A suppressor screen in chlamydomonas identifies novel components of the retinoblastoma tumor suppressor pathway

The retinoblastoma (RB) protein is a eukatyotic tumor suppressor and negative cell-cycle regulator. Chlamydomonas reinhardtii cells that lack the RB homolog MATS show loss of sire checkpoint control and deregulated cell-cycle progression leading to the production of tiny cells. We carried out an insertional mutagenesis screen to isolate bypass suppressers of mat3 (s)id mutants) that reverted the mat3 cell-size defect. Previously we,e reported that the loci encoding Chlamydomonas homologs of E2F and DP were frequently disrupted in this screen, indicating that the architecture of the canonical RB pathway is conserved in Chlamydomonas with MAT3/RB acting as a negative regulator upstream of E2F/DP. Here, we describe four novel smt mutants that moderately suppressed the cell-size checkpoint and cell-cycle phenotypes of mat3. As single mutants, three of the smt strains displayed no obvious phenotypes, and one had a slightly small phenotype. Strikingly; several seal double-mutant combinations synergized to cause enhanced suppression of mat3 and even to cause a large-cell phenotype that is comparable to that caused by loss of DP 1. Molecular characterization of one smt mutant revealed that suppression is due to a defect in a gene encoding a putative small ubiquitin-like modifier (SUMO) peptidase. Our results reveal a complex genetic network that lies downstream of MAT3/RB and implicate protein sumoylation as an important step for cell-cycle progression in cells that are missing MAf3/RB.