A suppressor screen in chlamydomonas identifies novel components of the retinoblastoma tumor suppressor pathway.
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.
Fang
Su-Chiung
SC
Plant Biology Laboratory, The Salk Institute, La Jolla, California 92037, USA.
Umen
James G
JG
D020418:Algal Proteins / Q000737:chemistry / Q000235:genetics; D000595:Amino Acid Sequence; D000818:Animals; D015139:Blotting, Southern; D002453:Cell Cycle / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D048429:Cell Size / Q000187:drug effects; D002696:Chlamydomonas / Q000166:cytology / Q000187:drug effects / Q000235:genetics*; D020090:Chromosome Segregation; D003433:Crosses, Genetic; D003624:Darkness; D004351:Drug Resistance / Q000187:drug effects; D005799:Genes, Dominant; D016153:Genes, Suppressor; D008040:Genetic Linkage / Q000187:drug effects; D005838:Genotype; D008957:Models, Genetic; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D010303:Paromomycin / Q000494:pharmacology; D015800:Protozoan Proteins / Q000235:genetics*; D016160:Retinoblastoma Protein / Q000235:genetics*
D020418:Algal Proteins; D015800:Protozoan Proteins; D016160:Retinoblastoma Protein; C432865:mat3 protein, Chlamydomonas; D010303:Paromomycin