Raf60, a novel component of the Rpd3 histone deacetylase complex required for Rpd3 activity in Saccharomyces cerevisiae
| Autor: | A.R. Colina, Dallan Young |
|---|---|
| Rok vydání: | 2005 |
| Předmět: |
Saccharomyces cerevisiae Proteins
Acid Phosphatase Blotting Western Molecular Sequence Data Saccharomyces cerevisiae Biology SAP30 Biochemistry Histone Deacetylases Mass Spectrometry Epitopes Gene Expression Regulation Fungal Histone H2A Immunoprecipitation Amino Acid Sequence RNA Messenger Cycloheximide Molecular Biology Derepression Protein Synthesis Inhibitors Histone deacetylase 5 HDAC11 Reverse Transcriptase Polymerase Chain Reaction HDAC10 Cell Biology Molecular biology Repressor Proteins Phenotype Histone deacetylase complex Chromatography Gel Histone deacetylase activity Gene Deletion Plasmids Protein Binding Transcription Factors |
| Zdroj: | The Journal of biological chemistry. 280(52) |
| ISSN: | 0021-9258 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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