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. 2002 Jul 1;22(13):5253-8.
doi: 10.1523/JNEUROSCI.22-13-05253.2002.

Association of the kinesin superfamily motor protein KIF1Balpha with postsynaptic density-95 (PSD-95), synapse-associated protein-97, and synaptic scaffolding molecule PSD-95/discs large/zona occludens-1 proteins

Hyejung Mok  1 , Hyewon Shin, Seho Kim, Jae-Ran Lee, Jiyoung Yoon, Eunjoon Kim
Affiliations

Association of the kinesin superfamily motor protein KIF1Balpha with postsynaptic density-95 (PSD-95), synapse-associated protein-97, and synaptic scaffolding molecule PSD-95/discs large/zona occludens-1 proteins

Hyejung Mok et al. J Neurosci. .

Abstract

Mutation in KIF1B, a kinesin superfamily motor protein, causes a peripheral neuropathy known as Charcot-Marie-Tooth disease type 2A (CMT2A). Little is known, however, about how a defective KIF1B gene leads to CMT2A. Here we report that KIF1Balpha, one of the two splice variants of KIF1B, directly interacts through its C-terminal postsynaptic density-95 (PSD-95)/discs large/zona occludens (PDZ) domain-binding motif with PDZ proteins including PSD-95/synapse-associated protein-90 (SAP90), SAP97, and synaptic scaffolding molecule (S-SCAM)-90 (SAP90). KIF1Balpha selectively interacts with PSD-95, SAP97, and S-SCAM in yeast two-hybrid, pull-down, and in vivo coimmunoprecipitation experiments. KIF1Balpha, SAP97, and S-SCAM are widely distributed to both dendrites and axons of cultured neurons and are enriched in the small membrane fraction of the brain. In the flotation assay, KIF1Balpha cofractionates and coimmunoprecipitates with PSD-95, SAP97, and S-SCAM. These results suggest that the PSD-95 family proteins and S-SCAM have a novel function as KIF1Balpha receptors, linking KIF1Balpha to its specific cargos, and are involved in peripheral neuropathies.

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Figures

Fig. 1.
Fig. 1.
Interaction of KIF1Bα with PSD-95 relatives and S-SCAM in the yeast two-hybrid and pull-down assays.A, Positive clones from the yeast two-hybrid screen of a rat brain cDNA library and their specific interaction with the C terminus of KIF1Bα. A mutant KIF1Bα C terminus (the last residue valine to alanine) loses its interaction with PDZ domains. The positive clones are indicated by lines underneath the domain structures. Numbers indicate PDZ domains.SH3, Src homology 3 domain; GK, guanylate kinase-like domain; WW, WW domain; CT, C terminal; WT, wild type; mt, mutant;HIS, histidine. B, Specific interaction of the KIF1Bα C terminus with PDZ domain(s) of PSD-95, SAP97, chapsyn-110 (Chap-110), and S-SCAM but not Shank1, GRIP2, and NHERF1. HIS3 activity: +++, >60%; ++, 30–60%; +, 10–30%; −, no significant growth. β-galactosidase (β-gal) +++, <45 min; ++, 45–90 min; +, 90–240 min; −, no significant activity.CT, C terminal; HIS, histidine.C, Pull-down assay. COS cell lysates singly transfected with PSD-95, SAP97, S-SCAM, GRIP2, and liprin-α1 were pulled down by GST-KIF1Bα C terminus (CT) (last 165 residues) or GST alone (negative control). GST-KIF1Bα C terminus specifically pulled down PSD-95, SAP97, and S-SCAM but not GRIP2 and liprin-α1. Input: 10%.
Fig. 2.
Fig. 2.
Overlapping distribution of KIF1Bα with PSD-95, SAP97, and S-SCAM in cultured neurons. A, Regions of KIF1Bα against which polyclonal antibodies were generated. The two KIF1B splice variants (KIF1Bα and KIF1Bβ) begin to diverge at the splice site. B, Characterization of the KIF1B antibodies by immunoblot analysis on COS cells and rat brain (S2 fraction) proteins. All three antibodies recognized a KIF1Bα band (130 kDa), and the 1161 antibody recognized an additional band of 200 kDa (KIF1Bβ). IB, Immunoblot. C–F, Overlapping subcellular distribution of KIF1Bα with PSD-95, SAP97, and S-SCAM in cultured hippocampal neurons (at 6 d in vitro). KIF1BBα (red, 1189 antibody) is distributed to both MAP2 (green)-positive dendrites (arrows) and MAP2-negative axons (arrowheads) (C1–C3). The KIF1Bα staining pattern (1189 antibody) is eliminated by preincubation of the antibody with an excess of free peptide (C4). SAP97 and S-SCAM, similar to KIF1Bα, are distributed to both MAP2-positive dendrites (arrows) and MAP2-negative axons (arrowheads) (E, F), whereas PSD-95 is distributed primarily to dendrites (D). Scale bar, 30 μm.
Fig. 3.
Fig. 3.
Subcellular fractionation pattern of KIF1Bα and its coimmunoprecipitation with PSD-95 relatives and S-SCAM in rat brain. A, Subcellular fractionation pattern of KIF1Bα, PSD-95, SAP97, and S-SCAM in rat brain. Subcellular fractions of adult rat brain were immunoblotted for KIF1Bα, PSD-95, SAP97, S-SCAM, the NR1 subunit of NMDA receptors (NR1), and synaptotagmin (Syntag). H, Total homogenate;P1, nuclei and large debris; S2, supernatant after P2 precipitation; P2, crude synaptosome;S3, cytosol; P3, small membrane;LP1, synaptosomal membrane; LS2, synaptic cytosol; LP2, synaptic vesicles. LP1, LS2, and LP2 lanes, 10 μg of proteins; all otherlanes, 20 μg of proteins. B, Coimmunoprecipitation of KIF1Bα with PSD-95 relatives and S-SCAM in rat brain. Lysates of the S2 fraction were immunoprecipitated (IP) with KIF1Bα antibody (1183) or guinea pig IgG (IgG, control) and immunoblotted for KIF1Bα, PSD-95 (pan-antibody, K28/86), S-SCAM, and GRIP. Input: 3%.
Fig. 4.
Fig. 4.
Cofractionation and biochemical association of KIF1Bα with PSD-95 relatives and S-SCAM in the flotation assay.A, Partition of KIF1Bα and associated proteins into heavy and light membranes. HM, Heavy membranes;LM, light membranes; Syntag, synaptotagmin. B, Flotation assay. Samples enriched with light membranes were loaded at the bottom of a discontinuous sucrose gradient and centrifuged for flotation. KIF1Bα was recovered primarily in light fractions (fractions 2–5) along with PSD-95, SAP97, S-SCAM, NR1, synaptotagmin (Syntag), and GRIP, but not Homer-1. In control experiments, addition of Triton X-100 to samples before flotation eliminated the flotation. C, Coimmunoprecipitation of KIF1Bα with PSD-95 relatives and S-SCAM in floated membranes. Floated fractions (2–5) in B were pooled, solubilized with detergent, and immunoprecipitated (IP) with KIF1Bα antibody (1183) or guinea pig IgG (Gp IgG, control). The immunoprecipitates were analyzed by immunoblotting with antibodies against KIF1Bα, PSD-95, SAP97, S-SCAM, synaptotagmin (Syntag), and GRIP. KIF1Bα coimmunoprecipitates with PSD-95, SAP97, and S-SCAM but not with synaptotagmin and GRIP. Input: 5%.
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