 1998
). When EGFP–CAST-1 alone was expressed, it colocalized with bassoon ( B, b). EGFP–CAST-2, which lacks only the COOH-terminal three aa (IWA) and does not bind RIM1, colocalized with bassoon ( B, c). In contrast, EGFP–CAST-6, which has the COOH-terminal three aa (IWA) and binds RIM1 but lacks the most NH2
-terminal region, was diffusely distributed and not clustered, as compared with bassoon ( B, d). EGFP–CAST-3, which lacks the third and fourth coiled-coil domains and the COOH-terminal three aa (IWA), colocalized with bassoon, whereas EGFP–CAST-4, which lacks the first and second coiled-coil domains and the COOH-terminal three aa (IWA) but contains the third and fourth coiled-coil domains, did not colocalize with bassoon (unpublished data). EGFP–CAST-5, which contains only the fourth coiled-coil domain and the COOH-terminal three aa (IWA), did not colocalize with bassoon either (unpublished data). These results indicate that CAST is synaptically localized in cultured neurons through its NH2
-terminal half containing at least the first and second coiled-coil domains in a manner independent of RIM1.
Figure 9. RIM1-independent colocalization of CAST with bassoon in primary cultured rat hippocampal neurons. (A) pEGFP constructs of CAST. The activities of CAST for colocalization with bassoon and for binding to RIM1 are summarized. The lysate of HEK293 cells expressing (more ...)
As for the mechanism of the localization of RIM1 in neurons, Myc–RIM1 was colocalized with bassoon ( A, a) and CAST (unpublished data), when it was expressed in cultured neurons. In contrast, a Myc-tagged deletion mutant (Myc–RIM1ΔPDZ), which lacks the PDZ domain and does not bind to CAST, was diffusely distributed and not clustered, as compared with bassoon ( A, b). When the Myc-tagged PDZ domains of RIM1 (Myc–RIM1 PDZ) and EGFP–CAST-1 were coexpressed, they were colocalized with bassoon ( A, c). An essentially similar result was obtained when Myc–RIM1 PDZ alone was expressed (unpublished data). Thus, the PDZ domain of RIM1 appears to play a role, at least partly, in the localization of RIM1 in cultured neurons.
Figure 10. Mechanism of localization of RIM1 in primary cultured rat hippocampal neurons. (A) Colocalization of RIM1 with bassoon in cultured neurons. Neurons at 7 d of culture were transfected with the indicated expression vectors, and stained with the anti-Myc (more ...)
We finally examined the role of CAST in the localization of RIM1. When EGFP–CAST-2 and Myc–RIM1 were coexpressed in cultured neurons, both the proteins were colocalized with synaptophysin ( B, a), consistent with the results in B, suggesting that Myc–RIM1 binds endogenous CAST and/or other presynaptic proteins. In contrast, when EGFP–CAST-6 and Myc–RIM1 were coexpressed in cultured neurons, both the proteins were diffusely localized and not clustered, as compared with synaptophysin ( B, b). However, Myc–RIM1 was often colocalized with synaptophysin even when EGFP–CAST-6 was diffusely localized (unpublished data). Taken together, it is likely that CAST plays a role, at least partly, in the localization of RIM1 in cultured neurons, but that another presynaptic protein(s) is additionally involved in this localization of RIM1.
Temporal and spatial localization of CAST during synapse formation
In the last set of experiments, we examined the temporal and spatial localization of CAST during synapse formation, using young primary cultured rat hippocampal neurons as well as rat brain tissue. Western blot analysis using rat brain homogenates of various developmental stages showed that the expression of CAST, as well as of RIM1, bassoon, and Munc13-1, was detected from early stages, and the levels of expression of these CAZ proteins did not significantly change during the developmental stages tested, although those of synaptophysin and PSD-95/SAP90 were sharply increased ( A).
Figure 11. Expression and localization of CAST at the early stages of synapse formation. (A) Temporal expression of the CAZ proteins during synapse formation. The homogenates (20 μg of protein each) from rat brain tissues at embryonic day (E) 18, through (more ...)
Immunofluorescence microscopic analysis of CAST in cultured neurons revealed that after 2 d of culture, when minor processes appeared, the immunoreactivity of CAST was detected as dotty signals in the cell body and fine processes, which was similar to that of bassoon (unpublished data). After 3–4 d of culture, when the axonal outgrowth was observed, the immunoreactivity of CAST was detected as dotty signals in the axon shaft and the growth cone ( B), which were partly colocalized with the signals of bassoon.
Because our anti-RIM1 Ab could not be used for immunofluorescence microscopic analysis of RIM1, we examined the localization of exogenously expressed RIM1 at the early stages of synapse formation. When EGFP–CAST-1 alone was first expressed, it was colocalized with bassoon ( C, a), consistent with the results in B. Moreover, EGFP–CAST-2, which lacks only the COOH-terminal three aa (IWA) and does not bind RIM1, was also colocalized with bassoon (unpublished data). When Myc–RIM1 alone was expressed, it was indeed colocalized with bassoon and CAST ( C, b and c). In contrast, Myc–RIM1ΔPDZ was diffusely localized and not clustered, as compared with bassoon ( C, d), suggesting that the PDZ domain of RIM1 is at least partly required for its clustering with bassoon at the early stages.
The expression of CAST at the early stages of synapse formation and its overlapping dotty signals with those of bassoon allowed us to speculate that CAST could be associated with vesicular membranes. To clarify the nature of the CAST signals, we first performed a sucrose gradient centrifugation assay using E18 rat brain. E18 rat brain homogenate was hypotonically lysed and subjected to ultracentrifugation at 100,000 g
to obtain the supernatant (S100) and pellet (P100) fractions. Like bassoon (Zhai et al., 2001
), CAST, RIM1, and Munc13-1 were mainly detected in the P100 fraction ( A). The P100 fraction was then layered on a discontinuous sucrose gradient of 0.3, 0.8, and 1.2 M. After the centrifugation, fractions were collected and analyzed by Western blotting. CAST, bassoon, RIM1, and Munc13-1, as well as synaptophysin, were found in 0.3 and 0.8 M layers ( A), containing light membranes (Zhai et al., 2001
). The essentially similar results were obtained by continuous sucrose gradient (0.3–1.2 M) ultracentrifugation ( B). CAST, bassoon, RIM1, and Munc13-1 were detected in the fractions similar to those of synaptophysin. Importantly, when the P100 fraction was treated with Triton X-100 before the centrifugation, CAST, as well as the other CAZ proteins, was recovered near the bottom fraction, whereas synaptophysin was recovered near the top fraction ( B). These results suggest that the similar behavior of CAST, RIM1, and Munc13-1 to that of bassoon and synaptophysin on sucrose gradient centrifugation is dependent on membrane integrity and that not only bassoon, but also CAST, RIM1, and Munc13-1, is associated with the light membranes.
Figure 12. Biochemical analysis of association of the CAZ proteins with the same vesicles. (A) Presence of the CAZ proteins in the light membrane fraction. (Left) P100 and S100 fractions from hypotonically lysed E18 rat brain homogenates were analyzed by Western (more ...)
It has recently been reported that bassoon and piccolo are associated with precursor vesicles for the active zone, which resemble classic dense core vesicles with a diameter of ~80 nm (Zhai et al., 2001
). To clarify that CAST is also associated with the same vesicles, we immunoisolated the vesicles from the light membrane fraction by the antibassoon Ab (Zhai et al., 2001
). Beads coated with irrelevant IgG, the antibassoon Ab, or the anti–CAST-2 Ab were incubated with the light membrane fraction and the bound proteins were analyzed by Western blotting using indicated Abs. CAST, but not synaptophysin, was coimmunoisolated with bassoon by the antibassoon Ab–coupled beads ( C). In addition, RIM1, but not Munc13-1, was coimmunoisolated. Consistently, bassoon and RIM1, but not Munc13-1, were coimmunoisolated with CAST by the anti–CAST-2 Ab–coupled beads. Together with the earlier observation that bassoon is a good marker for the precursor vesicles for the active zone (Zhai et al., 2001
), our biochemical and cell biological results suggest that at least some portions of CAST and RIM1 might also be associated with the same vesicles as those transporting bassoon. It remains unknown whether Munc13-1 is associated with the same vesicles but dissociates from the vesicles during the immunoisolation procedure or whether it is associated with different vesicles.