Molecular dissection and expression of the LdK39 kinesin in the human pathogen, Leishmania donovani.
In this study we show for the first time the intracellular distribution of a K39 kinesin homologue in Leishmania donovani, a medically important parasite of humans. Further, we demonstrated that this motor protein is expressed in both the insect and mammalian developmental forms (i.e. promastigote and amastigotes) of this organism. Moreover, in both of these parasite developmental stages, immunofluorescence indicated that the LdK39 kinesin accumulated at anterior and posterior cell poles and that it displayed a peripheral localization consistent with the cortical cytoskeleton. Using a molecular approach, we identified, cloned and characterized the first complete open reading frame for the gene (LdK39) encoding this large (> 358 kDa) motor protein in L. donovani. Based on these observations, we subsequently used a homologous episomal expression system to dissect and express the functional domains that constitute the native molecule. Cell fractionation experiments demonstrated that LdK39 was soluble and that it bound to detergent-extracted cytoskeletons of these parasites in an ATP-dependent manner. The cumulative results of these experiments are consistent with LdK39 functioning as an ATP-dependent kinesin which binds to and travels along the cortical cytoskeleton of this important human pathogen.
Gerald
Noel J
NJ
Cell Biology Section, Laboratory of Parasitic Diseases, NIAID/NIH, Bethesda, MD, USA.
Coppens
Isabelle
I
Dwyer
Dennis M
DM
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D000953:Antigens, Protozoan / Q000235:genetics* / Q000378:metabolism*; D003001:Cloning, Molecular; D003593:Cytoplasm / Q000378:metabolism; D003599:Cytoskeleton / Q000378:metabolism; D003847:Deoxyglucose / Q000737:chemistry; D003902:Detergents / Q000737:chemistry; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D016547:Kinesins / Q000235:genetics* / Q000378:metabolism*; D007893:Leishmania donovani / Q000737:chemistry / Q000472:pathogenicity / Q000502:physiology*; D008969:Molecular Sequence Data; D010802:Phylogeny; D017434:Protein Structure, Tertiary; D015800:Protozoan Proteins / Q000235:genetics* / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D019810:Sodium Azide / Q000737:chemistry
D000953:Antigens, Protozoan; D003902:Detergents; D015800:Protozoan Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D000255:Adenosine Triphosphate; D019810:Sodium Azide; D003847:Deoxyglucose; C079253:K39 antigen, Leishmania; D016547:Kinesins