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Eukaryotic Cell, August 2008, p. 1362-1372, Vol. 7, No. 8
1535-9778/08/$08.00+0     doi:10.1128/EC.00084-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Glutamylation on {alpha}-Tubulin Is Not Essential but Affects the Assembly and Functions of a Subset of Microtubules in Tetrahymena thermophila{triangledown} ,{dagger}

Dorota Wloga,1,{ddagger} Krzysztof Rogowski,1,2,{ddagger} Neeraj Sharma,1 Juliette Van Dijk,2 Carsten Janke,2 Bernard Eddé,3 Marie-Hélène Bré,4 Nicolette Levilliers,4 Virginie Redeker,5 Jianming Duan,6 Martin A. Gorovsky,6 Maria Jerka-Dziadosz,7 and Jacek Gaertig1*

Department of Cellular Biology, University of Georgia, Athens, Georgia 30602-2607,1 CRBM, CNRS, 34293 Montpellier, France,2 Université de Paris VI, 75252 Paris, France,3 Laboratoire de Biologie Cellulaire 4, UMR8080, CNRS, Université Paris-Sud, 91405 Orsay cedex, France,4 Laboratoire d'Enzymologie et de Biochimie Structurales, CNRS, 91198 Gif-sur-Yvette, France,5 Department of Biology, University of Rochester, Rochester, New York 14627,6 Department of Cell Biology, M. Nencki Institute of Experimental Biology, Polish Academy of Science, 02-093 Warsaw, Poland7

Received 7 March 2008/ Accepted 18 June 2008

Tubulin undergoes glutamylation, a conserved posttranslational modification of poorly understood function. We show here that in the ciliate Tetrahymena, most of the microtubule arrays contain glutamylated tubulin. However, the length of the polyglutamyl side chain is spatially regulated, with the longest side chains present on ciliary and basal body microtubules. We focused our efforts on the function of glutamylation on the {alpha}-tubulin subunit. By site-directed mutagenesis, we show that all six glutamates of the C-terminal tail domain of {alpha}-tubulin that provide potential sites for glutamylation are not essential but are needed for normal rates of cell multiplication and cilium-based functions (phagocytosis and cell motility). By comparative phylogeny and biochemical assays, we identify two conserved tubulin tyrosine ligase (TTL) domain proteins, Ttll1p and Ttll9p, as {alpha}-tubulin-preferring glutamyl ligase enzymes. In an in vitro microtubule glutamylation assay, Ttll1p showed a chain-initiating activity while Ttll9p had primarily a chain-elongating activity. GFP-Ttll1p localized mainly to basal bodies, while GFP-Ttll9p localized to cilia. Disruption of the TTLL1 and TTLL9 genes decreased the rates of cell multiplication and phagocytosis. Cells lacking both genes had fewer cortical microtubules and showed defects in the maturation of basal bodies. We conclude that glutamylation on {alpha}-tubulin is not essential but is required for efficiency of assembly and function of a subset of microtubule-based organelles. Furthermore, the spatial restriction of modifying enzymes appears to be a major mechanism that drives differential glutamylation at the subcellular level.

* Corresponding author. Mailing address: Department of Cellular Biology, 724 Biological Sciences Building, Athens GA 30602-2607. Phone: (706) 542-3409. Fax: (706) 542-4271. E-mail: jgaertig{at}cb.uga.edu

{triangledown} Published ahead of print on 27 June 2008.

{dagger} Supplemental material for this article may be found at http://ec.asm.org/.

{ddagger} These authors contributed equally to this work.

Eukaryotic Cell, August 2008, p. 1362-1372, Vol. 7, No. 8
1535-9778/08/$08.00+0     doi:10.1128/EC.00084-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Dave, D., Wloga, D., Sharma, N., Gaertig, J. (2009). DYF-1 Is Required for Assembly of the Axoneme in Tetrahymena thermophila. Eukaryot Cell 8: 1397-1406 [Abstract] [Full Text]  
  • Wloga, D., Strzyzewska-Jowko, I., Gaertig, J., Jerka-Dziadosz, M. (2008). Septins Stabilize Mitochondria in Tetrahymena thermophila. Eukaryot Cell 7: 1373-1386 [Abstract] [Full Text]  


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