Brain Cytoplasmic and Flagellar Outer Arm Dyneins Share a Highly Conserved Mr 8,000 Light Chain

doi:10.1074/jbc.271.32.19358

Brain Cytoplasmic and Flagellar Outer Arm Dyneins Share a Highly Conserved M_r 8,000 Light Chain*

From the Departments of ^‡ Biochemistry and
^¶ Neurology and
the ^∥ Center for Biomedical Imaging Technology, University of Connecticut Health Center, Farmington, Connecticut 06032-3305 and
the ^” Department of Cell Biology, University of Virginia Health Science Center, Charlottesville, Virginia 22908-0439

^§ To whom correspondence should be addressed:
Dept. of Biochemistry, University of Connecticut Health Center, 263 Farmington Ave., Farmington, CT 06032-3305.
Tel.: 860-679-3347; Fax: 860-679-3408; E-mail: king{at}panda.uchc.edu

Abstract

Sequence comparisons with the M_r 8,000 light chain from Chlamydomonas outer arm dynein revealed the presence of highly conserved homologues (up to 90% identity) in the expressed sequence tag data base (King, S. M. & Patel-King, R. S. (1995a) J. Biol. Chem. 270, 11445-11452). Several of these homologous sequences were derived from organisms and/or tissues that lack motile cilia/flagella, suggesting that these proteins may function in the cytoplasm. In Drosophila, lack of the homologous protein results in embryonic lethality (Dick, T., Ray, K., Salz, H. K. & Chia, W. (1996) Mol. Cell. Biol., 16, 1966-1977). Fractionation of mammalian brain homogenates reveals three distinct cytosolic pools of the homologous protein, one of which specifically copurifies with cytoplasmic dynein following both ATP-sensitive microtubule affinity/sucrose density gradient centrifugation and immunoprecipitation with a monoclonal antibody specific for the 74-kDa intermediate chain (IC74). Quantitative densitometry indicates that there is one copy of the M_r 8,000 polypeptide per IC74. Dual channel confocal immunofluorescent microscopy revealed that the M_r 8,000 protein is significantly colocalized with cytoplasmic dynein but not with kinesin in punctate structures (many of which are associated with microtubules) within mammalian oligodendrocytes. Thus, it appears that flagellar outer arm and brain cytoplasmic dyneins share a highly conserved light chain polypeptide that, at least in Drosophila, is essential for viability.

Footnotes

↵* This work was supported by a New Investigator award from the Patrick and Catherine Weldon Donaghue Medical Research Foundation (to S. M. K.) and by National Institutes of Health Grants GM 51293 (to S. M. K.), NS 19943 (to E. B.), NS 15190 (to J. H. C.), and NS 29996 (to K. K. P.). The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

DHC

dynein heavy chain

IC

intermediate chain

LC

light chain

LIC

light intermediate chain

TBS

Tris-buffered saline.
↵² We have attempted to use both the R4058 antibody and a second polyclonal antibody made against the M_r 8,000 Chlamydomonas protein to immunoprecipitate associated polypeptides from both rat brain homogenates and Chlamydomonas flagellar extracts. In neither case were any proteins (including the M_r 8,000 LC) obtained in the pellet.
↵³ K. K. Pfister and G. B. Witman, unpublished observations.
↵⁴ S. M. King, unpublished observation.
↵⁵ E. Barbarese, unpublished observations.
- Received December 11, 1995.
- Revision received April 15, 1996.