A Suppressor Screen in Chlamydomonas Identifies Novel Components of the Retinoblastoma Tumor Suppressor Pathway

Abstract

The retinoblastoma (RB) protein is a eukaryotic tumor suppressor and negative cell-cycle regulator. Chlamydomonas reinhardtii cells that lack the RB homolog MAT3 show loss of size checkpoint control and deregulated cell-cycle progression leading to the production of tiny cells. We carried out an insertional mutagenesis screen to isolate bypass suppressors of mat3 (smt mutants) that reverted the mat3 cell-size defect. Previously we 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 smt 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 DP1. 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 MAT3/RB. T HE retinoblastoma (RB) protein is a tumor suppressor and negative cell-cycle regulator that is conserved in animals, plants, green algae, and other eukaryotic lineages, but has been lost from yeasts and other fungi. In the past two decades intensive efforts have been made to understand how RB regulates cellcycle progression, cell proliferation, differentiation, and development.The canonical RB pathway involves the cell-cycleregulated interaction of RB or its homologs (also called pocket proteins) with a heterodimeric transcription factor composed of E2F and DP subunits. The RB-associated E2F/DP protein complex represses transcription of cellcycle genes, and this repression is released by removal of RB via phosphorylation. Subsequently, E2F/DPdependent transcription of cell-cycle genes allows S-phase entry and cell-cycle progression (Weinberg 1995;Harbour and Dean 2000;Knudsen and Knudsen 2006).Genetic screens in the fruit fly Drosophila melanogaster and in the roundworm Caenorhabditis elegans have led to new insights into the RB pathway, including the identification of functions that are cell-cycle independent (Lu and

Translation-independent circadian control of the cell cycle in a unicellular photosynthetic eukaryote

“…8). As shown in green algae 4,[13][14][15] , the DRBR C. merolae cell enters into the S phase even before growing to a certain threshold size (Fig. 6).…”

“…This observation led us to investigate two types of cell cycle regulators, one that accumulates in association with the cell growth and another that is regulated by circadian rhythms. A key regulator of the G1/S transition in eukaryotic cells, including eukaryotic algae 4,[13][14][15] , is the retinoblastoma (RB) tumour suppressor pathway 16 . RB represses the G1/S transition by binding to the E2F-DP heterodimer, which in turn binds certain specific cis elements of S-phase genes.…”

Defects in a New Class of Sulfate/Anion Transporter Link Sulfur Acclimation Responses to Intracellular Glutathione Levels and Cell Cycle Control

“…Our previous studies showed that the RB pathway in C. reinhardtii is important for size checkpoint control and size-mediated cell division (Umen and Goodenough, 2001;Fang et al, 2006). The C. reinhardtii RBR homolog is encoded by a single gene, MAT3.…”

“…mat3 mutants pass commitment at a smaller size than normal but remain in G1 for a normal period of time before entering S/M, where they undergo supernumerary cell divisions to produce tiny daughter cells. Mutations in the C. reinhardtii E2F1 and DP1 genes could suppress the mat3 size defect, indicating that the overall architecture of the RBR pathway is conserved in C. reinhardtii, with MAT3/RBR serving as a negative regulator of E2F-and DP-related proteins (Fang et al, 2006;Olson et al, 2010).…”

“…Besides mutations in E2F1 and DP1, several additional extragenic suppressors of mat3 (smt mutants) were isolated that are weaker suppressors than e2f1 and dp1 (Fang and Umen, 2008). smt15-1 mat3-4 double mutants are larger than mat3-4 mutants but smaller than wildtype cells.…”