Twenty-Five Dyneins in Tetrahymena: A Re-examination of the Multidynein Hypothesis David E. Wilkes, 1 Hadley E. Watson, 1 David R. Mitchell, 2 and David J. Asai 1 * 1 Department of Biology, Harvey Mudd College, Claremont, California 91711 2 Department of Cell and Developmental Biology, SUNY Upstate Medical University, Syracuse, New York 13210 Dyneins are responsible for essential movements in eukaryotic cells. The motor activity of each dynein complex resides in its complement of heavy chains. In the present study, we examined 136 heavy chain sequences from the completed genomes of 11 diverse model organisms, including examples from Viridiplantae, Excavata, Chromalveolata, and Metazoa. In many cases, we discovered dynein heavy chains previously not identified. For example, Tetrahymena expresses a total of 25 DYH genes rather than the previously identified 14. The Tetrahymena DYH genes are nonaxonemal DYH1 and DYH2; axonemal outer arm a, b, and g; axonemal two-headed inner arm 1a and 1b; and 18 single-headed inner arm heavy chains. The heavy chains divide into nine classes; six of these are highly conserved in sequence and number of isoforms in a given organism. The other three are single-headed inner arm dyneins, whose numbers vary significantly in different organisms. These findings lead to two conclusions. One, the last com- mon ancestor of all eukaryotes expressed nine different dynein heavy chains. Two, subsequent to the divergences leading to different organisms, additional dynein heavy chains emerged. These newer dyneins are not well conserved across species and the variation may reflect different motility requirements in different organisms. Together, these results suggest that each of the nine classes of dyneins is functionally distinct, but members within some of the classes are not special- ized. An understanding of the relationships among the various dynein heavy chains is important when deducing functions across species. Cell Motil. Cytoskel- eton 65: 342–351, 2008. ' 2008 Wiley-Liss, Inc. Key words: AAA protein; dynein; phylogeny; Tetrahymena INTRODUCTION Dyneins are microtubule-based multisubunit motor complexes that perform a variety of functions [see reviews Porter, 1996; Vale, 2003]. Each complex com- prises one or more heavy chain, and a set of smaller sub- units called intermediate, light intermediate, and light chains. In Tetrahymena, the dynein heavy chain is encoded by the DYH gene, and we will use this termi- nology throughout this article to simplify the presenta- tion. The large heavy chain (>4000 residues) is organ- ized into two domains: the N-terminal ca. one-third forms a tail that binds most of the other dynein subunits and so mediates the tethering of the dynein to its molecu- lar cargo; and the C-terminal ca. two-thirds form the *Correspondence to: David J. Asai, Department of Biology, Harvey Mudd College, 301 Platt Boulevard, Claremont, CA 91711-5990, USA. E-mail: david_asai@hmc.edu This article contains supplementary material available via the Internet at http://www.interscience.wiley.com/jpages/0886-1544/suppmat Contract grant sponsors: National Science Foundation, National Insti- tutes of Health, Howard Hughes Medical Institute. Received 28 November 2007; Accepted 2 January 2008 Published online 25 February 2008 in Wiley InterScience (www. interscience.wiley.com). DOI: 10.1002/cm.20264 ' 2008 Wiley-Liss, Inc. Cell Motility and the Cytoskeleton 65: 342–351 (2008)