Quantitative mass spectrometry of TATA binding protein-containing complexes and subunit phosphorylations during the cell cycle

<p>Abstract</p> <p>Background</p> <p>Progression through the cell cycle is accompanied by tightly controlled regulation of transcription. On one hand, a subset of genes is expressed in a cell cycle-dependent manner. On the other hand, a general inhibition of transcription occurs during mitosis.</p> <p>Genetic and genome-wide studies suggest cell cycle regulation at the level of transcription initiation by protein complexes containing the common DNA-binding subunit TATA binding protein (TBP). TBP is a key player in regulating transcription by all three nuclear RNA polymerases. It forms at least four distinct protein complexes with TBP-associated factors (TAFs): SL1, B-TFIID, TFIID, and TFIIIB. Some TAFs are known to remain associated with TBP during the cell cycle. Here we analyze all TAFs and their phosphorylation status during the cell cycle using a quantitative mass spectrometry approach.</p> <p>Results</p> <p>TBP protein complexes present in human cells at the G2/M and G1/S transitions were analyzed by combining affinity purification with quantitative mass spectrometry using stable isotope labeling with amino acids in cell culture (SILAC). Phosphorylations were mapped and quantified after enrichment of tryptic peptides by titanium dioxide. This revealed that subunit stoichiometries of TBP complexes remained intact, but their relative abundances in nuclear extracts changed during the cell cycle. Several novel phosphorylations were detected on subunits of the TBP complexes TFIID and SL1. G2/M-specific phosphorylations were detected on TAF1, TAF4, TAF7, and TAFI41/TAF1D, and G1/S-specific dephosphorylations were detected on TAF3. Many phosphorylated residues were evolutionary conserved from human to zebrafish and/or drosophila, and were present in conserved regions suggesting important regulatory functions.</p> <p>Conclusions</p> <p>This study provides the first quantitative proteomic analysis of human TBP containing protein complexes at the G2/M and G1/S transitions, and identifies new cell cycle-dependent phosphorylations on TAFs present in their protein complex. We speculate that phosphorylation of complex-specific subunits may be involved in regulating the activities of TBP protein complexes during the cell cycle.</p

Proteome analysis of yeast response to various nutrient limitations

We compared the response of Saccharomyces cerevisiae to carbon (glucose) and nitrogen (ammonia) limitation in chemostat cultivation at the proteome level. Protein levels were differentially quantified using unlabeled and (15)N metabolically labeled yeast cultures. A total of 928 proteins covering a wide range of isoelectric points, molecular weights and subcellular localizations were identified. Stringent statistical analysis identified 51 proteins upregulated in response to glucose limitation and 51 upregulated in response to ammonia limitation. Under glucose limitation, typical glucose-repressed genes encoding proteins involved in alternative carbon source utilization, fatty acids β-oxidation and oxidative phosphorylation displayed an increased protein level. Proteins upregulated in response to nitrogen limitation were mostly involved in scavenging of alternative nitrogen sources and protein degradation. Comparison of transcript and protein levels clearly showed that upregulation in response to glucose limitation was mainly transcriptionally controlled, whereas upregulation in response to nitrogen limitation was essentially controlled at the post-transcriptional level by increased translational efficiency and/or decreased protein degradation. These observations underline the need for multilevel analysis in yeast systems biology

Fate and removal of trace pollutants from an anion exchange spent brine during the recovery process of natural organic matter and salts

The results of this sampling campaign on pilot scale processes aim to evaluate the occurrence and behavior of trace organic micro-pollutants and metal elements during anion exchange treatment of surface water and the subsequent treatment of generated spent brine with two types of electrodialysis membrane pairs. This knowledge is relevant to assess the quality and reusability of secondary products created during brine treatment; specifically the excess of sodium chloride to be recycled onsite and the natural organic matter, mostly consisting of humic substances, which find multiple applications in the agricultural industry. This study highlights that (1) the attachment mechanism of organic micro-pollutants to anion exchange resin occurs through electrostatic interaction and the subsequent transfer through ion exchange membranes is restricted by size exclusion; and (2) the complexation of trace metals compounds with the natural organic matter partly explains their removal by anion exchange. Complexes remain stable during treatment of the brine with electrodialysis.BT/Environmental BiotechnologyOLD BT/Cell Systems Engineerin