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J. Biol. Chem., Vol. 282, Issue 18, 13780-13790, May 4, 2007

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An Essential Cell Cycle-regulated Nucleolar Protein Relocates to the Mitotic Spindle Where It Is Involved in Mitotic Progression in Trypanosoma brucei^*

From the Laboratoire de Microbiologie Cellulaire et Moléculaire et Pathogénicité, UMR/CNRS-5234, Université Victor Segalen Bordeaux 2, 33076 Bordeaux, France

TbNOP86 and TbNOP66 are two novel nucleolar proteins isolated in Trypanosoma brucei. They share 92.6% identity, except for an additional C-terminal domain of TbNOP86 of 182 amino acids in length. Both proteins are found in Trypanosomatidae, but similarity to other eukaryotic proteins could not be found. TbNOP86 and TbNOP66 are expressed at similar level in procyclic and bloodstream forms, although the relative level of expression of TbNOP66 is 11 times lower. TbNOP86 undergoes post-translational modifications, as it is found predominantly at 110 kDa compared with the predicted 86 kDa. Immunofluorescence of overexpressed ty-tagged TbNOP86 and TbNOP66 showed that both proteins accumulated in the nucleolus of G₁ cells. This was confirmed by the co-localization of an endogenous TbNOP86-myc with the nucleolar protein Nopp140. TbNOP86-ty localization is cell cycle-regulated, because it colocalizes with the mitotic spindle in mitotic cells. TbNOP86 is required for mitotic progression in both life stages as depleted cells are enriched in the G₂/M phase. In procyclic cells, a reduced growth rate is accompanied by an accumulation of zoids (0N1K), 2N1K, and multinucleated cells (xNyK). The 2N1K cells are blocked in late mitosis as nucleolar segregation is completed. TbNOP86 depletion in bloodstream form caused a drastic growth inhibition producing cells bearing two kinetoplasts and an enlarged nucleus (1N^*2K), followed by an accumulation of 2N2K cells with connected nuclei and xNyK cells. These studies of TbNOP86 provide a more comprehensive account of proteins involved in mitotic events in trypanosomes and should lead to the identification of partners with similar function.

Received for publication, January 26, 2007 , and in revised form, February 23, 2007.

^* This work was supported in part by the CNRS, the Fondation Recherche Médicale, and the Ministèredel'Éducation Nationale de la Recherche et de la Technologie. 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.

The on-line version of this article (available at http://www.jbc.org) contains supplemental data and Figs. S1-S3.

¹ A recipient of a Fonds de la Recherche en Santé du Québec postdoctoral fellowship. To whom correspondence should be addressed. Tel.: 33-557-574-804; Fax: 33-557-574-803; E-mail: nathalie.boucher{at}parasitmol.u-bordeaux2.fr.

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