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J. Biol. Chem., Vol. 276, Issue 52, 49117-49124, December 28, 2001

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A Novel C-terminal Kinesin Is Essential for Maintaining Functional Acidocalcisomes in Trypanosoma brucei^*

Sandrine Dutoya^§, Stephanie Gibert^§, Guillaume Lemercier, Xavier Santarelli^¶, Dominique Baltz, Theo Baltz, and Norbert Bakalara

From the  Laboratoire de Parasitologie Moléculaire UMR CNRS 5016, Université Victor Segalen and the ^¶ Ecole Supérieure des Technologies et Biomolécules de l'Université de Bordeaux II, Bordeaux II, 33076 France

Kinesins are cytoskeletal motor proteins that play roles in a variety of fundamental cellular processes including cell division and the anterograde transport of vesicles and organelles. We purified, cloned, and functionally characterized in Trypanosoma brucei a new member of the C-terminal kinesin family, TbKIFC1. Kinetic constants of the recombinant motor domain of TbKIFC1 were estimated at 0.56 µM for the microtubule dissociation constant (K_d) with a k_cat of 0.2 s¹. Immunolocalization analysis showed an association of TbKIFC1 with punctate structures. Because they were rapidly transported to the negative pole of the microtubule after NH₄Cl treatment, these structures were considered to be associated with acidic vesicles. To determine the role of the kinesin in vivo, we produced an inducible kinesin-deficient strain by double-stranded RNA interference methodology. Mutant cells were loaded with the fluorescent reagent fura2/acetoxymethylester to measure intracellular free calcium ([Ca²⁺]_i). The resting [Ca²⁺]_i was unchanged in mutant cells; however, alkalinization of acidic vesicles induced by NH₄Cl or nigericin was not followed by release of Ca²⁺. These data and the relative importance of the ionomycin-releasable and the ionomycin-plus-NH₄Cl-releasable Ca²⁺ pools suggest a lower Ca²⁺ content in acidocalcisomes and dysfunctional Ca²⁺ release.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank^TM/EMBL Data Bank with accession number(s) AF319546.

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