Identification of a novel F-box protein, Fbxo45. A, the amino acid sequence of mouse Fbxo45 compared with its Drosophila (DFsn) and C. elegans (FSN-1) homologues is shown. The conserved amino acids are highlighted in gray. The underlines indicate the signature sequences of the F-box and SPRY domains. B, domain structures of Fbxo45, DFsn, and FSN-1.

Fbxo45 protein expression. A, immunoblot analysis of Fbxo45 in adult mice is shown. B, subcellular distribution of Fbxo45 in the mouse brain is shown. Immunoblot analysis of Fbxo45, Munc13, SNAP25, synaptophysin (Syph), synaptotagmin (Syt), synaptogyrin (Syngr), or PSD-95 in the indicated fractions showed that Fbxo45 was enriched at the synaptic region, including the synaptosomal membrane fraction (LP1) and postsynaptic density (PSD). H, homogenate; P1, nuclear fraction; S1, crude synaptosomal fraction; S2, cytosolic synaptosomal fraction; P2, crude synaptosomal pellet fraction; LP2, synaptosomal vesicle fraction; SPM, synaptic plasma membrane fraction. C, immunocytochemistry of primary cultured rat hippocampal neurons expressing Fbxo45 is shown. Shown are Fbxo45 (green), VGlut1 (red), and PSD-95 (purple) (upper panels). Magnifications of the boxed areas in the upper panels are shown in the lower panels. Arrowheads indicate synapses, at which VGlut1 and PSD-95 were colocalized. Scale bars: 20 μm in the upper panels, 5 μm in the lower panels). D, shown is immunoelectron microscopy of endogenous Fbxo45. The lower panel shows a magnified image of the boxed area in the upper panel. S, synapse; Pre, presynaptic terminal; Post, postsynaptic terminal. Scale bars: 0.5 μm in the upper panel, 0.2 μm in the lower panel. E, localization of nanogold particles was quantitatively evaluated on each indicated structures (count/μm²). Asterisk, unpaired t test, p < 0.01.

Fbxo45 interacts with Skp1 and induces protein ubiquitination. A, shown is an immunoprecipitation (IP) assay of FLAG-Fbxo45 with endogenous Skp1 compared with FLAG-Musashi1 (Msi1) or FLAG-HuC as a negative control. IB, immunoblot. B, shown is ubiquitination activity of Fbxo45 in 293T cells. The high molecular mass, ubiquitinated proteins are labeled (Ub)n at the right.

Fbxo45 mRNA expression. A, shown is a Northern blot analysis of Fbxo45 mRNA in embryonic brain and adult tissues from mice. G3PDH, glyceraldehyde-3-phosphate dehydrogenase. B–G, shown is an in situ hybridization analysis of Fbxo45 in the brain. Sagittal sections of adult mouse brain were hybridized with an antisense or sense Fbxo45-specific cRNA probe. Fbxo45 expression was observed in the cerebellum (B–D) and hippocampus (E–G) as indicated by arrowheads. No signal was detected with the sense probe (D and G). Mo, Molecular layer; Pu, Purkinje cell layer; GrC, granule cell layer; DG, dentate gyrus. Scale bars: 400 μm in B and E, 50 μm in C, D, F, and G.

Regulation of synaptic activity by Fbxo45. A, shown is immunocytochemistry of cultured hippocampal neurons transfected with full-length of Fbxo45, vector control (Vec), siRNAs against Fbxo45 (siRNA Fbxo45-1, siRNA Fbxo45-2), or a control siRNA (siRNA Cont.) and EGFP. GFP (green), Synapsin-I (Syn; red), and PSD-95 (white) (scale bars: 5 μm). B, shown is quantification of the clusters that Synapsin-I and PSD-95 colocalized in the dendrites shown in A. Vec, n = 18; Fbxo45, n = 29; siRNA control, n = 24; siRNA Fbxo45-1, n = 20; siRNA Fbxo45-2, n = 11 dendrites. C, shown is a representative recording of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate receptor-mediated mEPSC from neurons transfected with full-length or deletion mutants of Fbxo45, vector control, siRNAs against Fbxo45, or a control siRNA. D, quantification is shown of the mEPSC frequency and amplitude under the conditions described in C. Vec, n = 11; Fbxo45, n = 9; Fbxo45(54–140aa), n = 9; Fbxo45(101–286aa), n = 9; siRNA control, n = 9; siRNA Fbxo45-1, n = 12; siRNA Fbxo45-2, n = 6 neurons; asterisk, unpaired t test, p < 0.05). E, shown is mEPSC frequency in neurons transfected with Fbxo45 or the control vector in 2, 5, or 10 mm extracellular Ca²⁺. (Vec, n = 11, 14, 14; Fbxo45, n = 12, 8, 13; siRNA control, n = 9, 6, 7; siRNA Fbxo45-1, n = 12, 9, 8; siRNA Fbxo45-2, n = 6, 6, 7 neurons (2, 5, 10 mm extracellular Ca²⁺); asterisk, unpaired t test, p < 0.05.

Involvement of Fbxo45 in the degradation of Munc13-1. A, Fbxo45 interacts with Munc13-1 through the SPRY domain. FLAG-tagged full-length or deletion mutants of Fbxo45 were co-expressed with Myc-Munc13-1 in 293T cells and then immunoprecipitated (IP) with an anti-Myc antibody. The precipitates were analyzed by immunoblotting (IB) using an anti-FLAG or anti-Myc antibody. Vec, vector. B, the level of Munc13-1 protein was decreased by Fbxo45 in a dose-dependent manner. In contrast, the RIM1 level was not affected by Fbxo45. Shown is quantification of the optical densities of the Munc13-1 and RIM1 bands. The protein levels, normalized to those of the corresponding EGFP bands, are shown as a percentage of the normalized value without Fbxo45 co-expression. Asterisk, p < 0.05, Mann-Whitney U test. C, shown is regulation of Munc13-1 stability by Fbxo45. Munc13-1 was co-expressed with Fbxo45 full-length, Fbxo45 deletion mutants, or Fbxo45 with MG132 and EGFP in 293T cells. D, shown is regulation of endogenous Munc13-1 protein stability by endogenous Fbxo45 in Neuro2A cells. siRNA control or siRNAs against Fbxo45 were transfected into Neuro2A cell. The protein stability was assessed by immunoblot analysis after incubation with cycloheximide for various times. Protein decay curves were determined from these studies. The optical density of the Munc13-1 or endogenous Munc13-1 bands was normalized to that of the corresponding EGFP or β-actin band, respectively, and is shown as a percentage of the normalized value for the 0-h time point. Asterisk, p < 0.05. Mann-Whitney U test.