A novel mouse kinesin of the UNC-104/KIF1 subfamily encoded by the Kif1b gene☆
Introduction
Kinesin and kinesin-related proteins are microtubule-associated motor proteins that move along polarized microtubules to transport a variety of intracellular cargo, e.g. organelles or membrane vesicles (Goldstein, 1993, Goodson et al., 1997, Hirokawa, 1998, Hirokawa et al., 1998, Moore and Endow, 1996). Kinesin is a heterotetramer composed of two heavy chains and two light chains. The classical kinesin heavy chain contains a highly conserved globular motor domain of about 350 amino acids that has both ATP-binding and microtubule binding sites (Goldstein, 1993). Adjacent to the globular motor domain is the stalk domain composed of alpha-helical regions that mediate coiled-coil interactions between dimers (Goldstein, 1993, Moore and Endow, 1996). The kinesin tail domain is composed of both heavy and light chains and is thought to bind specific organelles and thus determine cargo specificity.
The UNC-104/KIF1 subfamily of kinesin-related proteins comprises: C. elegans UNC-104 (Otsuka et al., 1991), mouse KIF1A (Aizawa et al., 1992, Okada et al., 1995) and its human orthologue anterograde transporter of synaptic vesicle (ATSV) (Furlong et al., 1996), mouse KIF1B (Nangaku et al., 1994), mouse KIF1C (partial sequence) (Nakagawa et al., 1997), rat KIF1D (Rogers et al., 1997) and its human ortholog KIF1C (Dorner et al., 1998), fish kinesins FKIF3 and FKIF8 (Bost-Usinger et al., 1997), and Drosophila kinesins KLP38B (Alphey et al., 1997) and Kinesin-73 (Li et al., 1997). These proteins all have a conserved UNC-104-like motor domain and a signature sequence called U104 (Ponting, 1995) that overlaps a forkhead associated domain (FHA). Both UNC-104 and KIF1A have pleckstrin homology (PH) domains at their C-termini. Rotary shadowing analysis suggests that both KIF1A (Okada et al., 1995) and KIF1B (Nangaku et al., 1994) exist as monomers, rather than dimers.
C. elegans UNC-104 is required for axonal transport of synaptic vesicles. Mutations in the C. elegans unc-104 gene result in uncoordinated and slow movement; axons have low numbers of synaptic vesicles and make few synapses (Hall and Hedgecock, 1991). KIF1A, like UNC-104, is found specifically in neurons and is enriched in axons. The cargo of KIF1A includes a subset of synaptic vesicle precursors that contain synaptophysin, synaptotagmin, and Rab3A (Okada et al., 1995). Targeted disruption of the mouse Kif1a gene results in decreased numbers of synaptic vesicles (Yonekawa et al., 1998), suggesting that KIF1A may play a role in transport of synaptic vesicles. However, other defects in these knockout mice suggest that KIF1A may perform additional functions. It has been suggested that KIF1B co-localizes with mitochondria and may transport mitochondria in neuronal cells (Nangaku et al., 1994). However, recent studies on targeted disruption of the Kif5b gene for the neuronal form of conventional kinesin heavy chain indicated abnormal perinuclear localization of mitochondria in the mutant mice, suggesting that kinesin KIF5B is involved in mitochondrial transport (Tanaka et al., 1998).
In this report, we present the molecular cloning of a 10 kb transcript that encodes a novel member of the UNC-104/KIF1 kinesin subfamily. The full-length cDNA encodes a 204 kDa protein that is similar in overall structure to KIF1A, including a C-terminal PH domain. Sequence identity between the 5′ portion of this cDNA and the cDNA for KIF1B (Nangaku et al., 1994), however, suggested that both cDNAs might be derived from a single gene, Kif1b. This model is supported by co-localization of the Kif1b gene and the gene for our novel kinesin on mouse Chromosome 4. The 10 kb cDNA encodes a protein that we designated KIF1Bp204, to distinguish it from the smaller kinesin, KIF1Bp130, encoded by the previously reported Kif1b cDNA (Nangaku et al., 1994). We examined the tissue distribution of transcripts for both KIF1Bp204 and KIF1Bp130 by Northern blot analysis of mouse tissues and identified additional alternative splicing events that increase the isoform diversity produced by the Kif1b gene.
Section snippets
Materials
Restriction enzymes, MMLV SuperScript II reverse transcriptase, T4 polynucleotide kinase, and Random Primers DNA Labeling Kit were from Life Sciences (Gaithersburg, MD). KlenTaq DNA polymerase, Marathon ready cDNA amplification kit, and the human RNA multi-tissue dot blot were from Clontech (La Jolla, CA). Radioactive nucleotides for random primer labeling in library screening ([α-32P]dCTP, >3000 Ci/mmol) and for riboprobe synthesis ([α-32P]UTP, >3000 Ci/mmol) were from DuPont-NEN (Boston, MA).
Cloning of KIF1Bp204
We isolated overlapping cDNAs encoding a novel mouse kinesin by screening a neonatal mouse brain cDNA library with a human cDNA, KH250. These cDNAs yielded a composite sequence of 9814 bp with an open reading frame (ORF) that lacked an ATG (Met) initiation codon. The 5′ end of the cDNA (bases 1–110) was obtained by RT-PCR of mouse brain RNA. The full-length 9924 bp cDNA (Fig. 1A) contained a 5451 bp ORF, followed by a long 4473 bp 3′ UTR. The deduced 1816-amino-acid protein had an estimated size of
Discussion
The mouse Kif1b gene on distal chromosome 4 encodes two kinesins, KIF1Bp130 and KIF1Bp204, that have identical N-terminal motor domains, but differ significantly in size and structure at their C-terminal halves. The first 706 amino acids of each kinesin are identical, except for a six-amino-acid insert seen only in KIF1Bp204. The identical N-terminal region includes the 350-amino-acid conserved motor domain characteristic of all members of the UNC-104/KIF1 kinesin subfamily, plus about 350
Acknowledgements
We thank Kristina Hunker for assistance with the SSCP analysis, Lucy Rowe and Mary Barter (The Jackson Laboratory, Bar Harbor, ME) for assistance with data analysis and the MapManager figure, and Drs Susan Brown and Ron Vale for helpful discussions of the data. This work was supported by National Institutes of Health grants DC02492 (M.I.L.), DC02982, and DC03049 (D.C.K.) and a grant from the National Organization for Hearing Research (M.I.L.).
During revision of this manuscript, another group
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The nucleotide sequence of the cDNA for the KIF1Bp204 kinesin has been deposited in the GenBank database under GenBank Accession No. AF090190.