Abstract. In neuronal axons, various kinds of membranous components are transported along microtubules bidirectionally. However, only two kinds of mechanochemical motor proteins, kinesin and brain dynein, had been identified as transporters of membranous organelles in mammalian neurons. Recently, a series of genes that encode proteins closely related to kinesin heavy chain were identified in several organisms including Schizosaccharomyces pombe, Aspergillus niddulans, Saccharomyces cerevisiae, Caenorhabditus elegans, and Drosophila. Most of these members of the kinesin family ale implicated in mechanisms of mitosis or meiosis. To address the mechanism of intracellular organelle transport at a molecular level, we have cloned and characterized five different members , that encode the microtubule-associated motor domain homologous to kinesin heavy chain, in murine brain tissue. Homology analysis of amino acid sequence indicated that KIF1 and KIF5 are mufine counterparts of uric/04 and kinesin heavy chain, respectively, while KIF2, KIF3, and KIF4 are as yet unidentified new species. Complete amino acid sequence of KIF3 revealed that KIF3 consists of NH2-terminal motor domain, central a-helical rod domain, and COOH-terminal globular domain. Complete amino acid sequence of KIF2 revealed that KIF2 consists of NH~-terminal globular domain, central motor domain, and COOH-terminal or-helical rod domain. This is the first identification of the kinesin-related protein which has its motor domain at the central part in its primary structure. Northern blot analysis revealed that KIFI, KIF3, and KIF5 are expressed almost exclusively in murine brain, whereas KIF2 and KIF4 are expressed in brain as well as in other tissues. All these members of the kinesin family are expressed in the same type of neurons, and thus each one of them may transport its specific organelle in the murine central nervous system.

Abstract. Neurofilaments are the major cytoskeletal elements in the axon that take highly ordered structures composed of parallel arrays of 10-nm filaments linked to each other with frequent cross-bridges, and they are believed to maintain a highly polarized neuronal cell shape. Here we report the function of rat NF-M in this characteristic neurofilament assembly. Transfection experiments were done in an insect Sf9 cell line lacking endogenous intermediate filaments.

Overlapping cDNA clones encoding the heavy chain of rat brain cytoplasmic dynein have been isolated. The isolated cDNA clones contain an open reading frame of 13,932 bp encoding 4644 aa (Mr, 532,213). The deduced protein sequence of the heavy chain of rat brain dynein shows significant similarity to sea urchin flageilar -dynein (27.0% identical) and toDictyostelium cytoplasmic dynein (53.5% identical) throughout the entire sequence. The heavy chain of rat brain (cytoplasmic) dynein contains four putative nudeotide-binding consensus sequences [GX4GK(T/S)] in the central one-third region that are highly similar to those of sea urchin and Dictyostelium dyneins. The N-terminal one-third of the heavy chain of rat brain (cytoplasmic) dynein shows high similarity (43.8% identical) to that of Dictyostelium cytoplasmic dynein but poor similarity (19.4% identical) to that of sea urchin flageliar dynein. These results suggested that the C-terminal two-thirds of the dynein molecule is conserved and plays an essential role in microtubule-dependent motility activity, whereas the N-terminal regions are different between cytoplasmic and flageilar dyneins.The nerve cell develops a polarized morphology composed of highly branched dendrites, a long axon, and synapses. Because of the lack of protein synthesis machinery in the axon, proteins in the axon and synapses must be transported down the axon after being synthesized in the cell body. Many proteins are transported bidirectionally in membranous organelles of various kinds by fast axonal flow (200-400 mm/ day), whereas other proteins such as cytoskeletal components are conveyed by slow axonal flow (0.5-2 mm/day). Electron microscopic studies of the neuronal cytoskeleton in vivo have suggested that microtubules and crossbridges between microtubules and membranous organelles form the structural basis for fast flow (1-3). Recently, two microtubule-activated ATPases, kinesin and brain dynein [cytoplasmic dynein, microtubule-associated protein (MAP) 1C], were identified as candidates for anterograde and retrograde molecular motors, respectively (4-7). In fact, kinesin is primarily associated with anterogradely moving membranous organelles in the axon, strongly supporting the hypothesis that kinesin is really an anterograde motor in vivo (8). Brain dynein is associated with both anterogradely and retrogradely moving membranous organelles (9), consistent with the suggestion that brain dynein is transported to the nerve terminal by fast flow and subsequently functions as a retrograde transport motor in vivo. Molecular genetic and ultrastructural approaches have dissected the molecular structure and functional domains of kinesin motors (10-12). However, detailed analysis of the primary structure of brain dynein hasThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. not yet been accomplished. To further elucidate the functi...

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