Extensive Mass Spectrometry-based Analysis of the Fission Yeast Proteome

Gunaratne, Jayantha; Schmidt, Alexander; Quandt, Andreas; Neo, Suat Peng; Saraç, Ömer; Gracia, Tannia; Loguercio, Salvatore; Ahrné, Erik; Xia, Rachel Li Hai; Tan, Keng Hwa; Lößner, Christopher; Bähler, Jürg; Beyer, Andreas; Blackstock, Walter; Aebersold, Ruedi

doi:10.1074/mcp.m112.023754

Cited by 30 publications

(30 citation statements)

References 57 publications

(81 reference statements)

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“…The 3753 proteins identified in our study represent 74% of the predicted fission yeast proteins, making it one of the most comprehensive published protein datasets of this organism to date. To enable triple-SILAC labeling and avoid quantification problems arising from arginine to proline conversion (38), we exclusively used SILAC labeling with lysine and digestion with endoproteinase Lys-C. Our data complement the recently published PeptideAtlas of the fission yeast proteome obtained using trypsin (19), and it increases the coverage of the S. pombe proteome from 3542 to a total of 4068 proteins.…”

Section: Discussionmentioning

confidence: 70%

“…We compared our absolute quantification to several studies that used different technologies to derive absolute protein amounts in S. pombe as follows: MS intensity-based absolute quantification (18,19,22) and fluorescence microscopybased quantitation (27,43). It has been reported that …”

Section: Functional Classes Of Fluctuating Proteins and Phosphorylatimentioning

confidence: 99%

“…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)(18)(19)(20)(21)(22); however, a comprehensive analysis of the S. pombe cell cycle is so far missing.…”

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confidence: 99%

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

Carpy

Krug

Graf

et al. 2014

Molecular & Cellular Proteomics

144

129

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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 ...

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Section: Discussionmentioning

confidence: 70%

Section: Functional Classes Of Fluctuating Proteins and Phosphorylatimentioning

confidence: 99%

See 1 more Smart Citation

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

Carpy

Krug

Graf

et al. 2014

Molecular & Cellular Proteomics

144

129

View full text Add to dashboard Cite

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“…Although the genome of the cyanobacterium S. elongatus is not as extensive as that in mammalian systems, this percentage is still impressive, especially when compared with the 60% to 70% reported for other, smaller genomes such as yeast (52).…”

Section: Discussionmentioning

confidence: 89%

Daily Rhythms in the Cyanobacterium Synechococcus elongatus Probed by High-resolution Mass Spectrometry–based Proteomics Reveals a Small Defined Set of Cyclic Proteins

Guerreiro

Benevento

Lehmann

et al. 2014

Molecular & Cellular Proteomics

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Circadian rhythms are self-sustained and adjustable cycles, typically entrained with light/dark and/or temperature cycles. These rhythms are present in animals, plants, fungi, and several bacteria. The central mechanism behind these "pacemakers" and the connection to the circadian regulated pathways are still poorly understood. The circadian rhythm of the cyanobacterium Synechococcus elongatus PCC 7942 (S. elongatus) is highly robust and controlled by only three proteins, KaiA, KaiB, and KaiC. This central clock system has been extensively studied functionally and structurally and can be reconstituted in vitro. These characteristics, together with a relatively small genome (2.7 Mbp), make S. elongatus an ideal model system for the study of circadian rhythms.Different approaches have been used to reveal the influence of the central S. elongatus clock on rhythmic gene expression, rhythmic mRNA abundance, rhythmic DNA topology changes, and cell division. However, a global analysis of its proteome dynamics has not been reported yet.To uncover the variation in protein abundances during 48 h under light and dark cycles (12:12 h), we used quantitative proteomics, with TMT 6-plex isobaric labeling. We queried the S. elongatus proteome at 10 different time points spanning a single 24-h period, leading to 20 time points over the full 48-h period.Employing multidimensional separation and high-resolution mass spectrometry, we were able to find evidence for a total of 82% of the S. elongatus proteome. Of the 1537 proteins quantified over the time course of the experiment, only 77 underwent significant cyclic variations. Interestingly, our data provide evidence for in-and outof-phase correlation between mRNA and protein levels for a set of specific genes and proteins. As a range of cyclic proteins are functionally not well annotated, this work provides a resource for further studies to explore the role of these proteins in the cyanobacterial circadian rhythm. Molecular & Cellular

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“…The presence of 10 ATG triplets in the interval between the prt2 start site and the annotated ATG of the putative SPBC8E4.02c ORF is consistent with prt2 being a lncRNA rather than an mRNA. In support of this conclusion, deep analyses by mass spectrometry of the protein contents of the fission yeast cell have failed to detect any peptides derived from the putative SPBC8E4.02c ORF (12,13), while readily detecting the Pho84 polypeptide at a level of ~12,000 molecules per cell (13).…”

Section: Resultsmentioning

confidence: 98%

A long noncoding (lnc)RNA governs expression of the phosphate transporter Pho84 in fission yeast and has cascading effects on the flanking prt lncRNA and pho1 genes

Garg

Sánchez

Shuman

et al. 2018

Journal of Biological Chemistry

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The expression of the phosphate transporter Pho84 in fission yeast Schizosaccharomyces pombe is repressed in phosphate-rich medium and induced during phosphate starvation. Two other phosphate-responsive genes in S. pombe (pho1 and tgp1) had been shown to be repressed in cis by transcription of a long noncoding (lnc) RNA from the upstream flanking gene, but whether pho84 expression is regulated in this manner is unclear. Here we show that repression of pho84 is enforced by transcription of the SPBC8E4.02c locus upstream of pho84 to produce a lncRNA that we name prt2 (pho-repressive transcript 2) We identify two essential elements of the prt2 promoter: HomolD box and TATA box, mutations of which inactivate the prt2 promoter and derepress the downstream pho84 promoter under phosphate-replete conditions. We find that prt2 promoter inactivation also elicits a cascade effect on the adjacent downstream prt (lncRNA) and pho1 (acid phosphatase) genes, whereby increased pho84 transcription down-regulates prt lncRNA transcription and thereby de-represses pho1. Our results establish a unified model for the repressive arm of fission yeast phosphate homeostasis, in which transcription of prt2, prt, and nc-tgp1 lncRNAs interferes with the promoters of the flanking pho84, pho1, and tgp1 genes, respectively. ___________________________________________

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Extensive Mass Spectrometry-based Analysis of the Fission Yeast Proteome

Cited by 30 publications

References 57 publications

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

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

Daily Rhythms in the Cyanobacterium Synechococcus elongatus Probed by High-resolution Mass Spectrometry–based Proteomics Reveals a Small Defined Set of Cyclic Proteins

A long noncoding (lnc)RNA governs expression of the phosphate transporter Pho84 in fission yeast and has cascading effects on the flanking prt lncRNA and pho1 genes

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