Raf60, a novel component of the Rpd3 histone deacetylase complex required for Rpd3 activity in Saccharomyces cerevisiae.
The Rpd3 histone deacetylase complex contains several previously characterized proteins, including Rpd3, Sin3, Sds3, Sap30, and Pho23. We purified the Rdp3 complex to near homogeneity using the tandem affinity purification method. Mass spectrometric analysis revealed the presence of a novel component, which we named Raf60. We showed that Myc-Raf60 co-fractionated with Rpd3-TAP by gel filtration chromatography, and both Myc-Rpd3 and Sin3 co-immunoprecipitated with HA-Raf60. In addition, HA-Raf60 immunoprecipitates displayed Rpd3-dependent histone deacetylase activity, and raf60 deletion resulted in loss of Rpd3 complex activity, as measured by in vitro assays. Furthermore, we found that raf60Delta cells exhibited phenotypes similar to those of rpd3Delta cells, including derepression of secreted acid phosphatase (Pho5), hypersensitivity to cycloheximide, and hypersensitivity to heat shock. Also, we found by reverse transcription-PCR that raf60Delta cells, similar to rpd3Delta cells, displayed elevated levels of PHO5 and INO1 mRNA. Our results demonstrate that Raf60 is a component of the Rpd3 histone deacetylase complex and that it is required for normal Rpd3 complex activity and repression of gene expression.
Colina
Ana R
AR
Department of Biochemistry & Molecular Biology, University of Calgary, Calgary, Alberta T2N 4N1, Canada.
Young
Dallan
D
D000135:Acid Phosphatase; D000595:Amino Acid Sequence; D015153:Blotting, Western; D002850:Chromatography, Gel; D003513:Cycloheximide / Q000494:pharmacology; D000939:Epitopes; D017353:Gene Deletion; D015966:Gene Expression Regulation, Fungal; D006655:Histone Deacetylases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D047468:Immunoprecipitation; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010641:Phenotype; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D012333:RNA, Messenger / Q000378:metabolism; D012097:Repressor Proteins / Q000737:chemistry / Q000502:physiology*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D014157:Transcription Factors / Q000737:chemistry / Q000502:physiology*
D000939:Epitopes; D011500:Protein Synthesis Inhibitors; D012333:RNA, Messenger; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D003513:Cycloheximide; D000135:Acid Phosphatase; C488916:PHO5 protein, S cerevisiae; C071665:RPD3 protein, S cerevisiae; C507989:Raf60 protein, S cerevisiae; D006655:Histone Deacetylases