RESUMEN
BACKGROUND: Liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) is widely used for quantitative proteomic investigations. The typical output of such studies is a list of identified and quantified peptides. The biological and clinical interest is, however, usually focused on quantitative conclusions at the protein level. Furthermore, many investigations ask complex biological questions by studying multiple interrelated experimental conditions. Therefore, there is a need in the field for generic statistical models to quantify protein levels even in complex study designs. RESULTS: We propose a general statistical modeling approach for protein quantification in arbitrary complex experimental designs, such as time course studies, or those involving multiple experimental factors. The approach summarizes the quantitative experimental information from all the features and all the conditions that pertain to a protein. It enables both protein significance analysis between conditions, and protein quantification in individual samples or conditions. We implement the approach in an open-source R-based software package MSstats suitable for researchers with a limited statistics and programming background. CONCLUSIONS: We demonstrate, using as examples two experimental investigations with complex designs, that a simultaneous statistical modeling of all the relevant features and conditions yields a higher sensitivity of protein significance analysis and a higher accuracy of protein quantification as compared to commonly employed alternatives. The software is available at http://www.stat.purdue.edu/~ovitek/Software.html.
Asunto(s)
Cromatografía Liquida/estadística & datos numéricos , Modelos Estadísticos , Proteínas/análisis , Espectrometría de Masas en Tándem/estadística & datos numéricos , Péptidos/análisis , Proteómica/estadística & datos numéricos , Programas InformáticosRESUMEN
The SIR2 homologues HST3 and HST4 have been implicated in maintenance of genome integrity in the yeast Saccharomyces cerevisiae. We find that Hst3 has NAD-dependent histone deacetylase activity in vitro and that it functions during S phase to deacetylate the core domain of histone H3 at lysine 56 (H3K56). In response to genotoxic stress, Hst3 undergoes rapid Mec1-dependent phosphorylation and is targeted for ubiquitin-mediated proteolysis, thus providing a mechanism for the previously observed checkpoint-dependent accumulation of Ac-H3K56 at sites of DNA damage. Loss of Hst3-mediated regulation of H3K56 acetylation results in a defect in the S phase DNA damage checkpoint. The pathway that regulates H3K56 acetylation acts in parallel with the Rad9 pathway to transmit a DNA damage signal from Mec1 to Rad53. We also observe that loss of Hst3 function impairs sister chromatid cohesion (SCC). Both S phase checkpoint and SCC defects are phenocopied by H3K56 point mutants. Our findings demonstrate that Hst3-regulated H3K56 acetylation safeguards genome stability by controlling the S phase DNA damage response and promoting SCC.
Asunto(s)
Cromátides/genética , Regulación Enzimológica de la Expresión Génica , Regulación Fúngica de la Expresión Génica , Histona Desacetilasas/fisiología , Histonas/metabolismo , Lisina/química , Proteínas de Saccharomyces cerevisiae/fisiología , Ciclo Celular , Proteínas de Ciclo Celular/metabolismo , Daño del ADN , Histona Desacetilasas/biosíntesis , Histona Desacetilasas/metabolismo , Péptidos y Proteínas de Señalización Intracelular , Modelos Biológicos , Fenotipo , Fosforilación , Proteínas Serina-Treonina Quinasas , Proteínas de Saccharomyces cerevisiae/biosíntesis , Proteínas de Saccharomyces cerevisiae/metabolismo , Ubiquitina/metabolismoRESUMEN
The ATP binding cassette (ABC) transporters are important in human health and disease and represent the largest family of transmembrane proteins; however, their highly hydrophobic nature complicates the use of standard biochemical approaches to identify interacting proteins. Here, we report the development of a modified version of the split-ubiquitin membrane yeast two-hybrid (MYTH) technology using genomically integrated "bait" constructs, hence the designation iMYTH. We used iMYTH in a library-screening format and identified six potential interacting partners of the yeast ABC transporter Ycf1p. Strains deleted for several of these genes result in arsenite sensitivity similar to a Deltaycf1 strain. Transport assays show that one of these, Tus1p, a guanine nucleotide exchange factor (GEF) for the small GTPase Rho1p, is a Rho1p-dependent-positive regulator of Ycf1p. Our study provides proof of principle that iMYTH is an ideal methodology to identify physiological interactors and regulators of ABC transporters and other yeast transmembrane proteins.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/metabolismo , Membrana Celular/metabolismo , Regulación Fúngica de la Expresión Génica , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Técnicas del Sistema de Dos Híbridos , Ubiquitina/metabolismo , Transportadoras de Casetes de Unión a ATP/genética , Transporte Biológico , Citosol/metabolismo , Factores de Intercambio de Guanina Nucleótido/metabolismo , Factores de Intercambio de Guanina Nucleótido/fisiología , Metales Pesados/farmacología , Modelos Biológicos , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/fisiología , Eliminación de Secuencia , Proteínas de Unión al GTP rho/metabolismoRESUMEN
Various modifications of the conventional yeast two-hybrid system have played an essential role in confirming or detecting protein-protein interactions among nuclear and cytoplasmic proteins. These approaches have permitted the identification of novel interaction partners, as well as provided hints as to their function. However, membrane proteins, such as receptor tyrosine kinases, G protein-coupled receptors, membrane-bound phosphatases, and transporters, which represent important classes of signaling molecules, are difficult to study using classical protein interaction assays because of their hydrophobic nature. Here, we describe a genetic system that allows the identification of integral membrane-interacting proteins. This so-called "split-ubiquitin membrane-based yeast two-hybrid assay" involves fusing the halves of ubiquitin to two interacting proteins, at least one of which is membrane bound. Upon interaction of these two proteins, the halves of ubiquitin are brought together, and the transcription factor that is fused to a membrane protein of interest is cleaved and released. The free transcription factor then enters the nucleus and activates transcription of reporter genes. We also describe how this technology is used to screen complementary DNA libraries to identify novel binding partners of a membrane protein of interest.
Asunto(s)
Biblioteca de Genes , Proteínas de la Membrana/química , Mapeo de Interacción de Proteínas/métodos , Ubiquitina/química , Sustitución de Aminoácidos , Animales , Proteínas Bacterianas/química , Clonación Molecular/métodos , Colorimetría/métodos , ADN Complementario/genética , Endopeptidasas/metabolismo , Escherichia coli , Proteína Vmw65 de Virus del Herpes Simple/química , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Indicadores y Reactivos , Isoleucina/química , Proteínas de la Membrana/genética , Proteínas de la Membrana/aislamiento & purificación , Mutagénesis Sitio-Dirigida , Plásmidos , Reacción en Cadena de la Polimerasa , Unión Proteica , Mapeo de Interacción de Proteínas/instrumentación , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/aislamiento & purificación , Saccharomyces cerevisiae , Selección Genética , Serina Endopeptidasas/química , Proteasas Ubiquitina-EspecíficasRESUMEN
Protein-protein interactions are essential in almost all biological processes, extending from the formation of cellular macromolecular structures and enzymatic complexes to the regulation of signal transduction pathways. It is assumed that approximately one-third of all proteins in eukaryotic cells are membrane associated. Because of their hydrophobic nature, the analysis of membrane-protein interactions is difficult to be studied in a conventional two-hybrid assay. We described here a new genetic method for in vivo detection of membrane-protein interactions in the budding yeast Saccharomyces cerevisiae. The system uses the split-ubiquitin approach based on the detection of the in vivo processing of a reconstituted split ubiquitin. On interaction of X and Y proteins, ubiquitin reconstitution occurs and leads to the proteolytic cleavage and subsequent release of a transcription factor that triggers the activation of a reporter system enabling easy detection. In this manner, and in contrast to the conventional yeast-two hybrid system in which interactions occur in the nucleus, the membrane-based yeast two-hybrid system represents an in vivo system that detects interactions between membrane proteins in their natural environment.
Asunto(s)
Proteínas de la Membrana/metabolismo , Mapeo de Interacción de Proteínas/métodos , Saccharomyces cerevisiae/genética , Técnicas del Sistema de Dos Híbridos , Ubiquitina/metabolismo , Secuencia de Aminoácidos , Animales , Humanos , Proteínas de la Membrana/genética , Datos de Secuencia Molecular , Unión ProteicaRESUMEN
Analysis of membrane protein interactions is difficult because of the hydrophobic nature of these proteins, which often renders conventional biochemical and genetic assays fruitless. This is a substantial problem because proteins that are integral or associated with membranes represent approximately one-third of all proteins in a typical eukaryotic cell. We have shown previously that the modified split-ubiquitin system can be used as a genetic assay for the in vivo detection of interactions between the two characterized yeast transmembrane proteins, Ost1p and Wbp1p. This so-called split-ubiquitin membrane yeast two-hybrid (YTH) system uses the split-ubiquitin approach in which reconstitution of two ubiquitin halves is mediated by a protein-protein interaction. Here we converted the split-ubiquitin membrane YTH system into a generally applicable in vivo screening approach to identify interacting partners of a particular mammalian transmembrane protein. We have demonstrated the effectiveness of this approach by using the mammalian ErbB3 receptor as bait and have identified three previously unknown ErbB3-interacting proteins. In addition, we have confirmed one of the newly found interactions between ErbB3 and the membrane-associated RGS4 protein by coimmunoprecipitating the two proteins from human cells. We expect the split-ubiquitin membrane YTH technology to be valuable for the identification of potential interacting partners of integral membrane proteins from many model organisms.
Asunto(s)
Proteínas RGS/metabolismo , Receptor ErbB-3/metabolismo , Técnicas del Sistema de Dos Híbridos , Ubiquitina/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Encéfalo , Línea Celular , Clonación Molecular , ADN Complementario/genética , Biblioteca de Genes , Vectores Genéticos/biosíntesis , Vectores Genéticos/genética , Humanos , Riñón/química , Riñón/embriología , Riñón/metabolismo , Sustancias Macromoleculares , Proteínas de la Membrana/biosíntesis , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Datos de Secuencia Molecular , Mapeo Peptídico , Mapeo de Interacción de Proteínas , Proteínas RGS/química , Proteínas RGS/genética , Ratas , Receptor ErbB-3/biosíntesis , Receptor ErbB-3/química , Receptor ErbB-3/genética , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/metabolismo , Ubiquitina/biosíntesis , Ubiquitina/genéticaRESUMEN
Secretory proteins are translocated across the endoplasmic reticulum (ER) membrane through a channel formed by three proteins, namely Sec61p, Sbh1p, and Sss1p (Johnson, A. E., and van Waes, M. A. (1999) Annu. Rev. Cell Dev. Biol. 15, 799-842). Sec61p and Sss1p are essential for translocation (Esnault, Y., Blondel, M. O., Deshaies, R. J., Schekman, R., and Kepes, F. (1993) EMBO J. 12, 4083-4093). Sec61p is a polytopic membrane protein that lines the protein translocation channel. The role of Sss1p is unknown. During import into the ER through the Sec61p channel, many proteins are N-glycosylated before translocation is completed. In addition, both the Sec61 channel and oligosaccharyl transferase (OST) copurify with ribosomes from rough ER, suggesting that OST is located in close proximity to the Sec61 channel (Gorlich, D., Prehn, S., Hartmann, E., Kalies, K.-U., and Rapoport, T. A. (1992) Cell 71, 489-503 and Wang, L., and Dobberstein, B. (1999) FEBS Lett. 457, 316-322). Here, we demonstrate a direct interaction between Sss1p and a subunit of OST, Wbp1p, using the split-ubiquitin system and co-immunoprecipitation. We generated mutants in the cytoplasmic domain of Sss1p that disturb the interaction with OST and are viable but display a translocation defect specific for proteins with glycosylation acceptor sites. Our data suggest that Sss1p coordinates translocation across the ER membrane and N-linked glycosylation of secretory proteins.
Asunto(s)
Retículo Endoplásmico/metabolismo , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Proteínas Fúngicas/fisiología , Glicoproteínas/metabolismo , Hexosiltransferasas , Proteínas de la Membrana/química , Proteínas de la Membrana/fisiología , Proteínas de Saccharomyces cerevisiae , Secuencia de Aminoácidos , Glicosilación , Proteínas de Transporte de Membrana , Datos de Secuencia Molecular , Transporte de Proteínas , Canales de Translocación SEC , Relación Estructura-Actividad , Transferasas/fisiologíaRESUMEN
The availability of completed genome sequences of several eukaryotic and prokaryotic species has shifted the focus towards the identification and characterization of all gene products that are expressed in a given organism. In order to cope with the huge amounts of data that have been provided by large-scale sequencing projects, high-throughout methodologies also need to be applied in the emerging field of proteomics. In this review, we discuss methods that have been recently developed in order to characterize protein interactions and their functional relevance on a large scale. We then focus on those methodologies that are suitable for the identification and characterization of protein-protein interactions, namely the yeast two-hybrid system and related methods. Several recent studies have demonstrated the power of automated approaches involving the yeast two-hybrid system in building so-called "interaction networks", which hold the promise of identifying the entirety of all interactions that take place between proteins expressed in a certain cell type or organism. We compare the yeast two-hybrid system with several other screening methods that have been developed to investigate interactions between proteins that are not amenable to conventional yeast two-hybrid screenings, such as transcriptional activators and integral membrane proteins. The eventual adaptation of such methods to a high-throughput format and their use in combination with automated yeast two-hybrid screenings will help in elucidating protein-protein interactions on a scale that would have been unthinkable just a few years ago.
Asunto(s)
Genoma Fúngico , Proteínas/química , Proteoma/química , Levaduras/genética , Levaduras/metabolismo , Modelos Biológicos , Activación Transcripcional , Técnicas del Sistema de Dos HíbridosRESUMEN
Interaction of two separately expressed halves of sucrose transporter SUT1 was detected by an optimized split-ubiquitin system. The halves reconstitute sucrose transport activity at the plasma membrane with affinities similar to the intact protein. The halves do not function independently, and an intact central loop is not required for membrane insertion, plasma membrane targeting, and transport. Under native conditions, the halves associate into higher molecular mass complexes. Furthermore, the N-terminal half of the low-affinity SUT2 interacts functionally with the C-terminal half of SUT1. Since the N terminus of SUT2 determines affinity for sucrose, the reconstituted chimera has lower affinity than SUT1. The split-ubiquitin system efficiently detects intramolecular interactions in membrane proteins, and can be used to dissect transporter structure.