A novel cyclin dependent kinase in Chlamydomonas that mediates cell size checkpoint control

Yubing Li1, Bradley Olson1, Garrett Anderson2 and James G. Umen1

1) Plant Biology Laboratory, The Salk Institute, La Jolla, CA 92037, USA 2) Stanford Law School, Stanford, CA 94305-8610, USA

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 control in Chlamydomonas is MAT3, a retinoblastoma (RB) tumor suppressor homolog. Using forward genetic screens we isolated two mutant alleles of a gene designated CDKG1 whose loss-of-function phenotype is large cells and which functions upstream of MAT3/RB in a linear genetic pathway. While previously identified mutants such as mat3 and dp1 affect both the Commitment and mitotic (S/M) cell-size checkpoints, cdkg1 was found to be specifically impaired for the S/M checkpoint and is the first mutant that separates the two size checkpoints. CDKG1 encodes a novel cyclin dependent kinase with a unique 90 amino acid N-terminal extension preceding its kinase domain. Nonetheless, Chlamydomonas CDKG1 functionally complements a yeast cdk1 temperature sensitive mutation indicating that it retains the core functions of a cell cycle CDK. CDKG1 interacts specifically with D-type cyclins in yeast two hybrid assays and can bind to D cyclins in vitro and phosphorylate its key substrate, MAT3/RB. CDKG1 mRNA was found to be cell cycle regulated with a peak during S/M, and CDKG1 was found to co-immunoprecipitate with MAT3/RB during division. CDKG1 cDNA expressed under the control of a constitutive promoter was found to cause a small-cell phenotype, opposite to that of a cdkg1 deletion, indicating that CDKG1 activity is rate-limiting for size checkpoint control. Together these data demonstrate a specific role for a CDK in controlling cell size that can be uncoupled from a general function in driving cell cycle progression, and they uncover a remarkable convergence between CDKG1 and CDK4/6 that are metazoan D-cyclin dependent RB kinases. The mechanism by which CDKG1 expression and kinase activity are coordinated during the cell cycle to regulate MAT3/RB and E2F1-DP1 is the subject of ongoing investigation.

e-mail address of presenting author: yuli@salk.edu