The DYNLT3 Light Chain Directly Links Cytoplasmic Dynein to a Spindle Checkpoint Protein, Bub3*
- Department of Cell Biology, School of Medicine, University of Virginia, Charlottesville, Virginia 22908
- 1 Supported by the NINDS, National Institutes of Health and the University of Virginia Cancer Center. To whom correspondence should be addressed: Dept. of Cell Biology, School of Medicine, University of Virginia, P. O. Box 800732, Charlottesville, VA 22908-0732. Tel.: 434-924-1912; Fax: 434-982-3912; E-mail: kkp9w{at}virginia.edu.
Abstract
Cytoplasmic dynein is the motor protein responsible for the intracellular transport of various organelles and other cargoes toward microtubule minus ends. However, it remains to be determined how dynein is regulated to accomplish its varied roles. The dynein complex contains six subunits, including three classes of light chains. The two isoforms of the DYNLT (Tctex1) family of light chains, DYNLT1 and DYNLT3, have been proposed to link dynein to specific cargoes. However, no specific binding partner had been found for the DYNLT3 light chain. We find that DYNLT3 binds to Bub3, a spindle checkpoint protein. Bub3 binds exclusively to DYNLT3 and not to the other dynein light chains. Glutathione S-transferase pull-down and co-immunoprecipitation assays demonstrate that Bub3 interacts with the cytoplasmic dynein complex. DYNLT3 is present on kinetochores at prometaphase, but not later mitotic stages, demonstrating that this dynein light chain, like Bub3 and other checkpoint proteins, is depleted from the kinetochore during chromosome alignment. Knockdown of DYNLT3 with small interference RNA increases the mitotic index, in particular, the number of cells in prophase/prometaphase. These results demonstrate that dynein binds directly to a component of the spindle checkpoint complex through the DYNLT3 light chain. Thus, DYNLT3 contributes to dynein cargo binding specificity. These data also suggest that the subpopulation of dynein, containing the DYNLT3 light chain, may be important for chromosome congression, in addition to having a role in the transport of checkpoint proteins from the kinetochore to the spindle pole.
Footnotes
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↵2 The cytoplasmic dynein 1 light chain subunit nomenclature is as follows. There are three functionally distinct light chain families in the cytoplasmic dynein complex and each family has at least two members (or isoforms). The names of the light chains all begin with DYN for dynein, followed by L for light chain, then additional letters that designate the families. The family names are based on the old common name of the first identified member of each family: DYNLT (T for the Tctex1 family); DYNLRB (RB for the Roadblock family); and DYNLL (L for the LC8 family). The different members (or isoforms) of the families are distinguished by adding numbers to the name, for example DYNLT1 and DYNLT3 are the two members of the DYNLT light chain family. In the text, a light chain polypeptide subunit is identified at first mention with its formal name followed by the old common name in parentheses, for example DYNLT3 (previously called rp3). This nomenclature has been endorsed by the Human Genome Organization Nomenclature Committee (HGNC) and the International Committee on Standardized Nomenclature for Mice (2).
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↵3 K. W.-H. Lo, J. M. Kogoy, and K. K. Pfister, unpublished observations.
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↵4 The abbreviations used are: GST, glutathione S-transferase; 3-AT, 3-amino-1,2,4-triazole; DAPI, 4′,6-diamidino-2-phenylindole; GFP, green fluorescent protein; EGFP, enhanced GFP; GSH, glutathione-Sepharose; PBS, phosphate-buffered saline; siRNA, small interfering RNA; NRK, normal rat kidney cell; HA, hemagglutinin.
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↵5 K. W.-H. Lo, J. M. Kogoy, and K. K. Pfister, manuscript in preparation.
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↵6 E. Logarinho, T. Resende, C. Torres, and H. Bousbaa, personal communication and abstract of American Society for Cell Biology meeting 2006.
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↵* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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- Received December 8, 2006.
- Revision received January 26, 2007.
