Functional analyses of the CIF1–CIF2 complex in trypanosomes identify the structural motifs required for cytokinesis

Abstract

Cytokinesis in trypanosomes occurs uni-directionally along the longitudinal axis from the cell anterior towards the cell posterior and requires a trypanosome-specific CIF1-CIF2 protein complex. However, little is known about the contribution of the structural motifs in CIF1 and CIF2 to complex assembly and cytokinesis. Here, we demonstrate that the two zinc-finger motifs but not the coiled-coil motif in CIF1 are required for interaction with the EF-hand motifs in CIF2. We further show that localization of CIF1 depends on the coiled-coil motif and the first zinc-finger motif and that localization of CIF2 depends on the EF-hand motifs. Deletion of the coiled-coil motif and mutation of either zinc-finger motif in CIF1 disrupts cytokinesis. Furthermore, mutation of either zinc-finger motif in CIF1 mislocalizes CIF2 to the cytosol and destabilizes CIF2, whereas deletion of the coiled-coil motif in CIF1 spreads CIF2 over to the new flagellum attachment zone and stabilizes CIF2. Together, these results uncover the requirement of the coiled-coil and zinc-finger motifs for CIF1 function in cytokinesis and for CIF2 localization and stability, providing structural insights into the functional interplay between the two cytokinesis regulators.

The CIF1 protein is a master orchestrator of trypanosome cytokinesis that recruits several cytokinesis regulators to the cytokinesis initiation site

“…S4C) or interaction with any of the known or unknown CIF1-interacting proteins, respectively. It is noteworthy that two previously identified CIF1-interating proteins, TbAUK1 and CIF2, are also recruited by CIF1 to the new FAZ tip at late anaphase and S phase, respectively (12,15). However, localization of the CIF1interacting protein CIF3 to the new FAZ tip is independent of CIF1, but the stability of CIF3 depends on the formation of the CIF1-CIF3 complex (14).…”

“…However, precisely how CIF1 stability is maintained by KPP1 remains unknown. Previous studies demonstrated that formation of the CIF1-CIF2 protein complex is necessary for maintaining CIF1 stability (13,15). It is thus possible that KPP1 may play a role in promoting CIF1-CIF2 complex assembly.…”

“…CIF1 contains a coiled-coil motif at the N terminus and two CCHC-type zinc-finger motifs at the C terminus ( Fig. 2A) (where CCHC is Cys-Cys-His-Cys), and the zinc-finger motifs are required for interaction with CIF2 and CIF3 (14,15). To test whether the zinc-finger motifs are required for interaction with the six new CIF1-interaction proteins, we carried out in vitro GST pulldown experiments.…”

“…CIF1 forms two separate protein complexes with two trypanosome-specific proteins, CIF2 (13) and CIF3 (14). The CIF1-CIF2 complex appears to function during S phase (13), and CIF1 is required for recruiting CIF2 to the new FAZ tip (15). The CIF1-CIF3 complex likely acts from G 2 phase to cytokinesis, and the two protein subunits exert distinct effects on each other, with CIF1 maintaining CIF3 stability and CIF3 maintaining CIF1 localization (14).…”

Functional analyses of an axonemal inner‐arm dynein complex in the bloodstream form of Trypanosoma brucei uncover its essential role in cytokinesis initiation

“…Regulation of the unusual cytokinesis in T. brucei is orchestrated by a novel signaling pathway composed of two evolutionarily conserved proteins kinases, the Polo‐like kinase homolog TbPLK and the Aurora B kinase homolog TbAUK1, and a cohort of trypanosome‐specific regulators, which act at the distal tip of the new FAZ and/or the cleavage furrow to drive cytokinesis initiation and furrow ingression (Li and Wang, 2006; Li et al ., 2009; McAllaster et al , ; Zhou et al , ; ; ; ; Hu et al , ; Hilton et al , ; Kurasawa et al , ). Among these trypanosome‐specific regulators are three cytokinesis initiation factors named CIF1, CIF2, and CIF3 (Zhou et al , ; ; Hu et al , ; Kurasawa et al , ). CIF1 and CIF2 are interdependent for maintaining their respective protein stability (Zhou et al , ), whereas CIF1 maintains CIF3 protein stability and CIF3 recruits CIF1 to the new FAZ tip (Kurasawa et al , ).…”