A novel C-terminal kinesin is essential for maintaining functional acidocalcisomes in Trypanosoma brucei.
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 microm for the microtubule dissociation constant (K(d)) with a k(cat) of 0.2 s(-1). Immunolocalization analysis showed an association of TbKIFC1 with punctate structures. Because they were rapidly transported to the negative pole of the microtubule after NH(4)Cl 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 ([Ca(2+)](i)). The resting [Ca(2+)](i) was unchanged in mutant cells; however, alkalinization of acidic vesicles induced by NH(4)Cl or nigericin was not followed by release of Ca(2+). These data and the relative importance of the ionomycin-releasable and the ionomycin-plus-NH(4)Cl-releasable Ca(2+) pools suggest a lower Ca(2+) content in acidocalcisomes and dysfunctional Ca(2+) release.
Dutoya
S
S
Laboratoire de Parasitologie Moléculaire UMR CNRS 5016, Université Victor Segalen, Bordeaux II, 33076 France.
Gibert
S
S
Lemercier
G
G
Santarelli
X
X
Baltz
D
D
Baltz
T
T
Bakalara
N
N
D000643:Ammonium Chloride / Q000378:metabolism; D000818:Animals; D002118:Calcium / Q000378:metabolism; D022162:Cytoplasmic Vesicles / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007150:Immunohistochemistry; D015759:Ionomycin / Q000494:pharmacology; D007476:Ionophores / Q000494:pharmacology; D016547:Kinesins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D008870:Microtubules / Q000378:metabolism; D020409:Molecular Motor Proteins / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009550:Nigericin / Q000494:pharmacology; D010641:Phenotype; D010802:Phylogeny; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D015800:Protozoan Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D013752:Tetracycline / Q000494:pharmacology; D014346:Trypanosoma brucei brucei / Q000187:drug effects / Q000235:genetics / Q000502:physiology* / Q000648:ultrastructure
D007476:Ionophores; C445315:KIFC1 protein, Trypanosoma brucei; D020409:Molecular Motor Proteins; D011500:Protein Synthesis Inhibitors; D015800:Protozoan Proteins; D000643:Ammonium Chloride; D015759:Ionomycin; D016547:Kinesins; D013752:Tetracycline; D009550:Nigericin; D002118:Calcium