In-depth profiling of post-translational modifications on the related transcription factor complexes TFIID and SAGA.
The basal transcription factor TFIID and the chromatin-modifying complex SAGA, which have several subunits in common, are crucial for transcription regulation. Here, we describe an in-depth profiling of post-translational modifications (PTMs) on both TFIID and SAGA from yeast. We took a multipronged approach using high-resolution mass spectrometry (LC-MS) in combination with the proteases Trypsin, Chymotrypsin and Glu-C. The cumulative peptide identification data, at a false discovery rate <1%, allowed us to cover most TFIID and SAGA subunit sequences to near completion. Additionally, for TFIID/SAGA subunits, we identified 118/102 unique phosphorylated and 54/61 unique lysine acetylated sites. Especially, several lysine residues on the SAGA subunits Spt7p and Sgf73p were found to be acetylated. Using a spectral counting approach, we found that the shared subunit TAF5p is phosphorylated to a significant greater extent in SAGA than in TFIID. Finally, we were able to map for the first time the cleavage site in Spt7p that is related to formation of the SAGA-like complex SLIK/SALSA. In general, our combination of tandem affinity enrichment, digestion with different proteases, extensive prefractionation and high-resolution LC-MS identifies a large number of PTMs of TFIID and SAGA/SLIK that might aid in future functional studies on these transcription factors.
Mischerikow
Nikolai
N
Biomolecular Mass Spectrometry and Proteomics Group, Bijvoet Centre for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands.
Spedale
Gianpiero
G
Altelaar
A F Maarten
AF
Timmers
H Th Marc
HT
Pijnappel
W W M Pim
WW
Heck
Albert J R
AJ
D000595:Amino Acid Sequence; D000818:Animals; D002853:Chromatography, Liquid / Q000379:methods; D015966:Gene Expression Regulation, Fungal; D051548:Histone Acetyltransferases / Q000235:genetics / Q000378:metabolism; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods; D008969:Molecular Sequence Data; D011499:Protein Processing, Post-Translational; D021122:Protein Subunits / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015534:Trans-Activators / Q000737:chemistry / Q000378:metabolism; D035362:Transcription Factor TFIID / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism
D021122:Protein Subunits; C507785:SAGA complex, S cerevisiae; C093107:SPT7 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D015534:Trans-Activators; D035362:Transcription Factor TFIID; D014157:Transcription Factors; D051548:Histone Acetyltransferases; C515833:Sgf73 protein, S cerevisiae