Article

Analysis of the budding yeast pH 4–7 proteome in meiosis

School of Biomolecular and Biomedical Science, University College Dublin, Belfield, Dublin, Ireland.
Proteomics (Impact Factor: 3.81). 02/2010; 10(3):506-19. DOI: 10.1002/pmic.200900561
Source: PubMed

ABSTRACT

Meiosis, the developmental programme generating haploid gametes from diploid precursors, requires two cell divisions and many innovations. In budding yeast, a large number of genes are expressed exclusively during meiosis while others are repressed compared to vegetative growth. Microarray analysis has shown that gene expression during meiosis is highly regulated, and has been used to classify yeast genes according to meiotic temporal expression pattern. In this study, we have begun to investigate the kinetics of meiotic protein expression using a proteomics approach. 2-D DIGE was used to characterise the temporal protein expression patterns of the budding yeast pH 4-7 proteome in meiosis. More than 1400 meiotic protein spots were visualised and at least 63 spots were temporally regulated during meiosis in a statistically significant manner. Gel spots with significant expression changes were excised and 26 unique proteins were identified using LC-MS/MS. The identified proteins could be classified into functional categories and the genes encoding a number of these were previously shown to be involved in yeast sporulation and meiosis. This data set was used to assemble the first differential 2-D PAGE map of budding yeast meiosis, which can be accessed through a web server. This work represents one of the first quantitative proteomic analyses of meiosis in yeast and will provide a valuable resource for future investigations.

Download full-text

Full-text

Available from: Julia Grassl, Mar 05, 2015
  • Source
    • "For protein identification by MS, preparative gels, each loaded with 450 g of protein, were silver-stained with an MS compatible stain (PlusOne Silver stain kit, GE Healthcare), omitting the use of glutaraldehyde[62]. Protein spots were excised from the gels and digested using a protocol similar to that of Grassl et al., 2010 but without the use of Ziptips[63]). Briefly, the gel spots were destained, reduced and alkylated, then dehydrated with ACN. The proteins were digested overnight with trypsin (Promega, modified sequencing grade) at 37C. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Psoriatic arthritis (PsA) can be treated using biologic therapies targeting biomolecules such as tumour necrosis factor alpha, interleukins (IL)-17 and IL-23. Whilst 70% PsA patients respond well to therapy, 30% patients show no or limited clinical improvement. Biomarkers that predict response to therapy would help to avoid unnecessary use of expensive biologics in non-responding patients and enable alternative treatments to be explored. Patient synovial-tissue samples from two clinical studies were analysed using Difference In-Gel Electrophoresis based proteomics to identify protein expression differences in response to anti-TNF-α treatment. Subsequent multiplexed multiple-reaction-monitoring (MRM) measurements were used to verify potential biomarkers. A total of 119 proteins were differentially expressed (p<0.05) in response to anti-TNF-α treatment and 25 proteins were differentially expressed (p<0.05) between "good responders" and "poor responders". From these differentially expressed proteins, MRM assays were developed for 4 proteins to explore their potential as treatment predictive biomarkers. Gel-based proteomics strategy has demonstrated differential protein expression in synovial tissue of PsA patients, in response to anti-TNF-α treatment. Development of multiplex MRM assays to these differentially expressed proteins has the potential to predict response to therapy and allow alternative, more effective treatments to be explored sooner. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.
    Full-text · Article · Jun 2015 · PROTEOMICS - CLINICAL APPLICATIONS
  • Source
    • "Apart from genome wide studies, few proteomic studies have also been carried out to identify proteins specifically during sporulation as well as to compare the expression of proteins at different stages of the meiotic cell cycle [10] [11] [12]. Since the uniqueness of chromosome segregation in meiosis over mitosis lies on the execution of meiosis I, we aimed to compare the proteome of the cells residing within meiosis I to that of the cells proceeding through the equivalent stage in mitosis which was not investigated in previous proteomic studies. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Unlabelled: Precise and timely segregation of genetic material and conservation of ploidy are the two foremost requirements for survival of a eukaryotic organism. Two highly regulated cell division processes, namely mitosis and meiosis are central to achieve this objective. The modes of chromosome segregation are distinct in these two processes that generate progeny cells of equal ploidy and half the ploidy in mitosis and meiosis, respectively. Additionally, the nutritional requirement and intracellular processing of biological cue also differ in these two processes. From this, it can be envisaged that proteome of mitotic and meiotic cells will differ significantly. Therefore, identification of proteins that differ in their level of expression between mitosis and meiosis would further reveal the mechanistic detail of these processes. In the present study, we have investigated the protein expression profile of mitosis and meiosis by comparing proteome of budding yeast cultures arrested at mitotic metaphase and metaphase-I of meiosis using proteomic approach. Approximately 1000 and 2000 protein spots were visualized on 2-DE and 2D-DIGE gels respectively, out of which 14 protein spots were significant in 2-DE and 22 in 2D-DIGE (p<0.05). All the significant spots were reproducible in all biological replicates and followed the same trend. Identification of the proteins from these spots revealed that nine proteins were common in both 2-DE and 2D-DIGE. These proteins are found to be involved in various cellular processes and pathways such as cytoskeleton function and cytokinesis, carbon, nitrogen, lipid metabolism, general translation and protein folding. Among these, our further study with the cytoskeletal proteins reveals that, compared to mitosis, an up-regulation of actin cytoskeleton and its negative regulator occurs in meiosis. Biological significance: Mitosis and meiosis are two different types of cell division cycles with entirely different outcomes with definite biological implication for almost all eukaryotic species. In this work, we investigated, for the first time, the differential proteomic profile of Saccharomyces cerevisiae culture arrested at mitotic metaphase (M) and metaphase-I (MI) of meiosis using 2-DE and 2D-DIGE. Our findings of up-regulation of actin and its negative regulator cofilin during meiosis suggest that the rate of actin cytoskeleton turnover is more in meiosis and actin cytoskeleton may play more crucial role during meiosis compared to mitosis. Present study also suggests that actin cytoskeleton and its regulators accumulated during meiosis by forming stable protein structure even though the corresponding mRNAs are degraded as cells enter into meiosis. This is in accordance with recent studies in higher eukaryotes where actin cytoskeleton is found to play vital role during meiotic chromosome segregation. Information generated by this study is significant to reveal that even though a cell that, unlike mitosis, is metabolically inactive with no isotropic bulging of membranes as buds (in meiosis) can require more actin cytoskeleton presumably to support nuclear movements.
    Full-text · Article · Jun 2014 · Journal of Proteomics
  • Source
    • "For the DIGE time course, five single-colony isolates were processed in parallel and synchrony of the replicates was assessed by DAPI staining and expression of the REC8 protein as previously described [23]. Time course samples from the three replicate cultures judged to be the most synchronous were selected for DIGE labelling and analysis; two of these replicates had been used in the pH 4–7 analysis [23] "
    [Show abstract] [Hide abstract]
    ABSTRACT: Meiosis is the cell division that generates haploid gametes from diploid precursors. To provide insight into the functional proteome of budding yeast during meiosis, a 2-D DIGE kinetic approach was used to study proteins in the pH 6-11 range. Nearly 600 protein spots were visualised and 79 spots exhibited statistically significant changes in abundance as cells progressed through meiosis. Expression changes of up to 41-fold were detected and protein sequence information was obtained for 48 spots. Single protein identifications were obtained for 21 spots including different gel mobility forms of 5 proteins. A large number of post-translational events are suggested for these proteins, including processing, modification and import. The data are incorporated into an online 2-DE map of meiotic proteins in budding yeast, which extends our initial DIGE investigation of proteins in the pH 4-7 range. Together, the analyses provide peptide sequence data for 84 protein spots, including 50 single-protein identifications and gel mobility isoforms of 8 proteins. The largest classes of identified proteins include carbon metabolism, protein catabolism, protein folding, protein synthesis and the oxidative stress response. A number of the corresponding genes are required for yeast meiosis and recent studies have identified similar classes of proteins expressed during mammalian meiosis. This proteomic investigation and the resulting protein reference map make an important contribution towards a more detailed molecular view of yeast meiosis.
    Full-text · Article · Dec 2010 · Proteomics
Show more