Abstract
To understand the mechanisms of transport for organelles in the axon, we isolated and sequenced the cDNA encoding KIF4 from murine brain, and characterized the molecule biochemically and immunocytochemically. Complete amino acid sequence analysis of KIF4 and ultrastructural studies of KIF4 molecules expressed in Sf9 cells revealed that the protein contains 1,231 amino acid residues (M(r) 139,550) and that the molecule (116-nm rod with globular heads and tail) consists of three domains: an NH2-terminal globular motor domain, a central alpha-helical stalk domain and a COOH-terminal tail domain. KIF4 protein has the property of nucleotide-dependent binding to microtubules, microtubule- activated ATPase activity, and microtubule plus-end-directed motility. Northern blot analysis and in situ hybridization demonstrated that KIF4 is strongly expressed in juvenile tissues including differentiated young neurons, while its expression is decreased considerably in adult mice except in spleen. Immunocytochemical studies revealed that KIF4 colocalized with membranous organelles both in growth cones of differentiated neurons and in the cytoplasm of cultured fibroblasts. During mitotic phase of cell cycle, KIF4 appears to colocalize with membranous organelles in the mitotic spindle. Hence we conclude that KIF4 is a novel microtubule-associated anterograde motor protein for membranous organelles, the expression of which is regulated developmentally.
Full Text
The Full Text of this article is available as a PDF (7.0 MB).
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Aizawa H., Sekine Y., Takemura R., Zhang Z., Nangaku M., Hirokawa N. Kinesin family in murine central nervous system. J Cell Biol. 1992 Dec;119(5):1287–1296. doi: 10.1083/jcb.119.5.1287. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Bayer S. A. Development of the hippocampal region in the rat. I. Neurogenesis examined with 3H-thymidine autoradiography. J Comp Neurol. 1980 Mar 1;190(1):87–114. doi: 10.1002/cne.901900107. [DOI] [PubMed] [Google Scholar]
- Bloom G. S., Wagner M. C., Pfister K. K., Brady S. T. Native structure and physical properties of bovine brain kinesin and identification of the ATP-binding subunit polypeptide. Biochemistry. 1988 May 3;27(9):3409–3416. doi: 10.1021/bi00409a043. [DOI] [PubMed] [Google Scholar]
- Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
- Brady S. T. A novel brain ATPase with properties expected for the fast axonal transport motor. Nature. 1985 Sep 5;317(6032):73–75. doi: 10.1038/317073a0. [DOI] [PubMed] [Google Scholar]
- Caviness V. S., Jr Time of neuron origin in the hippocampus and dentate gyrus of normal and reeler mutant mice: an autoradiographic analysis. J Comp Neurol. 1973 Sep 15;151(2):113–120. doi: 10.1002/cne.901510203. [DOI] [PubMed] [Google Scholar]
- Chandra R., Salmon E. D., Erickson H. P., Lockhart A., Endow S. A. Structural and functional domains of the Drosophila ncd microtubule motor protein. J Biol Chem. 1993 Apr 25;268(12):9005–9013. [PubMed] [Google Scholar]
- Chou P. Y., Fasman G. D. Prediction of protein conformation. Biochemistry. 1974 Jan 15;13(2):222–245. doi: 10.1021/bi00699a002. [DOI] [PubMed] [Google Scholar]
- Cole D. G., Cande W. Z., Baskin R. J., Skoufias D. A., Hogan C. J., Scholey J. M. Isolation of a sea urchin egg kinesin-related protein using peptide antibodies. J Cell Sci. 1992 Feb;101(Pt 2):291–301. doi: 10.1242/jcs.101.2.291. [DOI] [PubMed] [Google Scholar]
- Fujita S., Shimada M., Nakamura T. H3-thymidine autoradiographic studies on the cell proliferation and differentiation in the external and the internal granular layers of the mouse cerebellum. J Comp Neurol. 1966 Oct;128(2):191–208. doi: 10.1002/cne.901280206. [DOI] [PubMed] [Google Scholar]
- Garnier J., Osguthorpe D. J., Robson B. Analysis of the accuracy and implications of simple methods for predicting the secondary structure of globular proteins. J Mol Biol. 1978 Mar 25;120(1):97–120. doi: 10.1016/0022-2836(78)90297-8. [DOI] [PubMed] [Google Scholar]
- Goldstein L. S. The kinesin superfamily: tails of functional redundancy. Trends Cell Biol. 1991 Oct;1(4):93–98. doi: 10.1016/0962-8924(91)90036-9. [DOI] [PubMed] [Google Scholar]
- Goldstein L. S. With apologies to scheherazade: tails of 1001 kinesin motors. Annu Rev Genet. 1993;27:319–351. doi: 10.1146/annurev.ge.27.120193.001535. [DOI] [PubMed] [Google Scholar]
- Grafstein B., Forman D. S. Intracellular transport in neurons. Physiol Rev. 1980 Oct;60(4):1167–1283. doi: 10.1152/physrev.1980.60.4.1167. [DOI] [PubMed] [Google Scholar]
- Hagan I., Yanagida M. Kinesin-related cut7 protein associates with mitotic and meiotic spindles in fission yeast. Nature. 1992 Mar 5;356(6364):74–76. doi: 10.1038/356074a0. [DOI] [PubMed] [Google Scholar]
- Higgins D. G., Bleasby A. J., Fuchs R. CLUSTAL V: improved software for multiple sequence alignment. Comput Appl Biosci. 1992 Apr;8(2):189–191. doi: 10.1093/bioinformatics/8.2.189. [DOI] [PubMed] [Google Scholar]
- Hirokawa N. Axonal transport and the cytoskeleton. Curr Opin Neurobiol. 1993 Oct;3(5):724–731. doi: 10.1016/0959-4388(93)90144-n. [DOI] [PubMed] [Google Scholar]
- Hirokawa N. Cross-linker system between neurofilaments, microtubules, and membranous organelles in frog axons revealed by the quick-freeze, deep-etching method. J Cell Biol. 1982 Jul;94(1):129–142. doi: 10.1083/jcb.94.1.129. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hirokawa N., Pfister K. K., Yorifuji H., Wagner M. C., Brady S. T., Bloom G. S. Submolecular domains of bovine brain kinesin identified by electron microscopy and monoclonal antibody decoration. Cell. 1989 Mar 10;56(5):867–878. doi: 10.1016/0092-8674(89)90691-0. [DOI] [PubMed] [Google Scholar]
- Hirokawa N., Sato-Yoshitake R., Kobayashi N., Pfister K. K., Bloom G. S., Brady S. T. Kinesin associates with anterogradely transported membranous organelles in vivo. J Cell Biol. 1991 Jul;114(2):295–302. doi: 10.1083/jcb.114.2.295. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hirokawa N., Sato-Yoshitake R., Yoshida T., Kawashima T. Brain dynein (MAP1C) localizes on both anterogradely and retrogradely transported membranous organelles in vivo. J Cell Biol. 1990 Sep;111(3):1027–1037. doi: 10.1083/jcb.111.3.1027. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hogan C. J., Stephens L., Shimizu T., Cande W. Z. Physiological evidence for involvement of a kinesin-related protein during anaphase spindle elongation in diatom central spindles. J Cell Biol. 1992 Dec;119(5):1277–1286. doi: 10.1083/jcb.119.5.1277. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Howard J., Hudspeth A. J., Vale R. D. Movement of microtubules by single kinesin molecules. Nature. 1989 Nov 9;342(6246):154–158. doi: 10.1038/342154a0. [DOI] [PubMed] [Google Scholar]
- Kondo S., Sato-Yoshitake R., Noda Y., Aizawa H., Nakata T., Matsuura Y., Hirokawa N. KIF3A is a new microtubule-based anterograde motor in the nerve axon. J Cell Biol. 1994 Jun;125(5):1095–1107. doi: 10.1083/jcb.125.5.1095. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Koonce M. P., Grissom P. M., McIntosh J. R. Dynein from Dictyostelium: primary structure comparisons between a cytoplasmic motor enzyme and flagellar dynein. J Cell Biol. 1992 Dec;119(6):1597–1604. doi: 10.1083/jcb.119.6.1597. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kuznetsov S. A., Vaisberg E. A., Shanina N. A., Magretova N. N., Chernyak V. Y., Gelfand V. I. The quaternary structure of bovine brain kinesin. EMBO J. 1988 Feb;7(2):353–356. doi: 10.1002/j.1460-2075.1988.tb02820.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
- Lye R. J., Porter M. E., Scholey J. M., McIntosh J. R. Identification of a microtubule-based cytoplasmic motor in the nematode C. elegans. Cell. 1987 Oct 23;51(2):309–318. doi: 10.1016/0092-8674(87)90157-7. [DOI] [PubMed] [Google Scholar]
- Maeda K., Nakata T., Noda Y., Sato-Yoshitake R., Hirokawa N. Interaction of dynamin with microtubules: its structure and GTPase activity investigated by using highly purified dynamin. Mol Biol Cell. 1992 Oct;3(10):1181–1194. doi: 10.1091/mbc.3.10.1181. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Matsuura Y., Possee R. D., Overton H. A., Bishop D. H. Baculovirus expression vectors: the requirements for high level expression of proteins, including glycoproteins. J Gen Virol. 1987 May;68(Pt 5):1233–1250. doi: 10.1099/0022-1317-68-5-1233. [DOI] [PubMed] [Google Scholar]
- McDonald H. B., Stewart R. J., Goldstein L. S. The kinesin-like ncd protein of Drosophila is a minus end-directed microtubule motor. Cell. 1990 Dec 21;63(6):1159–1165. doi: 10.1016/0092-8674(90)90412-8. [DOI] [PubMed] [Google Scholar]
- Meluh P. B., Rose M. D. KAR3, a kinesin-related gene required for yeast nuclear fusion. Cell. 1990 Mar 23;60(6):1029–1041. doi: 10.1016/0092-8674(90)90351-e. [DOI] [PubMed] [Google Scholar]
- Mikami A., Paschal B. M., Mazumdar M., Vallee R. B. Molecular cloning of the retrograde transport motor cytoplasmic dynein (MAP 1C). Neuron. 1993 May;10(5):787–796. doi: 10.1016/0896-6273(93)90195-w. [DOI] [PubMed] [Google Scholar]
- Miller R. H., Lasek R. J. Cross-bridges mediate anterograde and retrograde vesicle transport along microtubules in squid axoplasm. J Cell Biol. 1985 Dec;101(6):2181–2193. doi: 10.1083/jcb.101.6.2181. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Navone F., Niclas J., Hom-Booher N., Sparks L., Bernstein H. D., McCaffrey G., Vale R. D. Cloning and expression of a human kinesin heavy chain gene: interaction of the COOH-terminal domain with cytoplasmic microtubules in transfected CV-1 cells. J Cell Biol. 1992 Jun;117(6):1263–1275. doi: 10.1083/jcb.117.6.1263. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Nislow C., Lombillo V. A., Kuriyama R., McIntosh J. R. A plus-end-directed motor enzyme that moves antiparallel microtubules in vitro localizes to the interzone of mitotic spindles. Nature. 1992 Oct 8;359(6395):543–547. doi: 10.1038/359543a0. [DOI] [PubMed] [Google Scholar]
- Olmsted J. B. Affinity purification of antibodies from diazotized paper blots of heterogeneous protein samples. J Biol Chem. 1981 Dec 10;256(23):11955–11957. [PubMed] [Google Scholar]
- Paschal B. M., Vallee R. B. Microtubule and axoneme gliding assays for force production by microtubule motor proteins. Methods Cell Biol. 1993;39:65–74. doi: 10.1016/s0091-679x(08)60161-2. [DOI] [PubMed] [Google Scholar]
- Paschal B. M., Vallee R. B. Retrograde transport by the microtubule-associated protein MAP 1C. Nature. 1987 Nov 12;330(6144):181–183. doi: 10.1038/330181a0. [DOI] [PubMed] [Google Scholar]
- Saitou N., Nei M. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol. 1987 Jul;4(4):406–425. doi: 10.1093/oxfordjournals.molbev.a040454. [DOI] [PubMed] [Google Scholar]
- Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Saunders W. S., Hoyt M. A. Kinesin-related proteins required for structural integrity of the mitotic spindle. Cell. 1992 Aug 7;70(3):451–458. doi: 10.1016/0092-8674(92)90169-d. [DOI] [PubMed] [Google Scholar]
- Sawin K. E., LeGuellec K., Philippe M., Mitchison T. J. Mitotic spindle organization by a plus-end-directed microtubule motor. Nature. 1992 Oct 8;359(6395):540–543. doi: 10.1038/359540a0. [DOI] [PubMed] [Google Scholar]
- Sawin K. E., Mitchison T. J., Wordeman L. G. Evidence for kinesin-related proteins in the mitotic apparatus using peptide antibodies. J Cell Sci. 1992 Feb;101(Pt 2):303–313. doi: 10.1242/jcs.101.2.303. [DOI] [PubMed] [Google Scholar]
- Schnapp B. J., Reese T. S. Dynein is the motor for retrograde axonal transport of organelles. Proc Natl Acad Sci U S A. 1989 Mar;86(5):1548–1552. doi: 10.1073/pnas.86.5.1548. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Schnapp B. J. Viewing single microtubules by video light microscopy. Methods Enzymol. 1986;134:561–573. doi: 10.1016/0076-6879(86)34121-1. [DOI] [PubMed] [Google Scholar]
- Scholey J. M., Heuser J., Yang J. T., Goldstein L. S. Identification of globular mechanochemical heads of kinesin. Nature. 1989 Mar 23;338(6213):355–357. doi: 10.1038/338355a0. [DOI] [PubMed] [Google Scholar]
- Scholey J. M., Porter M. E., Grissom P. M., McIntosh J. R. Identification of kinesin in sea urchin eggs, and evidence for its localization in the mitotic spindle. Nature. 1985 Dec 5;318(6045):483–486. doi: 10.1038/318483a0. [DOI] [PubMed] [Google Scholar]
- Schroer T. A., Steuer E. R., Sheetz M. P. Cytoplasmic dynein is a minus end-directed motor for membranous organelles. Cell. 1989 Mar 24;56(6):937–946. doi: 10.1016/0092-8674(89)90627-2. [DOI] [PubMed] [Google Scholar]
- Seals J. R., McDonald J. M., Bruns D., Jarett L. A sensitive and precise isotopic assay of ATPase activity. Anal Biochem. 1978 Oct 15;90(2):785–795. doi: 10.1016/0003-2697(78)90169-0. [DOI] [PubMed] [Google Scholar]
- Shelanski M. L., Gaskin F., Cantor C. R. Microtubule assembly in the absence of added nucleotides. Proc Natl Acad Sci U S A. 1973 Mar;70(3):765–768. doi: 10.1073/pnas.70.3.765. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Studier F. W., Rosenberg A. H., Dunn J. J., Dubendorff J. W. Use of T7 RNA polymerase to direct expression of cloned genes. Methods Enzymol. 1990;185:60–89. doi: 10.1016/0076-6879(90)85008-c. [DOI] [PubMed] [Google Scholar]
- Takemura R., Kanai Y., Hirokawa N. In situ localization of tau mRNA in developing rat brain. Neuroscience. 1991;44(2):393–407. doi: 10.1016/0306-4522(91)90064-u. [DOI] [PubMed] [Google Scholar]
- Tanaka Y., Kawahata K., Nakata T., Hirokawa N. Chronological expression of microtubule-associated proteins (MAPs) in EC cell P19 after neuronal induction by retinoic acid. Brain Res. 1992 Nov 20;596(1-2):269–278. doi: 10.1016/0006-8993(92)91557-u. [DOI] [PubMed] [Google Scholar]
- Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Vale R. D., Schnapp B. J., Reese T. S., Sheetz M. P. Movement of organelles along filaments dissociated from the axoplasm of the squid giant axon. Cell. 1985 Feb;40(2):449–454. doi: 10.1016/0092-8674(85)90159-x. [DOI] [PubMed] [Google Scholar]
- Vale R. D., Schnapp B. J., Reese T. S., Sheetz M. P. Organelle, bead, and microtubule translocations promoted by soluble factors from the squid giant axon. Cell. 1985 Mar;40(3):559–569. doi: 10.1016/0092-8674(85)90204-1. [DOI] [PubMed] [Google Scholar]
- Vernos I., Heasman J., Wylie C. Multiple kinesin-like transcripts in Xenopus oocytes. Dev Biol. 1993 May;157(1):232–239. doi: 10.1006/dbio.1993.1127. [DOI] [PubMed] [Google Scholar]
- Witman G. B. Isolation of Chlamydomonas flagella and flagellar axonemes. Methods Enzymol. 1986;134:280–290. doi: 10.1016/0076-6879(86)34096-5. [DOI] [PubMed] [Google Scholar]
- Wright B. D., Henson J. H., Wedaman K. P., Willy P. J., Morand J. N., Scholey J. M. Subcellular localization and sequence of sea urchin kinesin heavy chain: evidence for its association with membranes in the mitotic apparatus and interphase cytoplasm. J Cell Biol. 1991 May;113(4):817–833. doi: 10.1083/jcb.113.4.817. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Wright B. D., Terasaki M., Scholey J. M. Roles of kinesin and kinesin-like proteins in sea urchin embryonic cell division: evaluation using antibody microinjection. J Cell Biol. 1993 Nov;123(3):681–689. doi: 10.1083/jcb.123.3.681. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Yang J. T., Laymon R. A., Goldstein L. S. A three-domain structure of kinesin heavy chain revealed by DNA sequence and microtubule binding analyses. Cell. 1989 Mar 10;56(5):879–889. doi: 10.1016/0092-8674(89)90692-2. [DOI] [PubMed] [Google Scholar]
- Yang J. T., Saxton W. M., Stewart R. J., Raff E. C., Goldstein L. S. Evidence that the head of kinesin is sufficient for force generation and motility in vitro. Science. 1990 Jul 6;249(4964):42–47. doi: 10.1126/science.2142332. [DOI] [PubMed] [Google Scholar]
- Yen T. J., Li G., Schaar B. T., Szilak I., Cleveland D. W. CENP-E is a putative kinetochore motor that accumulates just before mitosis. Nature. 1992 Oct 8;359(6395):536–539. doi: 10.1038/359536a0. [DOI] [PubMed] [Google Scholar]
- Zhang Z., Tanaka Y., Nonaka S., Aizawa H., Kawasaki H., Nakata T., Hirokawa N. The primary structure of rat brain (cytoplasmic) dynein heavy chain, a cytoplasmic motor enzyme. Proc Natl Acad Sci U S A. 1993 Sep 1;90(17):7928–7932. doi: 10.1073/pnas.90.17.7928. [DOI] [PMC free article] [PubMed] [Google Scholar]