Pho23 is associated with the Rpd3 histone deacetylase and is required for its normal function in regulation of gene expression and silencing in Saccharomyces cerevisiae.
The Rpd3 histone deacetylase (HDAC) functions in a large complex containing many proteins including Sin3 and Sap30. Previous evidence indicates that the pho23, rpd3, sin3, and sap30 mutants exhibit similar defects in PHO5 regulation. We report that pho23 mutants like rpd3, sin3, and sap30 are hypersensitive to cycloheximide and heat shock and exhibit enhanced silencing of rDNA, telomeric, and HMR loci, suggesting that these genes are functionally related. Based on these observations, we explored whether Pho23 is a component of the Rpd3 HDAC complex. Our results demonstrate that Myc-Pho23 co-immunoprecipitates with HA-Rpd3 and HA-Sap30. Furthermore, similar levels of HDAC activity were detected in immunoprecipitates of HA-Pho23, HA-Rpd3, or HA-Sap30. In contrast, HDAC activity was not detected in immunoprecipitates of HA-Pho23 or HA-Sap30 from strains lacking Rpd3, suggesting that Rpd3 is the HDAC associated with these proteins. However, HDAC activity was detected in immunoprecipitates of HA-Sap30 or HA-Rpd3 from cells lacking Pho23, although levels were significantly lower than those detected in wild-type cells, indicating that Rpd3 activity is compromised in the absence of Pho23. Together, our genetic and biochemical studies provide strong evidence that Pho23 is a component of the Rpd3 HDAC complex, and is required for the normal function of this complex.
Loewith
R
R
Departments of Biochemistry & Molecular Biology and Oncology, University of Calgary Health Sciences Centre, Calgary, Alberta T2N4N1, Canada.
Smith
J S
JS
Meijer
M
M
Williams
T J
TJ
Bachman
N
N
Boeke
J D
JD
Young
D
D
D000135:Acid Phosphatase / Q000378:metabolism; D003513:Cycloheximide / Q000494:pharmacology; D004271:DNA, Fungal / Q000235:genetics; D004275:DNA, Ribosomal / Q000235:genetics; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism* / Q000502:physiology*; D015966:Gene Expression Regulation, Fungal; D020868:Gene Silencing; D006655:Histone Deacetylases / Q000235:genetics / Q000378:metabolism; D046911:Macromolecular Substances; D009154:Mutation; D009687:Nuclear Proteins; D010641:Phenotype; D012097:Repressor Proteins / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins; D016615:Telomere / Q000235:genetics; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism / Q000502:physiology*
D004271:DNA, Fungal; D004275:DNA, Ribosomal; D005656:Fungal Proteins; D046911:Macromolecular Substances; D009687:Nuclear Proteins; C432409:Pho23 protein, S cerevisiae; D012097:Repressor Proteins; C071731:SIN3 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D003513:Cycloheximide; D000135:Acid Phosphatase; C071665:RPD3 protein, S cerevisiae; D006655:Histone Deacetylases; C491869:SAP30 protein, S cerevisiae