| Chlamydomonas depends on size checkpoints to control its multiple fission cell cycle, but it is still unknown how cell size is sensed. A key component of size checkpoint control in Chlamydomonas is MAT3, a retinoblastoma (RB) tumor suppressor homolog. Using a forward genetic screen based on insertional mutagenesis we isolated three novel cell size control mutants, lrg1-1, lrg1-2 and tny1-1. lrg1-1 and lrg1-2, whose phenotype is large cells, were found to be alleles of CDKG1. tny1-1, whose phenotype is small cells, was found to be an allele of a putative RNA binding protein with tandem RRMs that is related to the Musashi family. Both proteins act upstream of MAT3/RB in a linear pathway. CDKG1 was able to complement a budding yeast cdc28 temperature sensitive allele indicating that CDKG1 is a cell cycle CDK. Two D-type cyclins from Chlamydomonas, CYCD2 and CYCD3, were able to interact with CDKG1 in a yeast two-hybrid assay. Both D-type cyclins contain the RB protein binding motif LxCxE meaning that MAT3/RB is a likely substrate of CDKG1. The yeast two-hybrid assay also revealed an interaction between CYCD3 and DP1, a protein that forms a stable complex with E2F1 and MAT3. We propose that CDKG1 is the Chlamydomonas analog of CDK4/6, an animal-specific CDK that binds D cyclins and phosphorylates RB. In Chlamydomonas the D cyclins would activate CDKG1 in response to cell size cues. In vitro reconstitution and immunopurification of CDKG1 complexes are being used in order to test whether MAT3, DP1 or E2F1 are substrates of CYCD3-CDKG1.
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