Absolute Proteome and Phosphoproteome Dynamics during the Cell Cycle of Schizosaccharomyces pombe (Fission Yeast)

Abstract

To quantify cell cycle-dependent fluctuations on a proteome-wide scale, we performed integrative analysis of the proteome and phosphoproteome during the four major phases of the cell cycle in Schizosaccharomyces pombe. In highly synchronized cells, we identified 3753 proteins and 3682 phosphorylation events and relatively quantified 65% of the data across all phases. Quantitative changes during the cell cycle were infrequent and weak in the proteome but prominent in the phosphoproteome. Protein phosphorylation peaked in mitosis, where the median phosphorylation site occupancy was 44%, about 2-fold higher than in other phases. We measured copy numbers of 3178 proteins, which together with phosphorylation site stoichiometry enabled us to estimate the absolute amount of protein-bound phosphate, as well as its change across the cell cycle. Our results indicate that 23% of the average intracellular ATP is utilized by protein kinases to phosphorylate their substrates to drive regulatory processes during cell division. Accordingly, we observe that phosphate transporters and phosphate-metabolizing enzymes are phosphorylated and therefore likely to be regulated in mitosis. Molecular & Cellular Proteomics 13: 10.1074/mcp.M113. 035824,[1925][1926][1927][1928][1929][1930][1931][1932][1933][1934][1935][1936] 2014.Cell replication involves a complex series of highly regulated and evolutionary conserved events, called the "cell cycle." Aberrations in the cell cycle have severe implications and can cause cancerous growth. A detailed understanding of the cell cycle and its regulation may identify additional targets for cancer therapy (1-3). The cell cycle has been the subject of previous proteomics studies. Olsen et al. (4) Schizosaccharomyces pombe (fission yeast) is a unicellular organism, which can easily be genetically manipulated and carries many cell cycle features similar to metazoan cells. It is an important model organism to study the cell cycle and its checkpoint controls (6). Recent global proteomics studies of yeasts and their cell cycle (7-13) have mainly focused on Saccharomyces cerevisiae (budding yeast), with only a few studies of fission yeast (14, 15), although the fission yeast cell cycle may be more representative of eukaryotic cell cycles (16). However, attention of the proteomics community toward S. pombe is increasing. Recent proteomics studies covered up to 4087 S. pombe proteins (71% of the predicted proteome) and 1544 phosphoproteins in both asynchronous and synchronized cell cultures (17-22); however, a comprehensive analysis of the S. pombe cell cycle is so far missing.Here, we use high resolution mass spectrometry in combination with stable isotope labeling by amino acids in the cell culture (SILAC) 1 method, termed super-SILAC (23), and intensity-based absolute quantification (iBAQ) (24) to measure relative and absolute dynamics of the proteome and phosphoproteome during the cell cycle of fission yeast. We estimate copy numbers for 3178 S. pombe proteins, and we combine these data with calculated ...

Quantitative Phosphoproteomics Reveals Pathways for Coordination of Cell Growth and Division by the Conserved Fission Yeast Kinase Pom1*

“…We next examined overlap in both phosphoproteins and phosphopeptides from our study versus four previous fission yeast phosphoproteomic studies ( Figs. 2A) (33)(34)(35)(36). Our data identified 460 new phosphoproteins that were previously not seen in the other datasets, while 482 phosphoproteins were reported in all five studies.…”

“…To this end, we combined our data with four previous studies (33)(34)(35)(36) to generate a large database that includes all mapped phosphorylation sites and phosphoproteins in a unified format (Tables S3 and S4). In generating this database, we adjusted amino acid positions by mapping all peptides back to most recent Uniprot/PomBase sequences.…”

“…To our knowledge, this represents the largest current dataset for the fission yeast phosphoproteome. We also combined our data with four previously published studies (33)(34)(35)(36) to generate a comprehensive view of phosphorylation in fission yeast. This combined database reports all phosphorylated residues for any given protein in a unified format, with the potential to serve as a resource for researchers in a number of fields.…”

“…Generation of Combined Database and Gene Ontology Analysis-To assemble a complete phosphoproteomics map for fission yeast, data from previous large-scale phosphoproteomics studies (33)(34)(35)(36) were integrated with our data using custom-written PERL (version 5.8.8) scripts, which can be provided upon request. Briefly, the formats and assignments of phosphopeptides from different studies were unified using the Uniprot gene name pairing table (http:// www.uniprot.org/docs/pombe).…”

The Fission Yeast Pre-mRNA-processing Factor 18 (prp18+) Has Intron-specific Splicing Functions with Links to G1-S Cell Cycle Progression

“…We speculate that either a modification or altered conformer of wild-type SpPrp18 may cause its altered mobility. A recent study of the proteome and phosphoproteome of S. pombe during various cell cycle stages hints at phosphorylated forms of SpPrp18 (Ser-115 residue) (46).…”