title: Novel interaction between Apc5p and Rsp5p in an intracellular signaling pathway in Saccharomyces cerevisiae.
abstract: The ubiquitin-targeting pathway is evolutionarily conserved and critical for many cellular functions. Recently, we discovered a role for two ubiquitin-protein ligases (E3s), Rsp5p and the Apc5p subunit of the anaphase-promoting complex (APC), in mitotic chromatin assembly in Saccharomyces cerevisiae. In the present study, we investigated whether Rsp5p and Apc5p interact in an intracellular pathway regulating chromatin remodeling. Our genetic studies strongly suggest that Rsp5p and Apc5p do interact and that Rsp5p acts upstream of Apc5p. Since E3 enzymes typically require the action of a ubiquitin-conjugating enzyme (E2), we screened E2 mutants for chromatin assembly defects, which resulted in the identification of Cdc34p and Ubc7p. Cdc34p is the E2 component of the SCF (Skp1p/Cdc53p/F-box protein). Therefore, we analyzed additional SCF mutants for chromatin assembly defects. Defective chromatin assembly extracts generated from strains harboring a mutation in the Cdc53p SCF subunit or a nondegradable SCF target, Sic1(Deltaphos), confirmed that the SCF was involved in mitotic chromatin assembly. Furthermore, we demonstrated that Ubc7p physically and genetically interacts with Rsp5p, suggesting that Ubc7p acts as an E2 for Rsp5p. However, rsp5CA and Deltaubc7 mutations had opposite genetic effects on apc5CA and cdc34-2 phenotypes. Therefore, the antagonistic interplay between Deltaubc7 and rsp5CA, with respect to cdc34-2 and apc5CA, indicates that the outcome of Rsp5p's interaction with Cdc34p and Apc5p may depend on the E2 interacting with Rsp5p.
authors.affiliation: Department of Medicine, Royal University Hospital, University of Saskatchewan, Saskatoon, Saskatchewan, Canada.
authors.forename: Terra G
authors.initials: TG
authors.lastname: Arnason
authors.forename: Marnie G
authors.initials: MG
authors.lastname: Pisclevich
authors.forename: Megan D
authors.initials: MD
authors.lastname: Dash
authors.forename: Gerald F
authors.initials: GF
authors.lastname: Davies
authors.forename: Troy A A
authors.initials: TA
authors.lastname: Harkness
mesh_terms: D019883:Alkyl and Aryl Transferases; D000483:Alleles; D064173:Anaphase-Promoting Complex-Cyclosome; D015153:Blotting, Western; D018797:Cell Cycle Proteins / Q000737:chemistry; D002843:Chromatin / Q000737:chemistry; D044842:Cullin Proteins / Q000737:chemistry; D004247:DNA / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D056827:Endosomal Sorting Complexes Required for Transport; D005821:Genetic Techniques; D005838:Genotype; D047468:Immunoprecipitation; D008938:Mitosis; D009154:Mutation; D009685:Nuclear Envelope / Q000378:metabolism; D010641:Phenotype; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000378:metabolism*; D019089:Stem Cell Factor / Q000378:metabolism; D025801:Ubiquitin / Q000737:chemistry; D044763:Ubiquitin-Conjugating Enzymes / Q000737:chemistry; D043743:Ubiquitin-Protein Ligase Complexes / Q000737:chemistry / Q000378:metabolism*; D044767:Ubiquitin-Protein Ligases / Q000737:chemistry
chemical_list: C100988:Cdc53 protein, S cerevisiae; D018797:Cell Cycle Proteins; D002843:Chromatin; D044842:Cullin Proteins; D056827:Endosomal Sorting Complexes Required for Transport; D029701:Saccharomyces cerevisiae Proteins; D019089:Stem Cell Factor; D025801:Ubiquitin; D004247:DNA; C577449:CDC34 protein, S cerevisiae; C577447:CDC34 protein, human; C471617:UBE2G2 protein, human; D044763:Ubiquitin-Conjugating Enzymes; D043743:Ubiquitin-Protein Ligase Complexes; D064173:Anaphase-Promoting Complex-Cyclosome; D044767:Ubiquitin-Protein Ligases; D019883:Alkyl and Aryl Transferases; C065203:CDC43 protein, S cerevisiae; C469287:RSP5 protein, S cerevisiae