Structural Domains of CIF3 Required for Interaction with Cytokinesis Regulatory Proteins and for Cytokinesis Initiation in Trypanosoma brucei.

Cytokinesis in Trypanosoma brucei occurs unidirectionally from the anterior toward the posterior through mechanisms distinct from those of its human host and is controlled by a signaling pathway comprising evolutionarily conserved and trypanosome-specific regulatory proteins. The mechanistic roles and the functional interplay of these cytokinesis regulators remain poorly understood. Here, we investigate the requirement of the structural motifs in the trypanosome-specific cytokinesis regulator CIF3 for the initiation of cytokinesis, the interaction with other cytokinesis regulators, and the recruitment of CIF3-interacting proteins to the cytokinesis initiation site. We demonstrate that the internal and C-terminal coiled-coil motifs, but not the N-terminal coiled-coil motif, of CIF3 play essential roles in cytokinesis and interact with distinct cytokinesis regulators. CIF3 interacts with TbPLK, CIF1, CIF4, and FPRC through the N-terminal and C-terminal coiled-coil motifs and with KAT80 through all three coiled-coil motifs. The C-terminal coiled-coil motif of CIF3 is required for the localization of CIF3 and all of its interacting proteins, and additionally, the internal coiled-coil motif of CIF3 is required for KAT80 localization. Conversely, all the CIF3-interacting proteins are required to maintain CIF3 at the cytokinesis initiation site at different cell cycle stages. These results demonstrate that CIF3 cooperates with multiple interacting partner proteins to promote cytokinesis in T. brucei.