Mutant/mutation
The mutant expresses a C-terminal GFP-tagged version of CRK5 

Protein (function)
From the Abstract: 'Cell cycle transitions are generally triggered by variation in the activity of cyclindependent kinases (CDKs) bound to cyclins. Malaria-causing parasites have a life cycle with unique cell-division cycles, and a repertoire of divergent CDKs and cyclins of poorly understood function and interdependency. We show that Plasmodium berghei CDK-related kinase 5 (CRK5), is a critical regulator of atypical mitosis in the gametogony and is required for mosquito transmission. It phosphorylates canonical CDK motifs of components in the pre-replicative complex and is essential for DNA replication. During a replicative cycle, CRK5 stably interacts with a single Plasmodium specific cyclin (SOC2), although we obtained no evidence of SOC2 cycling by transcription, translation or degradation. Our results provide evidence that during Plasmodium male gametogony, this divergent cyclin/CDK pair fills the functional space of other eukaryotic cell-cycle kinases controlling DNA replication.'

Phenotype
Expression not tested in detail. The tagged protein was localised to the nucleus in non-activated gametocytes and at 1 min after activation of male gametocytes showed an additional location at the mitotic spindle as suggested by co-localisation with tubulinExpression observed in oocysts. The mutant is used for mass-spec analyses of co-precipitated proteins in activated male gametocytes.

Additional information
- Previous attempts to disrupt P. berghei crk5 had suggested the gene is essential for asexual blood-stage proliferation (Tewari et al., 2010). However, using long sequence homology regions to replace crk5 with a T. gondii DHFR/TS resistance marker resistant KO-parasites were obtained.
- We tagged the endogenous crk5 gene with an AID/HA epitope tag to degrade the fusion protein in presence of auxin in a strain expressing the Tir1 protein (Philip and Waters, 2015). Addition of the AID/HA tag to the CRK5 C-terminus imposed a significant fitness cost, with a 2-fold decrease in exflagellation in the absence of auxin, but importantly, depletion of CRK5-AID/HA by one hour of auxin treatment of mature gametocytes prior to activation resulted in a dramatic reduction in exflagellation.

Evidence in this study is presented that:
- CRK5 is a key regulator of gametogony and sporogony in the mosquito
- Phosphoproteome kinetics point to direct phosphorylation of the pre-replicative complex by CRK5
- CRK5 is required for both S- and M- phases during P. berghei gametogony
- We observed an average 2-fold fewer polyploid (>2N) gametocytes 1 min post-activation , demonstrating a dependency on CRK5 for DNA replication during male gametogony.
- Cells depleted of CRK5 are unable to correctly assemble or maintain the spindle, with a greater than two-fold decrease of visible spindles at 1 min pa in absence of CRK5
- CRK5 is part of an atypical nuclear cyclin/CDK complex
- CRK5, SOC2 ((PBANKA_1442200)and CDKrs (PBANKA_0824400; (CDK regulatory subunit, CDKrs) have a similar location and complementary functions during gametogony. CDKrs is a protein related to Cks1 and CksHs2, two CDK-associated proteins in Saccharomyces cerevisiae and and human, respectively.
- CRK5, SOC2 and CDKrs have a similar location and complementary functions during gametogony
- SOC2 expression does not follow a temporal cyclin pattern during gametogony and the CSC complex (CRK5/SOC2/CDKrs complex) is stable during the first round of mitosis
- CSC is dynamically phosphorylated during the first round of replication


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