Isolation and analysis of microtubule motor proteins. 1994

W M Saxton
Department of Biology, Indiana University, Bloomington 47405.

Isolation of microtubule motor proteins is needed both for the discovery of new motors and for characterization of the products of motor-related genes. The sequences of motor-related genes cannot yet be used to predict the mechanochemical properties of the gene products. This was illustrated by the first kinesin-related gene product to be characterized. Protein expressed from the ncd gene moved toward the minus ends of microtubules (Walker et al., 1990; McDonald et al., 1990), while kinesin itself moves toward the plus ends. Until the relationship between mechanochemical function and amino acid sequence is more thoroughly understood, biochemical isolation and characterization of microtubule motor proteins will remain essential. Two approaches for getting useful quantities of microtubule motor proteins have been used: isolation from cytosol as described under Section II above and isolation from bacteria carrying cloned motor protein genes in expression vectors. Bacterial expression of functional microtubule motors has been successful to date in only a few cases (Yang et al., 1990; Walker et al., 1990, McDonald et al., 1990). Additional progress is expected with the expression of cloned genes from viral vectors in cultured eukaryotic cells, but broad success has not yet been reported. Biochemical isolation of motors from their natural cytosol has some distinct advantages. One can have confidence that a given motor will be folded properly and have normal post-translational modifications. In addition, if it exists in vivo as a heteromultimer, a microtubule motor isolated from its native cytosol will carry with it a normal complement of associated proteins. Studies of such associated proteins will be important in learning how motors accomplish their tasks in vivo. Drosophila cytosol should be a rich source of microtubule motors. Drosophila carry at least 11 and perhaps as many as 30 genes that are related to kinesin (Stewart et al., 1991; Endow and Hatsumi, 1991). The work of Tom Hays' lab indicates that Drosophila carry more than nine dynein related genes (Rasmussen et al., 1994). Relatively little effort to isolate the products of these genes from cytosol has been made. The only work that I am aware of has produced a kinesin-like microtubule motor (D.G. Cole, K.B. Sheehan, W.M. Saxton, and J.M. Scholey, in progress) that may be the Drosophila homolog of Xenopus eg5 (Sawin et al., 1992). This isolation was straightforward, and efforts to identify additional motors are almost assured of success.

UI MeSH Term Description Entries
D008297 Male Males
D008868 Microtubule Proteins Proteins found in the microtubules. Proteins, Microtubule
D008870 Microtubules Slender, cylindrical filaments found in the cytoskeleton of plant and animal cells. They are composed of the protein TUBULIN and are influenced by TUBULIN MODULATORS. Microtubule
D002458 Cell Fractionation Techniques to partition various components of the cell into SUBCELLULAR FRACTIONS. Cell Fractionations,Fractionation, Cell,Fractionations, Cell
D004331 Drosophila melanogaster A species of fruit fly frequently used in genetics because of the large size of its chromosomes. D. melanogaster,Drosophila melanogasters,melanogaster, Drosophila
D004625 Embryo, Nonmammalian The developmental entity of a fertilized egg (ZYGOTE) in animal species other than MAMMALS. For chickens, use CHICK EMBRYO. Embryonic Structures, Nonmammalian,Embryo, Non-Mammalian,Embryonic Structures, Non-Mammalian,Nonmammalian Embryo,Nonmammalian Embryo Structures,Nonmammalian Embryonic Structures,Embryo Structure, Nonmammalian,Embryo Structures, Nonmammalian,Embryo, Non Mammalian,Embryonic Structure, Non-Mammalian,Embryonic Structure, Nonmammalian,Embryonic Structures, Non Mammalian,Embryos, Non-Mammalian,Embryos, Nonmammalian,Non-Mammalian Embryo,Non-Mammalian Embryonic Structure,Non-Mammalian Embryonic Structures,Non-Mammalian Embryos,Nonmammalian Embryo Structure,Nonmammalian Embryonic Structure,Nonmammalian Embryos,Structure, Non-Mammalian Embryonic,Structure, Nonmammalian Embryo,Structure, Nonmammalian Embryonic,Structures, Non-Mammalian Embryonic,Structures, Nonmammalian Embryo,Structures, Nonmammalian Embryonic
D000818 Animals Unicellular or multicellular, heterotrophic organisms, that have sensation and the power of voluntary movement. Under the older five kingdom paradigm, Animalia was one of the kingdoms. Under the modern three domain model, Animalia represents one of the many groups in the domain EUKARYOTA. Animal,Metazoa,Animalia
D012617 Sea Urchins Somewhat flattened, globular echinoderms, having thin, brittle shells of calcareous plates. They are useful models for studying FERTILIZATION and EMBRYO DEVELOPMENT. Echinoidea,Sand-Dollar,Clypeasteroida,Sand Dollars,Clypeasteroidas,Dollar, Sand,Dollars, Sand,Echinoideas,Sand Dollar,Sand-Dollars,Sea Urchin,Urchin, Sea,Urchins, Sea
D013081 Sperm Motility Movement characteristics of SPERMATOZOA in a fresh specimen. It is measured as the percentage of sperms that are moving, and as the percentage of sperms with productive flagellar motion such as rapid, linear, and forward progression. Motilities, Sperm,Motility, Sperm,Sperm Motilities

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