Quantitative proteomics reveals regulation of dynamic components within TATA-binding protein (TBP) transcription complexes.
Affinity purification in combination with isotope labeling of proteins has proven to be a powerful method to discriminate specific from nonspecific interactors. However, in the standard SILAC (stable isotope labeling by amino acids in cell culture) approach dynamic components may easily be assigned as nonspecific. We compared two affinity purification protocols, which in combination revealed information on the dynamics of protein complexes. We focused on the central component in eukaryotic transcription, the human TATA-binding protein, which is involved in different complexes. All known TATA-binding protein-associated factors (TAFs) were detected as specific interactors. Interestingly one of them, BTAF1, exchanged significantly in cell extracts during the affinity purification. The other TAFs did not display this behavior. Cell cycle synchronization showed that BTAF1 exchange was regulated during mitosis. The combination of the two affinity purification protocols allows a quantitative approach to identify transient components in any protein complex.
Mousson
Florence
F
Department of Physiological Chemistry, University Medical Centre Utrecht, Universiteitsweg 100, 3584CG Utrecht, The Netherlands.
Kolkman
Annemieke
A
Pijnappel
W W M Pim
WW
Timmers
H Th Marc
HT
Heck
Albert J R
AJ
D002453:Cell Cycle / Q000235:genetics; D002846:Chromatography, Affinity / Q000379:methods*; D006367:HeLa Cells; D006801:Humans; D007553:Isotope Labeling; D013058:Mass Spectrometry; D008938:Mitosis / Q000235:genetics; D025941:Protein Interaction Mapping / Q000379:methods*; D021122:Protein Subunits / Q000378:metabolism; D040901:Proteomics / Q000379:methods*; D035381:TATA-Binding Protein Associated Factors / Q000032:analysis / Q000378:metabolism*; D035181:TATA-Box Binding Protein / Q000378:metabolism*; D014158:Transcription, Genetic
D021122:Protein Subunits; D035381:TATA-Binding Protein Associated Factors; D035181:TATA-Box Binding Protein