RESUMO
In the last decade several collections of Saccharomyces cerevisiae yeast strains have been created. In these collections every gene is modified in a similar manner such as by a deletion or the addition of a protein tag. Such libraries have enabled a diversity of systematic screens, giving rise to large amounts of information regarding gene functions. However, often papers describing such screens focus on a single gene or a small set of genes and all other loci affecting the phenotype of choice ('hits') are only mentioned in tables that are provided as supplementary material and are often hard to retrieve or search. To help unify and make such data accessible, we have created a Database of High Throughput Screening Hits (dHITS). The dHITS database enables information to be obtained about screens in which genes of interest were found as well as the other genes that came up in that screen - all in a readily accessible and downloadable format. The ability to query large lists of genes at the same time provides a platform to easily analyse hits obtained from transcriptional analyses or other screens. We hope that this platform will serve as a tool to facilitate investigation of protein functions to the yeast community.
Assuntos
Bases de Dados Genéticas , Biblioteca Gênica , Ensaios de Triagem em Larga Escala/métodos , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Mineração de Dados/métodos , Sistemas de Gerenciamento de Base de Dados , Deleção de Genes , Genoma Fúngico/genética , Proteínas de Fluorescência Verde/genética , FenótipoRESUMO
The endoplasmic reticulum (ER) is the entry site of proteins into the endomembrane system. Proteins exit the ER via coat protein II (COPII) vesicles in a selective manner, mediated either by direct interaction with the COPII coat or aided by cargo receptors. Despite the fundamental role of such receptors in protein sorting, only a few have been identified. To further define the machinery that packages secretory cargo and targets proteins from the ER to Golgi membranes, we used multiple systematic approaches, which revealed 2 uncharacterized proteins that mediate the trafficking and maturation of Pma1, the essential yeast plasma membrane proton ATPase. Ydl121c (Exp1) is an ER protein that binds Pma1, is packaged into COPII vesicles, and whose deletion causes ER retention of Pma1. Ykl077w (Psg1) physically interacts with Exp1 and can be found in the Golgi and coat protein I (COPI) vesicles but does not directly bind Pma1. Loss of Psg1 causes enhanced degradation of Pma1 in the vacuole. Our findings suggest that Exp1 is a Pma1 cargo receptor and that Psg1 aids Pma1 maturation in the Golgi or affects its retrieval. More generally our work shows the utility of high content screens in the identification of novel trafficking components.
Assuntos
ATPases Translocadoras de Prótons/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Vesículas Revestidas pelo Complexo de Proteína do Envoltório/metabolismo , Complexo de Golgi/metabolismo , Ligação Proteica , Transporte Proteico , ATPases Translocadoras de Prótons/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Transporte Vesicular/genéticaRESUMO
Pex3 has been proposed to be important for the exit of peroxisomal membrane proteins (PMPs) from the ER, based on the observation that PMPs accumulate at the ER in Saccharomyces cerevisiae pex3 mutant cells. Using a combination of microscopy and biochemical approaches, we show that a subset of the PMPs, including the receptor docking protein Pex14, localizes to membrane vesicles in S. cerevisiae pex3 cells. These vesicles are morphologically distinct from the ER and do not co-sediment with ER markers in cell fractionation experiments. At the vesicles, Pex14 assembles with other peroxins (Pex13, Pex17, and Pex5) to form a complex with a composition similar to the PTS1 import pore in wild-type cells. Fluorescence microscopy studies revealed that also the PTS2 receptor Pex7, the importomer organizing peroxin Pex8, the ubiquitin conjugating enzyme Pex4 with its recruiting PMP Pex22, as well as Pex15 and Pex25 co-localize with Pex14. Other peroxins (including the RING finger complex and Pex27) did not accumulate at these structures, of which Pex11 localized to mitochondria. In line with these observations, proteomic analysis showed that in addition to the docking proteins and Pex5, also Pex7, Pex4/Pex22 and Pex25 were present in Pex14 complexes isolated from pex3 cells. However, formation of the entire importomer was not observed, most likely because Pex8 and the RING proteins were absent in the Pex14 protein complexes. Our data suggest that peroxisomal membrane vesicles can form in the absence of Pex3 and that several PMPs can insert in these vesicles in a Pex3 independent manner.
Assuntos
Proteínas de Membrana/genética , Proteínas de Membrana Transportadoras/genética , Peroxinas/genética , Peroxissomos/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Proteínas de Arabidopsis/biossíntese , Proteínas de Arabidopsis/genética , Retículo Endoplasmático/genética , Retículo Endoplasmático/metabolismo , Proteínas de Membrana/biossíntese , Proteínas de Membrana Transportadoras/biossíntese , Peroxinas/biossíntese , Peroxissomos/metabolismo , Proteoma/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/biossíntese , Vesículas Transportadoras/genética , Vesículas Transportadoras/metabolismo , Enzimas de Conjugação de Ubiquitina/biossíntese , Enzimas de Conjugação de Ubiquitina/genéticaRESUMO
Differential signaling of the type I interferon receptor (IFNAR) has been correlated with the ability of its subunit, IFNAR1, to differentially recognize a large spectrum of different ligands, which involves intricate conformational re-arrangements of multiple interacting domains. To shed light onto the structural determinants governing ligand recognition, we compared the force-induced unfolding of the IFNAR1 ectodomain when bound to interferon and when free, using the atomic force microscope and steered molecular dynamics simulations. Unexpectedly, we find that IFNAR1 is easier to mechanically unfold when bound to interferon than when free. Analysis of the structures indicated that the origin of the reduction in unfolding forces is a conformational change in IFNAR1 induced by ligand binding.
Assuntos
Interferon Tipo I/química , Receptor de Interferon alfa e beta/química , Humanos , Microscopia de Força Atômica , Simulação de Dinâmica Molecular , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Desdobramento de Proteína , TermodinâmicaRESUMO
Sphingolipids (SLs) are essential components of cell membranes and are broad-range bioactive signaling molecules. SL levels must be tightly regulated as imbalances affect cellular function and contribute to pathologies ranging from neurodegenerative and metabolic disorders to cancer and aging. Deciphering how SL homeostasis is maintained and uncovering new regulators is required for understanding lipid biology and for identifying new targets for therapeutic interventions. Here we combine omics technologies to identify the changes of the transcriptome, proteome, and phosphoproteome in the yeast Saccharomyces cerevisiae upon SL depletion induced by myriocin. Surprisingly, while SL depletion triggers important changes in the expression of regulatory proteins involved in SL homeostasis, the most dramatic regulation occurs at the level of the phosphoproteome, suggesting that maintaining SL homeostasis demands rapid responses. To discover which of the phosphoproteomic changes are required for the cell's first-line response to SL depletion, we overlaid our omics results with systematic growth screens for genes required during growth in myriocin. By following the rate of SL biosynthesis in those candidates that are both affecting growth and are phosphorylated in response to the drug, we uncovered Atg9, Stp4, and Gvp36 as putative new regulators of SL homeostasis.
Assuntos
Ácido Aspártico Endopeptidases/genética , Proteínas Relacionadas à Autofagia/genética , Regulação Fúngica da Expressão Gênica , Proteínas de Membrana/genética , Proteínas de Transporte de Monossacarídeos/genética , Fosfoproteínas/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Antifúngicos/farmacologia , Ácido Aspártico Endopeptidases/metabolismo , Proteínas Relacionadas à Autofagia/metabolismo , Ácidos Graxos Monoinsaturados/farmacologia , Perfilação da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Homeostase/efeitos dos fármacos , Homeostase/genética , Proteínas de Membrana/metabolismo , Proteínas de Transporte de Monossacarídeos/metabolismo , Fosfoproteínas/metabolismo , Fosforilação/efeitos dos fármacos , Proteômica/métodos , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Transdução de Sinais , Esfingolipídeos/antagonistas & inibidores , Esfingolipídeos/biossínteseRESUMO
Autophagy plays a central role in the DNA damage response (DDR) by controlling the levels of various DNA repair and checkpoint proteins; however, how the DDR communicates with the autophagy pathway remains unknown. Using budding yeast, we demonstrate that global genotoxic damage or even a single unrepaired double-strand break (DSB) initiates a previously undescribed and selective pathway of autophagy that we term genotoxin-induced targeted autophagy (GTA). GTA requires the action primarily of Mec1/ATR and Rad53/CHEK2 checkpoint kinases, in part via transcriptional up-regulation of central autophagy proteins. GTA is distinct from starvation-induced autophagy. GTA requires Atg11, a central component of the selective autophagy machinery, but is different from previously described autophagy pathways. By screening a collection of â¼6,000 yeast mutants, we identified genes that control GTA but do not significantly affect rapamycin-induced autophagy. Overall, our findings establish a pathway of autophagy specific to the DNA damage response.
Assuntos
Autofagia/genética , Quebras de DNA de Cadeia Dupla , Dano ao DNA , Saccharomyces cerevisiae/genética , Transdução de Sinais/genética , Proteínas Relacionadas à Autofagia/genética , Proteínas Relacionadas à Autofagia/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Quinase do Ponto de Checagem 2/genética , Quinase do Ponto de Checagem 2/metabolismo , Reparo do DNA , DNA Fúngico/genética , DNA Fúngico/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismoRESUMO
Peroxisomes are cellular organelles with vital functions in lipid, amino acid and redox metabolism. The cellular formation and dynamics of peroxisomes are governed by PEX genes; however, the regulation of peroxisome abundance is still poorly understood. Here, we use a high-content microscopy screen in Saccharomyces cerevisiae to identify new regulators of peroxisome size and abundance. Our screen led to the identification of a previously uncharacterized gene, which we term PEX35, which affects peroxisome abundance. PEX35 encodes a peroxisomal membrane protein, a remote homolog to several curvature-generating human proteins. We systematically characterized the genetic and physical interactome as well as the metabolome of mutants in PEX35, and we found that Pex35 functionally interacts with the vesicle-budding-inducer Arf1. Our results highlight the functional interaction between peroxisomes and the secretory pathway.
Assuntos
Proteínas de Membrana/metabolismo , Peroxissomos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Epistasia Genética , Deleção de Genes , Genes Fúngicos , Microscopia , Saccharomyces cerevisiae/genéticaRESUMO
In eukaryotes, up to one-third of cellular proteins are targeted to the endoplasmic reticulum, where they undergo folding, processing, sorting and trafficking to subsequent endomembrane compartments. Targeting to the endoplasmic reticulum has been shown to occur co-translationally by the signal recognition particle (SRP) pathway or post-translationally by the mammalian transmembrane recognition complex of 40 kDa (TRC40) and homologous yeast guided entry of tail-anchored proteins (GET) pathways. Despite the range of proteins that can be catered for by these two pathways, many proteins are still known to be independent of both SRP and GET, so there seems to be a critical need for an additional dedicated pathway for endoplasmic reticulum relay. We set out to uncover additional targeting proteins using unbiased high-content screening approaches. To this end, we performed a systematic visual screen using the yeast Saccharomyces cerevisiae, and uncovered three uncharacterized proteins whose loss affected targeting. We suggest that these proteins work together and demonstrate that they function in parallel with SRP and GET to target a broad range of substrates to the endoplasmic reticulum. The three proteins, which we name Snd1, Snd2 and Snd3 (for SRP-independent targeting), can synthetically compensate for the loss of both the SRP and GET pathways, and act as a backup targeting system. This explains why it has previously been difficult to demonstrate complete loss of targeting for some substrates. Our discovery thus puts in place an essential piece of the endoplasmic reticulum targeting puzzle, highlighting how the targeting apparatus of the eukaryotic cell is robust, interlinked and flexible.
Assuntos
Retículo Endoplasmático/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/metabolismo , Células HEK293 , Humanos , Proteínas de Membrana/metabolismo , Proteínas de Transporte de Fosfato/metabolismo , Domínios Proteicos , Sinais Direcionadores de Proteínas , Transporte Proteico , Proteínas Ribossômicas/metabolismo , Partícula de Reconhecimento de Sinal/metabolismoRESUMO
To optimally perform the diversity of metabolic functions that occur within peroxisomes, cells must dynamically regulate peroxisome size, number and content in response to the cell state and the environment. Except for transcriptional regulation little is known about the mechanisms used to perform this complicated feat. Focusing on the yeast Saccharomyces cerevisiae, we used complementary high-content screens to follow changes in localization of most proteins during growth in oleate. We found extensive changes in cellular architecture and identified several proteins that colocalized with peroxisomes that had not previously been considered peroxisomal proteins. One of the newly identified peroxisomal proteins, Ymr018w, is a protein with an unknown function that is similar to the yeast and human peroxisomal targeting receptor Pex5. We demonstrate that Ymr018w is a new peroxisomal-targeting receptor that targets a subset of matrix proteins to peroxisomes. We, therefore, renamed Ymr018w, Pex9, and suggest that Pex9 is a condition-specific targeting receptor that enables the dynamic rewiring of peroxisomes in response to metabolic needs. Moreover, we suggest that Pex5-like receptors might also exist in vertebrates.
Assuntos
Ácido Oleico/farmacologia , Peroxissomos/metabolismo , Proteoma/metabolismo , Receptores de Superfície Celular/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/metabolismo , Modelos Biológicos , Transporte Proteico/efeitos dos fármacos , Proteômica , Saccharomyces cerevisiae/efeitos dos fármacosRESUMO
Upon amino acid (AA) starvation and TOR inactivation, plasma-membrane-localized permeases rapidly undergo ubiquitination and internalization via the vacuolar protein sorting/multivesicular body (VPS-MVB) pathway and are degraded in the yeast vacuole. We now show that specific Golgi proteins are also directed to the vacuole under these conditions as part of a Golgi quality-control (GQC) process. The degradation of GQC substrates is dependent upon ubiquitination by the defective-for-SREBP-cleavage (DSC) complex, which was identified via genetic screening and includes the Tul1 E3 ligase. Using a model GQC substrate, GFP-tagged Yif1, we show that vacuolar targeting necessitates upregulation of the VPS pathway via proteasome-mediated degradation of the initial endosomal sorting complex required for transport, ESCRT-0, but not downstream ESCRT components. Thus, early cellular responses to starvation include the targeting of specific Golgi proteins for degradation, a phenomenon reminiscent of the inactivation of BTN1, the yeast Batten disease gene ortholog.
Assuntos
Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Complexo de Golgi/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Complexo de Golgi/enzimologia , Transporte Proteico , Controle de Qualidade , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Transdução de Sinais , Enzimas de Conjugação de Ubiquitina/metabolismoRESUMO
Peroxisomes are ubiquitous and dynamic organelles that house many important pathways of cellular metabolism. In recent years it has been demonstrated that mitochondria are tightly connected with peroxisomes and are defective in several peroxisomal diseases. Indeed, these two organelles share metabolic routes as well as resident proteins and, at least in mammals, are connected via a vesicular transport pathway. However the exact extent of cross-talk between peroxisomes and mitochondria remains unclear. Here we used a combination of high throughput genetic manipulations of yeast libraries alongside high content screens to systematically unravel proteins that affect the transport of peroxisomal proteins and peroxisome biogenesis. Follow up work on the effector proteins that were identified revealed that peroxisomes are not randomly distributed in cells but are rather localized to specific mitochondrial subdomains such as mitochondria-ER junctions and sites of acetyl-CoA synthesis. Our approach highlights the intricate geography of the cell and suggests an additional layer of organization as a possible way to enable efficient metabolism. Our findings pave the way for further studying the machinery aligning mitochondria and peroxisomes, the role of the juxtaposition, as well as its regulation during various metabolic conditions. More broadly, the approaches used here can be easily applied to study any organelle of choice, facilitating the discovery of new aspects in cell biology.
Assuntos
Mitocôndrias/metabolismo , Peroxissomos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/metabolismo , Acetilcoenzima A/metabolismo , Retículo Endoplasmático/metabolismo , Mutagênese , Transporte Proteico , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
Single-molecule manipulation methods provide a powerful means to study protein transitions. Here we combined single-molecule force spectroscopy and steered molecular-dynamics simulations to study the mechanical properties and unfolding behavior of the small enzyme acylphosphatase (AcP). We find that mechanical unfolding of AcP occurs at relatively low forces in an all-or-none fashion and is decelerated in the presence of a ligand, as observed in solution measurements. The prominent energy barrier for the transition is separated from the native state by a distance that is unusually long for alpha/beta proteins. Unfolding is initiated at the C-terminal strand (beta(T)) that lies at one edge of the beta-sheet of AcP, followed by unraveling of the strand located at the other. The central strand of the sheet and the two helices in the protein unfold last. Ligand binding counteracts unfolding by stabilizing contacts between an arginine residue (Arg-23) and the catalytic loop, as well as with beta(T) of AcP, which renders the force-bearing units of the protein resistant to force. This stabilizing effect may also account for the decelerated unfolding of ligand-bound AcP in the absence of force.
Assuntos
Hidrolases Anidrido Ácido/química , Simulação de Dinâmica Molecular , Análise Espectral , Animais , Fenômenos Biomecânicos , Cinética , Ligantes , Desnaturação Proteica , Dobramento de Proteína , Estrutura Secundária de Proteína , Soluções , AcilfosfataseRESUMO
Adaptability of oxygenic photosynthetic organisms to fluctuations in light spectral composition and intensity is conferred by state transitions, short-term regulatory processes that enable the photosynthetic apparatus to rapidly adjust to variations in light quality. In green algae and higher plants, these processes are accompanied by reversible structural rearrangements in the thylakoid membranes. We studied these structural changes in the thylakoid membranes of Arabidopsis thaliana chloroplasts using atomic force microscopy, scanning and transmission electron microscopy, and confocal imaging. Based on our results and on the recently determined three-dimensional structure of higher-plant thylakoids trapped in one of the two major light-adapted states, we propose a model for the transitions in membrane architecture. The model suggests that reorganization of the membranes involves fission and fusion events that occur at the interface between the appressed (granal) and nonappressed (stroma lamellar) domains of the thylakoid membranes. Vertical and lateral displacements of the grana layers presumably follow these localized events, eventually leading to macroscopic rearrangements of the entire membrane network.