Molecular characterization and expression of a novel kinesin which localizes with the kinetoplast in the human pathogen, Leishmania donovani

Noel J. Gerald, Isabelle Coppens, Dennis M. Dwyer

Research output: Contribution to journalArticlepeer-review

Abstract

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.

Original languageEnglish (US)
Pages (from-to)269-280
Number of pages12
JournalCell Motility and the Cytoskeleton
Volume65
Issue number4
DOIs
StatePublished - Apr 2008

Keywords

  • GFP
  • Kinesin
  • Kinetoplast
  • Leishmania
  • Protozoan parasite

ASJC Scopus subject areas

  • Structural Biology
  • Cell Biology

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