An essential farnesylated kinesin in Trypanosoma brucei

Abstract

Kinesins are a family of motor proteins conserved throughout eukaryotes. In our present study we characterize a novel kinesin, Kinesin CaaX, orthologs of which are only found in the kinetoplastids and not other eukaryotes. Kinesin CaaX has the CVIM amino acids at the C-terminus, and CVIM was previously shown to be an ideal signal for protein farnesylation in T. brucei. In this study we show Kinesin CaaX is farnesylated using radiolabeling studies and that farnesylation is dependent on the CVIM motif. Using RNA interference, we show Kinesin CaaX is essential for T. brucei proliferation. Additionally RNAi Kinesin CaaX depleted T. brucei are 4 fold more sensitive to the protein farneysltransferase (PFT) inhibitor LN-59, suggesting that Kinesin CaaX is a target of PFT inhibitors' action to block proliferation of T. brucei. Using tetracycline-induced exogenous tagged Kinesin CaaX and Kinesin CVIMdeletion (non-farnesylated Kinesin) expression lines in T. brucei, we demonstrate Kinesin CaaX is farnesylated in T. brucei cells and this farnesylation has functional effects. In cells expressing a CaaX-deleted version of Kinesin, the localization is more diffuse which suggests correct localization depends on farnesylation. Through our investigation of cell cycle, nucleus and kinetoplast quantitation and immunofluorescence assays an important role is suggested for Kinesin CaaX in the separation of nuclei and kinetoplasts during and after they have been replicated. Taken together, our work suggests Kinesin CaaX is a target of PFT inhibition of T. brucei cell proliferation and Kinesin CaaX functions through both the motor and farnesyl groups. © 2011 Engelson et al.