Unicellular Eukaryotic Organisms Research Articles

The Evolution of COP9 Signalosome in Unicellular and Multicellular Organisms.

The COP9 signalosome (CSN) is a highly conserved protein complex, recently being crystallized for human. In mammals and plants the COP9 complex consists of nine subunits, CSN 1–8 and CSNAP. The CSN regulates the activity of culling ring E3 ubiquitin and plays central roles in pleiotropy, cell cycle, and defense of pathogens. Despite the interesting and essential functions, a thorough analysis of the CSN subunits in evolutionary comparative perspective is missing. Here we compared 61 eukaryotic genomes including plants, animals, and yeasts genomes and show that the most conserved subunits of eukaryotes among the nine subunits are CSN2 and CSN5. This may indicate a strong evolutionary selection for these two subunits. Despite the strong conservation of the protein sequence, the genomic structures of the intron/exon boundaries indicate no conservation at genomic level. This suggests that the gene structure is exposed to a much less selection compared with the protein sequence. We also show the conservation of important active domains, such as PCI (proteasome lid-CSN-initiation factor) and MPN (MPR1/PAD1 amino-terminal). We identified novel exons and alternative splicing variants for all CSN subunits. This indicates another level of complexity of the CSN. Notably, most COP9-subunits were identified in all multicellular and unicellular eukaryotic organisms analyzed, but not in prokaryotes or archaeas. Thus, genes encoding CSN subunits present in all analyzed eukaryotes indicate the invention of the signalosome at the root of eukaryotes. The identification of alternative splice variants indicates possible “mini-complexes” or COP9 complexes with independent subunits containing potentially novel and not yet identified functions.

Multiphase Biomineralization: Enigmatic Invasive Siliceous Diatoms Produce Crystalline Calcite

Diatoms are considered unicellular eukaryotic organisms exclusively depositing biogenic silica. Heretofore there has been no report of calcification by these algae. Here it is shown that calcium carbonate within the stalks ofDidymosphenia geminata, a nuisance species that has prolifically colonized streams and rivers globally, is biogenic in origin and occurs as a network of calcite nanofibers. The nanofibrous framework in the mineralized polysaccharide matrix imparts mechanical support to the stalks, providing stability in variable flow conditions. The results demonstrate thatD. geminatapossesses cellular and periplasmic carbonic‐anhydrases that contribute to carbon fixation and biomineralization, respectively. The activity of external carbonic‐anhydrase was more than 50% of the total activity, which points to its role in anchoring this bioeroding diatom on hard surfaces. The first evidence of multiphase biomineralization by diatoms that deposit both biogenic silica and crystalline biogenic calcite which are imparting distinct functional advantage to the organism is provided.

Ionic Conductivity as a Tool To Study Biocidal Activity of Sulfobetaine Micelles against Saccharomyces cerevisiae Model Cells.

Zwitterionic sulfobetaine surfactants are used in pharmaceutical or biomedical applications for the solubilization and delivery of hydrophobic molecules in aqueous medium or in biological environments. In a screening on the biocidal activity of synthetic surfactants on microbial cells, remarkable results have emerged with sulfobetaine amphiphiles. The interaction between eight zwitterionic sulfobetaine amphiphiles and Saccharomyces cerevisiae model cells was therefore analyzed. S. cerevisiae yeast cells were chosen, as they are a widely used unicellular eukaryotic model organism in cell biology. Conductivity measurements were used to investigate the interaction between surfactant solution and cells. Viable counts measurements were performed, and the mortality data correlated with the conductivity profiles very well, in terms of the inflection points (IPs) observed in the curves and in terms of supramolecular properties of the aggregates. A Fourier transform infrared (FTIR)-based bioassay was then performed to determine the metabolomic stress-response of the cells subjected to the action of zwitterionic surfactants. The surfactants showed nodal concentration (IPs) with all the techniques in their activities, corresponding to the critical micellar concentrations of the amphiphiles. This is due to the pseudocationic behavior of sulfobetaine micelles, because of their charge distribution and charge densities. This behavior permits the interaction of the micellar aggregates with the cells, and the structure of the surfactant monomers has impact on the mortality and the metabolomic response data observed. On the other hand, the concentrations that are necessary to provoke a biocidal activity do not promote these amphiphiles as potential antimicrobial agents. In fact, they are much higher than the ones of cationic surfactants.

Proteomic analyses of early response of unicellular eukaryotic microorganism Tetrahymena thermophila exposed to TiO2 particles

Key biological functions involved in cell survival have been studied to understand the difference between the impact of exposure to TiO2 nanoparticles (TiO2-NPs) and their bulk counterparts (bulk-TiO2). By selecting a unicellular eukaryotic model organism and applying proteomic analysis an overview of the possible impact of exposure could be obtained. In this study, we investigated the early response of unicellular eukaryotic protozoan Tetrahymena thermophila exposed to TiO2-NPs or bulk-TiO2 particles at subtoxic concentrations for this organism. The proteomic analysis based on 2DE + nLC-ESI-MS/MS revealed 930 distinct protein spots, among which 77 were differentially expressed and 18 were unambiguously identified. We identified alterations in metabolic pathways, including lipid and fatty acid metabolism, purine metabolism and energetic metabolism, as well as salt stress and protein degradation. This proteomic study is consistent with our previous findings, where the early response of T. thermophila to subtoxic concentrations of TiO2 particles included alterations in lipid and fatty acid metabolism and ion regulation. The response to the lowest TiO2-NPs concentration differed significantly from the response to higher TiO2-NPs concentration and both bulk-TiO2 concentrations. Alterations on the physiological landscape were significant after exposure to both nano- and bulk-TiO2; however, no toxic effects were evidenced even at very high exposure concentrations. This study confirms the relevance of the alteration of the lipid profile and lipid metabolism in understanding the early impact of TiO2-NPs in eukaryotic cells, for example, phagocytosing cells like macrophages and ciliated cells in the respiratory epithelium.

Label-free quantitative analysis of the casein kinase 2-responsive phosphoproteome of the marine minimal model species Ostreococcus tauri.

Casein kinase 2 (CK2) is a protein kinase that phosphorylates a plethora of cellular target proteins involved in processes including DNA repair, cell cycle control, and circadian timekeeping. CK2 is functionally conserved across eukaryotes, although the substrate proteins identified in a range of complex tissues are often different. The marine alga Ostreococcus tauri is a unicellular eukaryotic model organism ideally suited to efficiently study generic roles of CK2 in the cellular circadian clock. Overexpression of CK2 leads to a slow circadian rhythm, verifying functional conservation of CK2 in timekeeping. The proteome was analysed in wild‐type and CK2‐overexpressing algae at dawn and dusk, revealing that differential abundance of the global proteome across the day is largely unaffected by overexpression. However, CK2 activity contributed more strongly to timekeeping at dusk than at dawn. The phosphoproteome of a CK2 overexpression line and cells treated with CK2 inhibitor was therefore analysed and compared to control cells at dusk. We report an extensive catalogue of 447 unique CK2‐responsive differential phosphopeptide motifs to inform future studies into CK2 activity in the circadian clock of more complex tissues. All MS data have been deposited in the ProteomeXchange with identifier PXD000975 (http://proteomecentral.proteomexchange.org/dataset/PXD000975).

Melatonin regulates antioxidative mechanisms in microalgae Chlamydomonas reinhardtii (Volvocales, Chlorophyceae)

:The role of melatonin (MLT) in micro- and macroalgae is the subject of continuous discussion. In eukaryotic unicellular organisms, the physiological role of MLT seems to differ from those of higher plants and mammals. Experimental data on the role of MLT in algae suggest that it acts mainly as an antioxidant. In the present study, the physiological role of MLT in phototrophic organisms was studied by measuring its effects on periodically controlled processes (cell division and HSP70B expression) as well as mechanisms involved in oxidative stress protection (carotenoids, FeSOD and MnSOD expression). The presence of MLT in Chlamydomonas reinhardtii was assessed and quantified using analytical thin-layer chromatography followed by fluorescence measurements. These data reveal that photoperiod had a significant influence on the processes and mechanisms studied in C. reinhardtii. Moreover, MLT had the greatest impact on C. reinhardtii under conditions promoting photo-oxidative stress. Addition of MLT to the growth medium down-regulated carotenoid content and the expression of FeSOD and MnSOD. These results support the idea that MLT has a physiological role as a direct and indirect antioxidant agent.

Phylogenomics of non-model ciliates based on transcriptomic analyses.

Ciliates are one of the oldest living eukaryotic unicellular organisms, widely distributed in the waters around the world. As a typical marine oligotrich ciliate, Strombidium sulcatum plays an important role in marine food webs and energy flow. Here we report the first deep sequencing and analyses of RNA-Seq data from Strombidium sulcatum. We generated 42,640 unigenes with an N50 of 1,451 bp after de novo assembly and removing rRNA, mitochondrial and bacteria contaminants. We employed SPOCS to detect orthologs from S. sulcatum and 17 other ciliates, and then carried out the phylogenomic reconstruction using 127 single copy orthologs. In phylogenomic analyses, concatenated trees have similar topological structures with concordance tree on the class level. Together with phylogenetic networks analysis, it aroused more doubts about the placement of Protocruzia, Mesodinium and Myrionecta. While epiplasmic proteins are known to be related to morphological characteristics, we found the potential relationship between gene expression of epiplasmic proteins and morphological characteristics. This work supports the use of high throughput approaches for phylogenomic analysis as well as correlation analysis between expression level of target genes and morphological characteristics.Electronic supplementary materialThe online version of this article (doi:10.1007/s13238-015-0147-3) contains supplementary material, which is available to authorized users.

Establishing Chlamydomonas reinhardtii as an industrial biotechnology host

Microalgae constitute a diverse group of eukaryotic unicellular organisms that are of interest for pure and applied research. Owing to their natural synthesis of value-added natural products microalgae are emerging as a source of sustainable chemical compounds, proteins and metabolites, including but not limited to those that could replace compounds currently made from fossil fuels. For the model microalga, Chlamydomonas reinhardtii, this has prompted a period of rapid development so that this organism is poised for exploitation as an industrial biotechnology platform. The question now is how best to achieve this? Highly advanced industrial biotechnology systems using bacteria and yeasts were established in a classical metabolic engineering manner over several decades. However, the advent of advanced molecular tools and the rise of synthetic biology provide an opportunity to expedite the development of C. reinhardtii as an industrial biotechnology platform, avoiding the process of incremental improvement. In this review we describe the current status of genetic manipulation of C. reinhardtii for metabolic engineering. We then introduce several concepts that underpin synthetic biology, and show how generic parts are identified and used in a standard manner to achieve predictable outputs. Based on this we suggest that the development of C. reinhardtii as an industrial biotechnology platform can be achieved more efficiently through adoption of a synthetic biology approach.Significance StatementChlamydomonas reinhardtii offers potential as a host for the production of high value compounds for industrial biotechnology. Synthetic biology provides a mechanism to generate generic, well characterised tools for application in the rational genetic manipulation of organisms: if synthetic biology principles were adopted for manipulation of C. reinhardtii, development of this microalga as an industrial biotechnology platform would be expedited.

Sexual reproduction of human fungal pathogens.

We review here recent advances in our understanding of sexual reproduction in fungal pathogens that commonly infect humans, including Candida albicans, Cryptococcus neoformans/gattii, and Aspergillus fumigatus. Where appropriate or relevant, we introduce findings on other species associated with human infections. In particular, we focus on rapid advances involving genetic, genomic, and population genetic approaches that have reshaped our view of how fungal pathogens evolve. Rather than being asexual, mitotic, and largely clonal, as was thought to be prevalent as recently as a decade ago, we now appreciate that the vast majority of pathogenic fungi have retained extant sexual, or parasexual, cycles. In some examples, sexual and parasexual unions of pathogenic fungi involve closely related individuals, generating diversity in the population but with more restricted recombination than expected from fertile, sexual, outcrossing and recombining populations. In other cases, species and isolates participate in global outcrossing populations with the capacity for considerable levels of gene flow. These findings illustrate general principles of eukaryotic pathogen emergence with relevance for other fungi, parasitic eukaryotic pathogens, and both unicellular and multicellular eukaryotic organisms.

Bioinformatic and proteomic analysis of bulk histones reveals PTM crosstalk and chromatin features.

Systems analysis of chromatin has been constrained by complex patterns and dynamics of histone post-translational modifications (PTMs), which represent major challenges for both mass spectrometry (MS) and immuno-based approaches (e.g., chromatin immuno-precipitation, ChIP). Here we present a proof-of-concept study demonstrating that crosstalk among PTMs and their functional significance can be revealed via systematic bioinformatic and proteomic analysis of steady-state histone PTM levels from cells under various perturbations. Using high resolution tandem MS, we quantified 53 modification states from all core histones and their conserved variants in the unicellular eukaryotic model organism Tetrahymena. By correlating histone PTM patterns across 15 different conditions, including various physiological states and mutations of key histone modifying enzymes, we identified 5 specific chromatin states with characteristic covarying histone PTMs and associated them with distinctive functions in replication, transcription, and DNA repair. In addition to providing a detailed picture on histone PTM crosstalk at global levels, this work has established a novel bioinformatic and proteomic approach, which can be adapted to other organisms and readily scaled up to allow increased resolution of chromatin states.

Respiration in Heterotrophic Unicellular Eukaryotic Organisms

Surface:volume quotient, mitochondrial volume fraction, and their distribution within cells were investigated and oxygen gradients within and outside cells were modelled. Cell surface increases allometrically with cell size. Mitochondrial volume fraction is invariant with cell size and constitutes about 10% and mitochondria are predominantly found close to the outer membrane. The results predict that for small and medium sized protozoa maximum respiration rates should be proportional to cell volume (scaling exponent ≈1) and access to intracellular O2 is not limiting except at very low ambient O2-tensions. Available data do not contradict this and some evidence supports this interpretation. Cell size is ultimately limited because an increasing fraction of the mitochondria becomes exposed to near anoxic conditions with increasing cell size. The fact that mitochondria cluster close to the cell surface and the allometric change in cell shape with increasing cell size alleviates the limitation of aerobic life at low ambient O2-tension and for large cell size.

Autophagy, plant senescence, and nutrient recycling

Large numbers of publications have appeared over the last few years, dealing with the molecular details of the regulation and process of the autophagy machinery in animals, plants, and unicellular eukaryotic organisms. This strong interest is caused by the fact that the autophagic process is involved in the adaptation of organisms to their environment and to stressful conditions, thereby contributing to cell and organism survival and longevity. In plants, as in other eukaryotes, autophagy is associated with longevity as mutants display early and strong leaf senescence symptoms, however, the exact role of autophagy as a pro-survival or pro-death process is unclear. Recently, evidence that autophagy participates in nitrogen remobilization has been provided, but the duality of the role of autophagy in leaf longevity and/or nutrient recycling through cell component catabolism remains. This review aims to give an overview of leaf senescence-associated processes from the physiological point of view and to discuss relationships between nutrient recycling, proteolysis, and autophagy. The dual role of autophagy as a pro-survival or pro-death process is discussed.

Phosphoproteomic Analysis of Protein Phosphorylation Networks in Tetrahymena thermophila, a Model Single-celled Organism

Tetrahymena thermophila is a widely used unicellular eukaryotic model organism in biological research and contains more than 1000 protein kinases and phosphatases with specificity for Ser/Thr/Tyr residues. However, only a few dozen phosphorylation sites in T. thermophila are known, presenting a major obstacle to further understanding of the regulatory roles of reversible phosphorylation in this organism. In this study, we used high-accuracy mass-spectrometry-based proteomics to conduct global and site-specific phosphoproteome profiling of T. thermophila. In total, 1384 phosphopeptides and 2238 phosphorylation sites from 1008 T. thermophila proteins were identified through the combined use of peptide prefractionation, TiO2 enrichment, and two-dimensional LC-MS/MS analysis. The identified phosphoproteins are implicated in the regulation of various biological processes such as transport, gene expression, and mRNA metabolic process. Moreover, integrated analysis of the T. thermophila phosphoproteome and gene network revealed the potential biological functions of many previously unannotated proteins and predicted some putative kinase-substrate pairs. Our data provide the first global survey of phosphorylation in T. thermophila using a phosphoproteomic approach and suggest a wide-ranging regulatory scope of this modification. The provided dataset is a valuable resource for the future understanding of signaling pathways in this important model organism.

Interventions against nutrient-sensing pathways represent an emerging new therapeutic approach for diabetic nephropathy

Autophagy has evolved as a stress response that allows unicellular eukaryotic organisms to survive in starved conditions by regulating energy homeostasis and/or by protein and organelle quality control. The diabetes-induced accumulation of damaged proteins and organelles results in the development and progression of diabetic nephropathy. In contrast, autophagy machinery is activated by calorie restriction and environmental stress in proximal tubular cells, and is maintained at a high level in podocytes, suggesting its crucial role in the pathogenesis of diabetic nephropathy. However, its role in diabetic nephropathy has not been fully known. Here, we will discuss the role of autophagy and its involvement in the pathogenesis of diabetic nephropathy.

Exploring the N-glycosylation Pathway in Chlamydomonas reinhardtii Unravels Novel Complex Structures

Chlamydomonas reinhardtii is a green unicellular eukaryotic model organism for studying relevant biological and biotechnological questions. The availability of genomic resources and the growing interest in C. reinhardtii as an emerging cell factory for the industrial production of biopharmaceuticals require an in-depth analysis of protein N-glycosylation in this organism. Accordingly, we used a comprehensive approach including genomic, glycomic, and glycoproteomic techniques to unravel the N-glycosylation pathway of C. reinhardtii. Using mass-spectrometry-based approaches, we found that both endogenous soluble and membrane-bound proteins carry predominantly oligomannosides ranging from Man-2 to Man-5. In addition, minor complex N-linked glycans were identified as being composed of partially 6-O-methylated Man-3 to Man-5 carrying one or two xylose residues. These findings were supported by results from a glycoproteomic approach that led to the identification of 86 glycoproteins. Here, a combination of in-source collision-induced dissodiation (CID) for glycan fragmentation followed by mass tag-triggered CID for peptide sequencing and PNGase F treatment of glycopeptides in the presence of (18)O-labeled water in conjunction with CID mass spectrometric analyses were employed. In conclusion, our data support the notion that the biosynthesis and maturation of N-linked glycans in the endoplasmic reticulum and Golgi apparatus occur via a GnT I-independent pathway yielding novel complex N-linked glycans that maturate differently from their counterparts in land plants.

Recovery of soil unicellular eukaryotes: An efficiency and activity analysis on the single cell level

Eukaryotic unicellular organisms are an important part of the soil microbial community, but they are often neglected in soil functional microbial diversity analysis, principally due to the absence of specific investigation methods in the special soil environment. In this study we used a method based on high-density centrifugation to specifically isolate intact algal and yeast cells, with the aim to analyze them with flow cytometry and sort them for further molecular analysis such as deep sequencing. Recovery efficiency was tested at low abundance levels that fit those in natural environments (104 to 106 cells per g soil). Five algae and five yeast morphospecies isolated from soil were used for the testing. Recovery efficiency was between 1.5 to 43.16% and 2 to 30.2%, respectively, and was dependent on soil type for three of the algae. Control treatments without soil showed that the majority of cells were lost due to the method itself (58% and 55.8% respectively). However, the cell extraction technique did not much compromise cell vitality because a fluorescein di-acetate assay indicated high viability percentages (73.3% and 97.2% of cells, respectively). The low abundant algae and yeast morphospecies recovered from soil were cytometrically analyzed and sorted. Following, their DNA was isolated and amplified using specific primers. The developed workflow enables isolation and enrichment of intact autotrophic and heterotrophic soil unicellular eukaryotes from natural environments for subsequent application of deep sequencing technologies.

An Interview with Professor Michael Shapira

A n I nterview with P rofessor M ichael S hapira B S J Kuntal Chowdhary, Manraj Gill, Mariko Nakamura, Kaitlyn Kraybill-Voth, Atiriya Hari, Ali Palla The BSJ Interviews Crew had the opportunity to interview Professor Michael Shapira. Professor Shapira received his B.Sc. and his Ph.D. in biochemistry and molecular biology from the Hebrew University in Jerusalem. Following receipt of his doctorate, Professor Shapira moved to the Genetics department at Stanford University School of Medicine, where he trained with David Botstein and Man-Wah Tan as a Life Sciences Research Foundation postdoctoral fellow. Currently, Professor Shapira’s research is focused on understanding the fundamentals of host-pathogen interactions in the context of the whole organism. Through the use of his unique model organism, the soil nematode Caenorhabditis elegans, Professor Shapira’s current point of interest at the lab is pathogen recognition. BSJ: To start things off, how did you get interested in your research? Prof. Shapira: Well, things have happened as they sometimes do in science. I wasn’t always focusing on C. elegans; I used what seemed to be the most appropriate system at the time to answer a biological question. As my interests in stress and environmental stress conditions evolved to consider genome-wide responses, I moved to work with a eukaryotic unicellular model organism, in which analysis could be more complete, but actually, I was more interested in studying similar stress responses in multicellular organisms. By chance, a nearby lab was working with C. elegans, studying how it dealt with infection. I figured that that was a type of stress, and the system seemed overall very attractive: typically you study a one-sided response to stress, but when studying host-pathogen interactions, you have two sides that are responding to each other. I became increasingly fascinated with this host-pathogen interaction story. Again, nothing really stays the same, I started with stress and moved to the host-pathogen interactions, and before you know it, I’m back to stress. 65 • B erkeley S cientific J ournal • S tress • F all 2013 • V olume 18 • I ssue 1

Identification of Site Specific Diatom at Yamuna River of Allahabad

In present study water sample from four sites of Yamuna River were collected for identification of diatom. Collected water sample were digested with acid digestion and examined under high power microscope after slide preparation. After examination it was found that total 41 diatom species was found at four sites of river. Out of 41 diatom species 10 diatom species were found as site specific. These site specific diatoms can be used as marker for site identification in cases of suspected drowning as well as for other forensic purposes. I. Introduction: Diatoms are microscopic unicellular eukaryotic organisms ranging in size from approximately 5 microns to 1000 microns and of the most common types of phytoplankton. The word 'diatom' means 'cut in two' and is derived from the Greek :(dia) = 'through' + (temnein) = ' to cut', i.e. 'cut in half'. A characteristic feature of diatoms cell is that they are encased within a cell wall made of silica (hydrated silicon dioxide) called a Frustule and is found in almost aquatic environment including fresh and marine waters, soils, in fact almost anywhere in moist environment. There are more than 200 genera of living diatoms and it is estimated that there are approximately 100,000 extant species (Hasle et al., 1997), either free-floating, planktonic forms or attached to substrate. Diatoms grow as single cells, or form simple filaments/colonies. They form the base of aquatic food webs in marine and fresh water habitats. Diatoms are divided into two orders. The Centrales (now called the Biddulphiales) which have valve striae arranged basically in relation to a point, an annulus or a centrals areola and tend to appear radially symmetrical, and the Pennales (now called Bacillariales) which have valve striae arranged in relation to a line and tend to appear bilaterally symmetrical. The valve face of diatom frustules is ornamented with pores (areolae),spines, hyaline areas and other distinguishing morphological features. It is these skeletal features, which are used to classify and describe diatoms, which is an advantage in terms of paleontology since the same features are used to define extant species as extinct ones. Diatoms have several as a unique forensic marker in deciding the cause of death as drowning and in solving drowning related cases where medical officers fail ascertain the cause of death especially in cases when skeletonised and putrefied dead bodies are recovered. II. Methodology: 500ml to 1000ml water sample were collected in thoroughly washed bottle and then bottles were tightly fitted with cap & labelled with the location of sampling site, along with the date, time, and month. Water samples were collected at the bank, mid & across the river. Water samples were collected in different month continuously at month February to May .The water samples were collected from 4-different sites (Site1 (old bridge), Site2 (new bridge), Site3 (boat club) and Site4 (Sangam)) of Yamuna River at Allahabad. The collected water samples were brought to the laboratory for examination of diatoms. 500ml water sample was taken into the beaker from each bottles and 10 ml of Lugol's iodine was added in beaker .Lugol's iodine was used as preservative of water sample and left it for overnight. According to Ludes et al., (1996), 50ml of Conc. HNO3, KMnO4 & H2O2 was added in 500ml water sample in 5:2:3 ratios. They oxidize the organic matter present in the water sample except diatom cell wall because cell wall is resistant to them. The next day samples were taken in Tarson tubes & centrifuged at 1500rpm for 5-7 minutes and the supernant were discarded. This step was repeated till full of the water sample contained in the beaker was centrifuged. The Pellets were formed at the bottom of Tarson tube, the pellets were taken with the dropper and transferred on microscopic slide, left it for drying on the hot plate and cover it with cover slip then observed under compound microscope at 10x ,45x and 100x (oil immersion) magnification Taylor et al.,(2007). The same steps were repeated for all the samples from sites S1 to S4 for morphological examination of diatoms.

Proteome-wide Analysis of Lysine Acetylation Suggests its Broad Regulatory Scope in Saccharomyces cerevisiae

Post-translational modification of proteins by lysine acetylation plays important regulatory roles in living cells. The budding yeast Saccharomyces cerevisiae is a widely used unicellular eukaryotic model organism in biomedical research. S. cerevisiae contains several evolutionary conserved lysine acetyltransferases and deacetylases. However, only a few dozen acetylation sites in S. cerevisiae are known, presenting a major obstacle for further understanding the regulatory roles of acetylation in this organism. Here we use high resolution mass spectrometry to identify about 4000 lysine acetylation sites in S. cerevisiae. Acetylated proteins are implicated in the regulation of diverse cytoplasmic and nuclear processes including chromatin organization, mitochondrial metabolism, and protein synthesis. Bioinformatic analysis of yeast acetylation sites shows that acetylated lysines are significantly more conserved compared with nonacetylated lysines. A large fraction of the conserved acetylation sites are present on proteins involved in cellular metabolism, protein synthesis, and protein folding. Furthermore, quantification of the Rpd3-regulated acetylation sites identified several previously known, as well as new putative substrates of this deacetylase. Rpd3 deficiency increased acetylation of the SAGA (Spt-Ada-Gcn5-Acetyltransferase) complex subunit Sgf73 on K33. This acetylation site is located within a critical regulatory domain in Sgf73 that interacts with Ubp8 and is involved in the activation of the Ubp8-containing histone H2B deubiquitylase complex. Our data provides the first global survey of acetylation in budding yeast, and suggests a wide-ranging regulatory scope of this modification. The provided dataset may serve as an important resource for the functional analysis of lysine acetylation in eukaryotes.

L’autophagie chez les plantes : mécanismes, régulations et fonctions

Large numbers of publications investigating the molecular details, the regulation and the physiological roles of autophagic processes have appeared over the last few years, dealing with animals, plants and unicellular eukaryotic organisms. This strong interest is caused by the fact that autophagic processes are ubiquitous in eukaryotic organisms. They are involved in the adaptation of organisms to their environment and to stressful conditions, thereby contributing to cell and organism survival and longevity. This review article aims to describe the discovery of autophagy, the molecular details of this complex process, its regulation, and its specific functions in plants.