Kinesin superfamily motor protein KIF17 and mLin-10 in NMDA receptor- containing vesicle transport

Science

Volumn 288, Issue 5472, 2000, Pages 1796-1802

  Setou, Mitsutoshi ^a   Nakagawa, Terunaga ^a   Seog, Dae Hyun ^a   Hirokawa, Nobutaka ^a  

^a UNIVERSITY OF TOKYO   (Japan)

Author keywords

[No Author keywords available]

Indexed keywords

KINESIN; N METHYL DEXTRO ASPARTIC ACID RECEPTOR;

ARTICLE; CELL STRUCTURE; INTRACELLULAR TRANSPORT; MICROTUBULE; NERVE CELL DIFFERENTIATION; NERVE CELL PLASTICITY; NONHUMAN; NUCLEOTIDE SEQUENCE; PRIORITY JOURNAL; PROTEIN ANALYSIS; PROTEIN TRANSPORT; SYNAPSE;

AMINO ACID MOTIFS; AMINO ACID SEQUENCE; ANIMALS; BINDING SITES; BIOLOGICAL TRANSPORT; CAENORHABDITIS ELEGANS PROTEINS; CLONING, MOLECULAR; DENDRITES; DIMERIZATION; KINESIN; MALE; MEMBRANE PROTEINS; MICE; MICROTUBULES; MODELS, BIOLOGICAL; MOLECULAR MOTOR PROTEINS; MOLECULAR SEQUENCE DATA; MOLECULAR WEIGHT; ORGANELLES; PRECIPITIN TESTS; PROTEIN BINDING; PROTEINS; RECEPTORS, N-METHYL-D-ASPARTATE; RECOMBINANT PROTEINS; TWO-HYBRID SYSTEM TECHNIQUES;

EID: 0034625631     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.288.5472.1796     Document Type: Article

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24. 2-terminal peptide (αGluRε No59) (33) were provided by M. Mishina. PSD-95, NR2B, synaptotagmin, mLin-2, and mLin-10 mAbs were purchased from Transduction Laboratory, anti-substance P receptor was from Novus, anti-glycine receptor and NR1 mAb from Chemicon, and anti-GABAARβ2 from Santa Cruz. All the protein detection in immunoprecipitants was confirmed by all available antibodies: anti-mLin-10, NR1 antibodies (αGluRζ1 No43 and NR1 mAb), and NR2B antibodies (αGluRε No34, αGluRε No59, and NR2B mAb). The data shown in Figs. 4 and 5 are mAb blots.
25. 2-terminal, His-tagged full-length KIF17-expressing baculovirus was constructed using the Fast-Bac system (Gibco Life Technologies). High-Five cells (Invitrogen) were infected with the virus for 60 hours in Grace Insect Medium (Gibco Life Technologies) and harvested and purified as described (18).
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28. For tissue distribution analysis, 4-week-old male mouse total homogenate in RIPA was used; 20 μg of protein was loaded per lane. For neuronal distribution analysis, 5 μg of protein was loaded per lane.
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30. The floating assay and electron microscopy study were performed as described (18), with slight modification. The postnuclear supernatant of gray matter in IM-Ac (18) buffer was centrifuged with a continuous gradient from 40% to 0% Nycodenz for 4 hours at 65,000 rpm in a Beckman NVTi65 rotor, and 32 fractions were collected. The fraction that contained the highest concentration of KIF17 (26th) (∼0.3 ml) was incubated overnight with 1 μg of anti-KIF17 or 1 μg of control preimmune IgG at 4°C and then with 10 μl of second antibody-coated magnetic beads (Dynal) for 12 hours at 4°C. Then the sample was washed five times and processed for further analysis.
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34. + colonies. Two colonies encoded fragments of mLin-10 gene (457-840), which codes for a portion 99% identical to the corresponding portion of rat Lin-10 (Mint1). cDNA segments were amplified by the polymerase chain reaction (PCR) with specific primers and subcloned into pLexA 01 pB42AD.
35. Purified GST-fused COOH-terminus of KIF17 (939-1038) was immobilized on a BIACore CM5 sensor chip, equilibrated with 50 mM Hepes-NaOH (pH 8.0) containing 100 mM NaCl, and superfused with purified mLin-10 (650-840 with His6 on NH2-terminal) at a flow rate of 10 μl/min. Binding activities (in resonance units) were measured as the difference between the baseline value determined 20 s before sample injection and the measurements taken at the indicated time points. All experiments were performed at 25°C with BIACORE3000. Data were analyzed with the BIA evaluation program, version 3.0 [BIACORE; M. Irie et al., Science 277, 1511 (1997)].
36. A pull-down assay was performed with the post-nuclear supernatant obtained from 2-week-old mouse brain lysates in HBST [10 mM Hepes (pH 7.4), 150 mM NaCl, 0.1% Triton X-100, and proteinase inhibitors] and 10 μg of immobilized GST fusion KIF17 tail region recombinant protein or 10 μg of KIFC2 1-433 protein. After 12 hours of incubation, the beads were washed five times and immunoblotted with anti-mLin-10. Recombinant proteins were expressed in E. coli strain BL21(DE3) (Stratagene) with the pGEX vector (Amersham Pharmacia Biotech) and purified by 10 μl of Glutathione Sepharose Fast Flow (Amersham Pharmacia Biotech).
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41. A 4-week-old male mouse brain was dissected for use as reported (18) in HBST buffer. Lysates of the brains (0.5 ml) were incubated with 20 μl of anti-KIF17 serum and 10 μl of protein A-Sepharose Fast Flow at 4°C overnight and were washed five times with 0.5 ml of HBST. Sample was loaded at 5 μl per lane. The NR2B was solubilized ∼5% in 0.1% Triton, ∼5% in RIPA, and ∼30% in 1% deoxycholate (Doc) (28). The amount of immunoprecipitated NR2B by KIF17 is ∼5% of the lysates in 0.1% Triton, ∼5% in RIPA, and less in 1% Doc (28). There may be at least two populations of NR2B, with one population incorporated and anchored at the postsynaptic sites, and the other in the vesicles in the cytoplasm (some of which are transported by KIF17, forming complex with mLin-10, mLin-7, and mLin-2). Our immunofluorescence study indicates that strong permeabilization preserves synaptic NMDA receptors but affects the localization of cytoplasmic NMDA receptors, which suggests that strong detergent treatment affects the preservation of cytoplasmic NMDA receptors (28). From these results, we chose 0.1% Triton in this study.
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52. We thank T. Südhof for anti-mLin-10; Y. Hata for anti-mLin-7; M. Mishina for the anti-NR subunits; M. Watanabe, M. Mishina, T. Takahashi, T. Nakata, and Y. Kanai for discussions and advice; K. Yamamoto, L. Guillaud, Y. Ikeda, Y. Okada, M. Kikkawa, S. Nonaka, H. Sato, H. Fukuda, and M. Sugaya for technical assistance; and other members of the Hirokawa lab for technical assistance, stimulating discussions, and valuable advice throughout. Supported by the Japanese Society for Promotion of Science Research Fellowship for Young Scientists (T.N. and D.-H.S.) and by a Special Grant-in-Aid for Center of Excellence from the Japan Ministry of Education, Science, Sports and Culture (N.H.).