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J. Biol. Chem., Vol. 276, Issue 52, 49117-49124, December 28, 2001
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, and
From the 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 (Kd) with
a kcat of 0.2 s
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
1.
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 GenBankTM/EMBL Data Bank with accession number(s) AF319546.
§ These authors contributed equally to this work.
To whom correspondence should be addressed: Laboratoire
d'Immunologie et Parasitologie Moléculaire, B.P. 12, Université Bordeaux II, 146 rue Léo-Saignat, 33076 Bordeaux
Cedex, France. Tel.: 33-5-57571014; Fax: 33-5-57571015; E-mail:
bakalara@hippocrate.u-bordeaux2.fr.
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