KIF3A Is a New Microtubule-Based Anterograde Motor in the Nerve Axon

KIF3A Is a New Microtubule-based Anterograde Motor in the Nerve Axon Satoru Kondo, Reiko Sato-Yoshilake, Yasuko Noda, Hiroyuki Aizawa, Takao Nakata, Yoshiharu Matsuura,* and Nobutaka Hirokawa Department of Anatomy and Cell Biology, School of Medicine, University of Tokyo, Hongo, Tokyo 113, Japan; and *Laboratory of Hepatitis Viruses, Department of Virology II, National Institute of Health, Shinjuku-ku, Tokyo 162, Japan Abstract. Neurons are highly polarized cells com- complex axonal transports seems quite likely. Here we posed of dendrites, cell bodies, and long axons. Be- characterized a new member of the kinesin super- cause of the lack of protein synthesis machinery in family, KIF3A (50-nm rod with globular head and tail), axons, materials required in axons and synapses have and found that it is localized in neurons, associated to be transported down the axons after synthesis in the with membrane organdie fractions, and accumulates cell body. Fast anterograde transport conveys different with anterogradely moving membrane organelles after kinds of membranous organelles such as mitochondria ligation of peripheral nerves. Furthermore, native and precursors of synaptic vesicles and axonal mem- KIF3A (a complex of 80/85 KIF3A heavy chain and a branes, while organdies such as endosomes and au- 95-kD polypeptide) revealed microtubule gliding activ- tophagic prelysosomal organelles are conveyed retro- ity and baculovirus-expressed KIF3A heavy chain gradely. Although kinesin and dynein have been demonstrated microtubule plus end-directed (antero- identified as good candidates for microtubule-based grade) motility in vitro. These findings strongly sug- anterograde and retrograde transporters, respectively, gest that KIF3A is a new motor protein for the antero- the existence of other motors for performing these grade fast axonal transport. 'EORO~S are highly polarized cells composed of cell glass surface or translocate Latex beads on microtubules in bodies and long cytoplasmic processes (i.e., an vitro; the movement of microtubule segments and Latex N axon and dendrites). Because of the lack of protein beads showed that kinesin is a plus end-directed motor pro- synthesis machinery in the axon and because many materials tein (Vale et al., 1985; Saxton et al., 1988). Vesicle transport are synthesized in the cell body, highly developed intracellu- was affected by the introduction of antibodies against kinesin lar transport mechanisms are required for the maintenance into the axoplasm (Brady et al., 1990). Immunofluorescence of the axon. Axonal transports are divided into two flows, microscopy studies of cultured cells (Hollenbeck, 1989; fast and slow, with the fast flow possessing both anterograde Pfister et al., 1989) showed the colocalization with vesicle- and retrograde directions. Fast anterogradely transported like structures, and further study using ligated peripheral materials are tubulovesicular structures, membrane-asso- nerves showed that kinesin was abundantly associated with ciated proteins, neuropeptides, neurotransmitters, and as- anterogradely transported membrane-bound organeUes (Hi- sociated enzymes, whereas prelysosomal vesicles, multi- rokawa et al., 1991). Genetic and in vivo analyses of the kine- vesicular bodies, multilamelar bodies, growth factors, and sin heavy chain have been performed in Drosophila. The recycled proteins are transported retrogradely. Mitochondria mutants defective in the kinesin heavy chain of Drosophila are transported bidirectionally at a different fast velocity caused behavioral abnormality and, although there was no (Grafstein and Forman, 1980; Brady, 1991; Hirokawa, 1991; reduction in neurite length, this suggested the requirement Hollenbeck, 1993). These functions must be carried out by of kinesin for normal neuromuscular function (Saxton et al., motor proteins. 1991). Furthermore, the same kinesin mutation in Drosoph- Kinesin is a motor enzyme first identified in the squid axo- ila affected the action potential propagation in axons and plasm and now isolated from a variety of cell types (Scholey neurotransmitter release at nerve terminals (Gho et al., et al., 1985; Vale et al., 1985; Neighbors et al., 1988; Sax- 1992). In the axon various different kinds of membranous or- ton et al., 1988). Kinesin could move microtubules along a ganeUes are conveyed bidirectionally at different rates. In ad- dition, there is also a variety of structures of crossbridges be- Address all correspondence to N. Hirokawa, Department of Anatomy and tween membranous organelles and microtubules in vivo that Cell Biology, School of Medicine, University of Tokyo, Hongo, Tokyo, 113 are candidates for motor molecules (see Hirokawa, 1982; Japan. Hirokawa et al., 1985; Hirokawa and Yorifuji, 1986). These © The Rockefeller University Press, 0021-9525/94/06/1095/13 $2.00 The Journal of Cell Biology, Volume 125, Number 5, June 1994 1095-1107 1095 data indicated that kinesin is a good candidate for a fast ax- et al., 1992), was reintroduced into pBluescript SK(*) at the EcoRI site onal transporter, but also suggested the existence of other (pBS/KIF3A). A transfer vector was constructed using the pAcYM1 transfer vector (Matsuura et al., 1987) which contains the viral polyhedrin promoter motors mediating the fast axonal transport. for the expression of the cloned eDNA insert. To generate pAcYM1/KIF3A, Recently, several genes were discovered whose predicted the EcoRV site was introduced just upstream of the initial ATG start codon products were related to kinesin (for reviews see Endow, of the KIF3A eDNA in the pBS/KIF3A by PeR method. The KIF3A eDNA 1991; Goldstein, 1991; Bloom, 1992; Hirokawa, 1993). The was excised by digestion with EcoRV and EcoRI and repaired with the region of their similarity corresponds to the ldnesin motor Klenow fragment of DNA polymerase I. This EcoRV-EcoRI fragment en- codes the entire coding region of KIF3A including the termination codon. domain which includes a putative ATP-binding site and a re- The blunt-ended EcoRV-EcoRI fragment was ligated into the dephosphory- gion that can bind to microtubules (Yang et al., 1989; Enos lated SmaI site of the pAcYMI transfer vector. The junction sites were se- and Morris, 1990; Hagan and Yamigada, 1990; Kosik et al., quenced and determined to contain the ATG codon and termination codon 1990; McDonald and Goldstein, 1990; Meluh and Rose, of KIF3A. 1990; Zhang et al., 1990; Endow and Hatsumi, 1991; Ot- suka et al., 1991). This superfamily of kinesin-like, micro- Transfection and Selection of Recombinant Virus tubule-based motor proteins appears to have diverse motility The insect cell line Spodopetra frugiperda (Sfg) 1 was propagated in TC100 functions such as mitosis (Scholey et al., 1985; Leslie et al., medium (GIBCO BRL, Gaithersburg, MD) supplemented with bactotryp- 1987; Enos and Morris, 1990; Hagan and Yanagida, 1990; rose broth and 10% fetal bovine serum (Summers and Smith, 1987). AcRlV23.1acZ virus DNA (Kitts et al., 1990) was used because the replace- Hoyt et al., 1992; Roof et al., 1992) and meiosis (Endow et ment of the fl-galactosidase gene by eDNA in the pAcYM1 vector can be al., 1990; McDonald and Goldstein, 1990; Zhang et al., detected in the presence of X-gal. Sf9 cells were transfected with mixtures 1990). From these findings of the diversity of ldnesin, the of linearized AcRP23.1acZ DNA and pACYMI/KIF3A by lipofemion possibility exists that there are some unknown axonal trans- (GIBCO BRL) to generate recombinant baculovirus AcKIF3A containing KIF3A eDNA resulting from homologous recombination. After 4 d of incu- porters that are kinesin-like. Indeed, the UNC-104 from bation at 27°C, supernatant fluids were harvested and assayed for recom- nematode Caenorhabditis elegans, a novel member of the binant virus by infecting Sf9 monolayers and overlaying them with agarose. kinesin superfamily with a kinesin-like motor domain, has A few days later, recombinant plaques were selected in the presence of the been demonstrated to be a putative neuron-specific motor substrate X-gal. The plaques were picked out and the recombinant virus was used for the anterograde axonal transport of synaptic vesicles purified by tv~ rounds of screening in the presence of X-gal and grown to high titer stocks by serial amplification. The purified recombinant virus (Hall and Hedgecock, 1991). stock of high titration (107 plaque-forming U/ml) was used to infect Therefore, we speculated about the existence of several monolayers of Sf9 cells for the subsequent recombinant KIF3A expression. other new members of kinesin-like proteins in mammalian neuronal cells. We have previously cloned five different Expression and Purification of KIF3A Protein members from their homology to the kinesin motor domain For expression of the KIF3A protein, Sf9 cells were infected with AcKIF3A by PCR method (Aizawa et al., 1992). KIF3A (formerly at a multiplicity of 1-10 PFU/cell and incubated at 27°C for 72 h. Cells were designated as KIF3) (701 amino acids) is a member of this harvested by centrifugation and washed once with PBS. Each 10 mi of cell Idnesin superfamily and is an as yet unidentified new species. culture was resuspended in 1 ml of PEM buffer (0.1 M Pipes, pH 6.8, 1 mM EGTA, and 1 mM MgCh) supplemented with 1 mM DTT, 1 mM PMSE Northern blot analysis showed that KIF3A is predominantly and 10 ~g/ml leupeptin. The cells were then lysed with a glass-teflon ho- expressed in brain, although trace amounts of KIF3A tran- mogenizer and clarified with a Beckman TL100 ultracentrifuge at

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