The Crk4-Cyc4 complex regulates G2/M transition in Toxoplasma gondii.

A versatile division of apicomplexan parasites and a dearth of conserved regulators have hindered the progress of apicomplexan cell cycle studies. While most apicomplexans divide in a multinuclear fashion, Toxoplasma gondii tachyzoites divide in the traditional binary mode. We previously identified five Toxoplasma CDK-related kinases (Crk). Here, we investigated TgCrk4 and its cyclin partner TgCyc4. We demonstrated that TgCrk4 regulates conventional G2 phase processes, such as repression of chromosome rereplication and centrosome reduplication, and acts upstream of the spindle assembly checkpoint. The spatial TgCyc4 dynamics supported the TgCrk4–TgCyc4 complex role in the coordination of chromosome and centrosome cycles. We also identified a dominant TgCrk4–TgCyc4 complex interactor, TgiRD1 protein, related to DNA replication licensing factor CDT1 but played no role in licensing DNA replication in the G1 phase. Our results showed that TgiRD1 also plays a role in controlling chromosome and centrosome reduplication. Global phosphoproteome analyses identified TgCrk4 substrates, including TgORC4, TgCdc20, TgGCP2, and TgPP2ACA. Importantly, the phylogenetic and structural studies suggest the Crk4–Cyc4 complex is limited to a minor group of the binary dividing apicomplexans. Synopsis: Apicomplexan parasite division can yield between two and thousands of progenies, and how this process is regulated remains a mystery. This study identifies a presumed missing G2 phase, and the complex TgCrk4-TgCyc4-TgiRD1 that regulates G2 phase, in Toxoplasma gondii. Expression of the CDK-related kinase TgCrk4, of the cyclin TgCyc4, and of the inhibitor of the DNA and centrosome reduplication TgiRD1 is essential for binary division of T. gondii tachyzoites. Removing TgCrk4 and TgiRD1 triggers the reduplication of chromosomes and centrosomes, revealing their role as guardians of the "once per cycle" duplication rule. Although distantly related to fungal and metazoan CDT1, TgiRD1 does not license DNA replication in G1 phase. Comparative genomic studies imply that expression of the Crk4-Cyc4 complex is restricted to apicomplexan parasites undergoing binary division. A previously unrecognized G2 phase in apicomplexan parasites undergoing binary division depends on a novel CDK-related kinase, an atypical cyclin, and a Cdt1-related inhibitor of re-duplication. [ABSTRACT FROM AUTHOR]