CYC1

TgCyc1 is a parasite cyclin H-class protein that functions as the activating partner of a Cdk-related kinase to control mitotic progression during Toxoplasma gondii endodyogeny (PMID:35130726). It was first identified as an interactor of the cdc2-related kinase TgCRK2 and can functionally substitute for a canonical G1/S cyclin, rescuing a cyclin-deficient S. cerevisiae strain (PMID:11897124). Its expression is constitutive rather than periodic, as TgCYC1 mRNA levels do not vary across the tachyzoite cell cycle (PMID:11897124). In its characterized cellular role, TgCyc1 forms a complex with the atypical kinase TgCrk6, and loss of either component produces identical mitotic defects, establishing that the two act cooperatively (PMID:35130726). The TgCrk6/TgCyc1 complex regulates metaphase and the spindle assembly checkpoint, specifically governing the centromere-associated network during mitosis coupled to cytokinesis (PMID:35130726).

1. 2002 Medium

   Establishing which kinase TgCyc1 partners with was the first step in assigning it a cell-cycle role; a yeast two-hybrid screen identified TgCRK2 as its interactor, placing TgCyc1 within the parasite Cdk-cyclin machinery.

   Evidence GAL4-based yeast two-hybrid screen of 3.2 million transformants

   PMID:11897124

   Open questions at the time

   • Single Y2H interaction without reciprocal validation
   • Functional consequence of the TgCRK2 interaction not tested
2. 2002 Medium

   To ask whether TgCyc1 acts as a periodic cell-cycle timer, its transcript was measured across synchronized parasites; mRNA levels were invariant, indicating regulation is not achieved at the transcript level.

   Evidence mRNA quantification in synchronized tachyzoite populations

   PMID:11897124

   Open questions at the time

   • Does not address protein-level or activity-level cell-cycle regulation
   • Single method, single lab
3. 2002 Medium

   To test whether TgCyc1 retains genuine cyclin function, it was expressed in a cyclin-deficient yeast; it rescued the G1/S defect, demonstrating conserved cyclin activity in a heterologous system.

   Evidence Heterologous complementation in S. cerevisiae cyclin-deficient strain DL1

   PMID:11897124

   Open questions at the time

   • Heterologous rescue does not establish the native G1/S role in Toxoplasma
   • Does not identify the native cognate kinase
4. 2022 High

   Defining TgCyc1's native functional partner and phenotype resolved its endogenous role: it forms a complex with TgCrk6, and knockdown of either produces identical mitotic defects, establishing cooperative kinase-cyclin action in endodyogeny.

   Evidence Co-immunoprecipitation/proteomics with conditional knockdowns of both components and quantitative fluorescence microscopy

   PMID:35130726

   Open questions at the time

   • Direct kinase-activation mechanism by TgCyc1 not biochemically dissected
   • Relationship to the earlier TgCRK2 interaction unresolved
5. 2022 High

   Placing the complex in a pathway showed that TgCrk6/TgCyc1 governs metaphase and the spindle assembly checkpoint, specifically the centromere-associated network during mitosis coupled to cytokinesis.

   Evidence Conditional knockdown, quantitative microscopy of mitotic structures, global proteomics and phosphoproteomics

   PMID:35130726

   Open questions at the time

   • Direct substrates phosphorylated by the complex not individually validated
   • How the complex is activated at metaphase onset is unknown

• How TgCyc1's two reported kinase associations (TgCRK2 and TgCrk6) are reconciled, and the mechanism by which the cyclin activates its kinase to enforce the spindle checkpoint, remain unresolved.
• No structural model of the cyclin-kinase complex
• Direct catalytic substrates unmapped
• Relationship between TgCRK2 and TgCrk6 associations unexplained