Molecular characterization and expression of a novel kinesin which localizes with the kinetoplast in the human pathogen, Leishmania donovani.
Using a variety of molecular and cell biological approaches, we identified, characterized and expressed a novel kinesin, LdK39B in the protozoan pathogen Leishmania donovani. Results of RT-PCR revealed two distinct LdK39B products with different splice leader mini-exon sites, indicative of two potentially mature mRNA transcripts. Analyses indicated that LdK39B had a calculated molecular mass of >261, 327 Da and contained multiple amino acid repeat units. Several GFP-LdK39B fusion constructs were generated and used for episomal-expression in these parasites. Results of confocal and immunoelectron microscopy indicated that the GFP-LdK39B-fusion proteins localized to a region adjacent to the flagellar pocket and the kinetoplast i.e. the mitochondrial-DNA containing organelle that is physically tethered to the flagellar basal bodies. Sub-cellular fractionation results showed that GFP-LdK39B proteins were insoluble in nature and remained tightly associated with purified flagella/kinetoplasts following their extraction with detergent and high salts. Our cumulative results suggest that the LdK39B may play a scaffold-like role in facilitating and maintaining the unique spatial/structural association between the flagellum-basal body-kinetoplast complex in these parasites.
Gerald
Noel J
NJ
Cell Biology Section, Lab of Parasitic Diseases, NIAID/NIH, Bethesda, Maryland 20892-0425, USA.
Coppens
Isabelle
I
Dwyer
Dennis M
DM
D000818:Animals; D001483:Base Sequence; D018105:DNA, Kinetoplast / Q000378:metabolism*; D016547:Kinesins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D007893:Leishmania donovani / Q000166:cytology / Q000378:metabolism* / Q000648:ultrastructure; D008856:Microscopy, Fluorescence; D016253:Microscopy, Immunoelectron; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism
D018105:DNA, Kinetoplast; D011993:Recombinant Fusion Proteins; D016547:Kinesins