Skip to main content
NCBI home page
Protein Science : A Publication of the Protein Society logoLink to Protein Science : A Publication of the Protein Society
. 1999 Dec;8(12):2705–2710. doi: 10.1110/ps.8.12.2705

Cloning and expression of kinesins from the thermophilic fungus Thermomyces lanuginosus.

R Sakowicz 1, S Farlow 1, L S Goldstein 1
PMCID: PMC2144245  PMID: 10631986

Abstract

The motor domain regions of three novel members of the kinesin superfamily TLKIF1, TLKIFC, and TLBIMC were identified in a thermophilic fungus Thermomyces lanuginosus. Based on sequence similarity, they were classified as members of the known kinesin families Unc104/KIF1, KAR3, and BIMC. TLKIF1 was subsequently expressed in Escherichia coli. The expression level was high, and the protein was mostly soluble, easy to purify, and enzymatically active. TLKIF1 is a monomeric kinesin motor, which in a gliding motility assay displays a robust plus-directed microtubule movement up to 2 microm/s. The discovery of TLKIF1 also demonstrates that a family of kinesin motors not previously found in fungi may in fact be used in this group of organisms.

Full Text

The Full Text of this article is available as a PDF (911.1 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Altschul S. F., Madden T. L., Schäffer A. A., Zhang J., Zhang Z., Miller W., Lipman D. J. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 1997 Sep 1;25(17):3389–3402. doi: 10.1093/nar/25.17.3389. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Barton N. R., Goldstein L. S. Going mobile: microtubule motors and chromosome segregation. Proc Natl Acad Sci U S A. 1996 Mar 5;93(5):1735–1742. doi: 10.1073/pnas.93.5.1735. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Basaveswara Rao V., Sastri N. V., Subba Rao P. V. Purification and characterization of a thermostable glucoamylase from the thermophilic fungus Thermomyces lanuginosus. Biochem J. 1981 Feb 1;193(2):379–387. doi: 10.1042/bj1930379. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Berg O. G., Cajal Y., Butterfoss G. L., Grey R. L., Alsina M. A., Yu B. Z., Jain M. K. Interfacial activation of triglyceride lipase from Thermomyces (Humicola) lanuginosa: kinetic parameters and a basis for control of the lid. Biochemistry. 1998 May 12;37(19):6615–6627. doi: 10.1021/bi972998p. [DOI] [PubMed] [Google Scholar]
  5. Cherry J. M., Ball C., Weng S., Juvik G., Schmidt R., Adler C., Dunn B., Dwight S., Riles L., Mortimer R. K. Genetic and physical maps of Saccharomyces cerevisiae. Nature. 1997 May 29;387(6632 Suppl):67–73. [PMC free article] [PubMed] [Google Scholar]
  6. Cohn S. A., Saxton W. M., Lye R. J., Scholey J. M. Analyzing microtubule motors in real time. Methods Cell Biol. 1993;39:75–88. doi: 10.1016/s0091-679x(08)60162-4. [DOI] [PubMed] [Google Scholar]
  7. Enos A. P., Morris N. R. Mutation of a gene that encodes a kinesin-like protein blocks nuclear division in A. nidulans. Cell. 1990 Mar 23;60(6):1019–1027. doi: 10.1016/0092-8674(90)90350-n. [DOI] [PubMed] [Google Scholar]
  8. Grummt M., Pistor S., Lottspeich F., Schliwa M. Cloning and functional expression of a 'fast' fungal kinesin. FEBS Lett. 1998 May 1;427(1):79–84. doi: 10.1016/s0014-5793(98)00399-8. [DOI] [PubMed] [Google Scholar]
  9. Harauz G., Flannigan D. Structure of ribosomes from Thermomyces lanuginosus by electron microscopy and image processing. Biochim Biophys Acta. 1990 Apr 19;1038(2):260–267. doi: 10.1016/0167-4838(90)90214-z. [DOI] [PubMed] [Google Scholar]
  10. Hirokawa N. Kinesin and dynein superfamily proteins and the mechanism of organelle transport. Science. 1998 Jan 23;279(5350):519–526. doi: 10.1126/science.279.5350.519. [DOI] [PubMed] [Google Scholar]
  11. Jacobs C. W., Adams A. E., Szaniszlo P. J., Pringle J. R. Functions of microtubules in the Saccharomyces cerevisiae cell cycle. J Cell Biol. 1988 Oct;107(4):1409–1426. doi: 10.1083/jcb.107.4.1409. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Kiefer J. R., Mao C., Braman J. C., Beese L. S. Visualizing DNA replication in a catalytically active Bacillus DNA polymerase crystal. Nature. 1998 Jan 15;391(6664):304–307. doi: 10.1038/34693. [DOI] [PubMed] [Google Scholar]
  13. Kull F. J., Sablin E. P., Lau R., Fletterick R. J., Vale R. D. Crystal structure of the kinesin motor domain reveals a structural similarity to myosin. Nature. 1996 Apr 11;380(6574):550–555. doi: 10.1038/380550a0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Li H. P., Liu Z. M., Nirenberg M. Kinesin-73 in the nervous system of Drosophila embryos. Proc Natl Acad Sci U S A. 1997 Feb 18;94(4):1086–1091. doi: 10.1073/pnas.94.4.1086. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Mandai K., Nakanishi H., Satoh A., Obaishi H., Wada M., Nishioka H., Itoh M., Mizoguchi A., Aoki T., Fujimoto T. Afadin: A novel actin filament-binding protein with one PDZ domain localized at cadherin-based cell-to-cell adherens junction. J Cell Biol. 1997 Oct 20;139(2):517–528. doi: 10.1083/jcb.139.2.517. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Nangaku M., Sato-Yoshitake R., Okada Y., Noda Y., Takemura R., Yamazaki H., Hirokawa N. KIF1B, a novel microtubule plus end-directed monomeric motor protein for transport of mitochondria. Cell. 1994 Dec 30;79(7):1209–1220. doi: 10.1016/0092-8674(94)90012-4. [DOI] [PubMed] [Google Scholar]
  17. Nazar R. N., Wong W. M., Abrahamson J. L. Nucleotide sequence of the 18-25 S ribosomal RNA intergenic region from a thermophile, Thermomyces lanuginosus. J Biol Chem. 1987 Jun 5;262(16):7523–7527. [PubMed] [Google Scholar]
  18. O'Connell M. J., Meluh P. B., Rose M. D., Morris N. R. Suppression of the bimC4 mitotic spindle defect by deletion of klpA, a gene encoding a KAR3-related kinesin-like protein in Aspergillus nidulans. J Cell Biol. 1993 Jan;120(1):153–162. doi: 10.1083/jcb.120.1.153. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Ohkura H., Török T., Tick G., Hoheisel J., Kiss I., Glover D. M. Mutation of a gene for a Drosophila kinesin-like protein, Klp38B, leads to failure of cytokinesis. J Cell Sci. 1997 Apr;110(Pt 8):945–954. doi: 10.1242/jcs.110.8.945. [DOI] [PubMed] [Google Scholar]
  20. Okada Y., Hirokawa N. A processive single-headed motor: kinesin superfamily protein KIF1A. Science. 1999 Feb 19;283(5405):1152–1157. doi: 10.1126/science.283.5405.1152. [DOI] [PubMed] [Google Scholar]
  21. Okada Y., Yamazaki H., Sekine-Aizawa Y., Hirokawa N. The neuron-specific kinesin superfamily protein KIF1A is a unique monomeric motor for anterograde axonal transport of synaptic vesicle precursors. Cell. 1995 Jun 2;81(5):769–780. doi: 10.1016/0092-8674(95)90538-3. [DOI] [PubMed] [Google Scholar]
  22. Pierce D. W., Vale R. D. Assaying processive movement of kinesin by fluorescence microscopy. Methods Enzymol. 1998;298:154–171. doi: 10.1016/s0076-6879(98)98016-8. [DOI] [PubMed] [Google Scholar]
  23. Ponting C. P. AF-6/cno: neither a kinesin nor a myosin, but a bit of both. Trends Biochem Sci. 1995 Jul;20(7):265–266. doi: 10.1016/s0968-0004(00)89040-4. [DOI] [PubMed] [Google Scholar]
  24. Prasad R., Gu Y., Alder H., Nakamura T., Canaani O., Saito H., Huebner K., Gale R. P., Nowell P. C., Kuriyama K. Cloning of the ALL-1 fusion partner, the AF-6 gene, involved in acute myeloid leukemias with the t(6;11) chromosome translocation. Cancer Res. 1993 Dec 1;53(23):5624–5628. [PubMed] [Google Scholar]
  25. Sablin E. P., Kull F. J., Cooke R., Vale R. D., Fletterick R. J. Crystal structure of the motor domain of the kinesin-related motor ncd. Nature. 1996 Apr 11;380(6574):555–559. doi: 10.1038/380555a0. [DOI] [PubMed] [Google Scholar]
  26. Sakowicz R., Berdelis M. S., Ray K., Blackburn C. L., Hopmann C., Faulkner D. J., Goldstein L. S. A marine natural product inhibitor of kinesin motors. Science. 1998 Apr 10;280(5361):292–295. doi: 10.1126/science.280.5361.292. [DOI] [PubMed] [Google Scholar]
  27. Schlacher A., Holzmann K., Hayn M., Steiner W., Schwab H. Cloning and characterization of the gene for the thermostable xylanase XynA from Thermomyces lanuginosus. J Biotechnol. 1996 Aug 20;49(1-3):211–218. doi: 10.1016/0168-1656(96)01516-7. [DOI] [PubMed] [Google Scholar]
  28. Spudich J. A. Optical trapping: Motor molecules in motion. Nature. 1990 Nov 22;348(6299):284–285. doi: 10.1038/348284a0. [DOI] [PubMed] [Google Scholar]
  29. Steinberg G., Schliwa M. The Neurospora organelle motor: a distant relative of conventional kinesin with unconventional properties. Mol Biol Cell. 1995 Nov;6(11):1605–1618. doi: 10.1091/mbc.6.11.1605. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Vale R. D., Fletterick R. J. The design plan of kinesin motors. Annu Rev Cell Dev Biol. 1997;13:745–777. doi: 10.1146/annurev.cellbio.13.1.745. [DOI] [PubMed] [Google Scholar]

Articles from Protein Science : A Publication of the Protein Society are provided here courtesy of The Protein Society

RESOURCES