Purification and identification of p80 as synapsin I. (A) Schematic of the purification strategy for p80. (B) Final step in p80 purification. Protein overlays of aliquots (50 μl) of the final chromatographic separation reveal that p80 comigrates with an 80-kDa band seen on Coomassie staining, indicating that the Coomassie-stained band is p80. (C) Alignment of p80-derived peptides with synapsin I. Two tryptic peptides derived from p80 align exactly with rat synapsin I a/b. (D) Confirmation that p80 is synapsin I. Synapsin I purified by immunoprecipitation was labeled by ³²P-PTB in the overlay assay and exhibited the same electrophoretic mobility as p80 from cortical homogenates.

NOS, CAPON, and synapsin I form a ternary complex in transfected cells. COS7 cells were cotransfected with the indicated plasmids, and the lysates were incubated with glutathione-agarose. After extensive washing of the resin, bound nNOS was detected by immunoblot. nNOS adhered to the resin only in the presence of GST-synapsin 200–705 and CAPON, but not in the presence of only one, demonstrating the existence of a ternary complex composed of nNOS, CAPON, and synapsin I.

Identification of an 80-kDa CAPON PTB domain-binding protein, p80. (A) Schematic representation of CAPON. CAPON has a C-terminal PDZ domain-binding consensus sequence that binds nNOS. The N terminus contains a PTB domain. A fusion protein comprising GST, two PKA consensus phosphorylation sites, and the CAPON-PTB domain was phosphorylated in vitro with [³²P]ATP and PKA. The radiolabeled protein, ³²P-PTB, was used in overlay assays. (B) Protein overlays by using ³²P-PTB detects a prominent 80-kDa protein in rat brain cortex homogenates (50 μg per lane). (C) p80 is detected in neuronal tissues, whereas it is undetectable in peripheral tissues.

Characterization of the CAPON/Synapsin I interaction. (A) ³²P-PTB binds both synapsin Ia and synapsin Ib. Purified bovine synapsin I (0.1 μg) containing an approximate molar ratio of 1:2 for the Ia and Ib isoforms, respectively, was assayed for binding by overlay with ³²P-PTB. Binding of both isoforms indicates that the CAPON-binding region lies within residues 1–659 of the two alternatively spliced isoforms. (B) A domain consisting of amino acids 200–300 of synapsin I is the common region of interaction with CAPON. The indicated truncations of synapsin I were expressed as GST-fusion proteins in COS-7 cells along with an expression plasmid encoding CAPON. Following incubation of the lysates with glutathione agarose, complexes of synapsin I and CAPON were detected by immunoblot of protein retained on glutathione agarose. (C) Schematic diagram of the results in B. (D) CAPON interacts with synapsins I, II, and III. Each synapsin was expressed as a myc-tagged fusion protein in COS-7 cells and immunoprecipitated with an anti-myc antibody. The purified synapsins were subjected to overlay assay by using the ³²P-PTB probe (Upper). An anti-myc blot of the immunoprecipitated synapsins is shown to compare the relative expression of the different synapsin isoforms (Lower). (E) CAPON is detected in synapsin I immunoprecipitates. Cerebellar membranes were solubilized with 1% Triton X-100 and then incubated with anti-synapsin I antibodies or the control antibody against PKC-zeta (Upper). Only immunoprecipitates prepared by using anti-synapsin I antibodies contained appreciable amounts of CAPON. Blots were stained with Coomassie blue to visualize the heavy chain of the immunoprecipitating antibody to confirm that approximately equivalent amounts of antibody were used in immunoprecipitation experiments (Lower).

Subcellular localization of CAPON and nNOS is altered in synapsin I^−/−, synapsin II^−/− double knockout mice. (A) Subcellular fractionation of rat brain. Subcellular fractions from rat brain were blotted for synapsin I, nNOS, and CAPON as indicated. (B) Subcellular fractions (50 μg) derived from wild-type (W) or double knockout mice (K) were blotted with synapsin I, nNOS, CAPON, or FAK-specific antibodies. CAPON and nNOS levels were increased in S2, P3, SG1, SG3, and SG4 fractions of knockout mice relative to wild-type mice. Levels of FAK were not changed in wild-type vs. double knockout mice. H, homogenate; S1, postnuclear supernatant; S2, supernatant of P2; P2, crude synaptosomes; LP1, crude synaptic plasma membranes; LS1, supernatant of LP1; LP2, crude synaptic vesicles; LS2, supernatant of LP2 (synaptosol); SG1-SG4, sucrose gradient fractions of LP2 in which SG2 is enriched in synaptic vesicles and SG4 in small synaptic membranes; SV, synaptic vesicles purified from the SG2 fraction by controlled-pore glass chromatography; SSV, salt-treated synaptic vesicles prepared by elution of the SV fraction with 200 mM NaCl; S3, supernatant of P3 after high-speed centrifugation of S2 (cytosol); P3, microsomes. Subcellular fractionations were performed twice with essentially identical results.