RESUMEN
Macromolecular assemblies involving membrane proteins (MPs) serve vital biological roles and are prime drug targets in a variety of diseases. Large-scale affinity purification studies of soluble-protein complexes have been accomplished for diverse model organisms, but no global characterization of MP-complex membership has been described so far. Here we report a complete survey of 1,590 putative integral, peripheral and lipid-anchored MPs from Saccharomyces cerevisiae, which were affinity purified in the presence of non-denaturing detergents. The identities of the co-purifying proteins were determined by tandem mass spectrometry and subsequently used to derive a high-confidence physical interaction map encompassing 1,726 membrane protein-protein interactions and 501 putative heteromeric complexes associated with the various cellular membrane systems. Our analysis reveals unexpected physical associations underlying the membrane biology of eukaryotes and delineates the global topological landscape of the membrane interactome.
Asunto(s)
Proteínas de la Membrana/metabolismo , Mapas de Interacción de Proteínas , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Membrana Celular/química , Membrana Celular/metabolismo , Quitina Sintasa/metabolismo , Detergentes , Retículo Endoplásmico/metabolismo , Aparato de Golgi/metabolismo , Espectrometría de Masas , Proteínas de la Membrana/análisis , Proteínas de la Membrana/química , Unión Proteica , Mapeo de Interacción de Proteínas , Proteoma/análisis , Proteoma/química , Proteoma/metabolismo , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/citología , Proteínas de Saccharomyces cerevisiae/análisis , Proteínas de Saccharomyces cerevisiae/químicaRESUMEN
Protein complexes and protein-protein interactions are essential for almost all cellular processes. Here, we establish a mammalian affinity purification and lentiviral expression (MAPLE) system for characterizing the subunit compositions of protein complexes. The system is flexible (i.e. multiple N- and C-terminal tags and multiple promoters), is compatible with Gateway cloning, and incorporates a reference peptide. Its major advantage is that it permits efficient and stable delivery of affinity-tagged open reading frames into most mammalian cell types. We benchmarked MAPLE with a number of human protein complexes involved in transcription, including the RNA polymerase II-associated factor, negative elongation factor, positive transcription elongation factor b, SWI/SNF, and mixed lineage leukemia complexes. In addition, MAPLE was used to identify an interaction between the reprogramming factor Klf4 and the Swi/Snf chromatin remodeling complex in mouse embryonic stem cells. We show that the SWI/SNF catalytic subunit Smarca2/Brm is up-regulated during the process of induced pluripotency and demonstrate a role for the catalytic subunits of the SWI/SNF complex during somatic cell reprogramming. Our data suggest that the transcription factor Klf4 facilitates chromatin remodeling during reprogramming.
Asunto(s)
Proteínas Cromosómicas no Histona/metabolismo , Lentivirus/metabolismo , Células Madre Pluripotentes/metabolismo , Proteómica/métodos , Factores de Transcripción/metabolismo , Secuencia de Aminoácidos , Animales , Línea Celular , Reprogramación Celular/genética , Cromatografía de Afinidad , Humanos , Factor 4 Similar a Kruppel , Factores de Transcripción de Tipo Kruppel/metabolismo , Ratones , Datos de Secuencia Molecular , Complejos Multiproteicos/metabolismo , Células Madre Pluripotentes/citología , Unión Proteica , Transcripción GenéticaRESUMEN
Identification of protein-protein interactions often provides insight into protein function, and many cellular processes are performed by stable protein complexes. We used tandem affinity purification to process 4,562 different tagged proteins of the yeast Saccharomyces cerevisiae. Each preparation was analysed by both matrix-assisted laser desorption/ionization-time of flight mass spectrometry and liquid chromatography tandem mass spectrometry to increase coverage and accuracy. Machine learning was used to integrate the mass spectrometry scores and assign probabilities to the protein-protein interactions. Among 4,087 different proteins identified with high confidence by mass spectrometry from 2,357 successful purifications, our core data set (median precision of 0.69) comprises 7,123 protein-protein interactions involving 2,708 proteins. A Markov clustering algorithm organized these interactions into 547 protein complexes averaging 4.9 subunits per complex, about half of them absent from the MIPS database, as well as 429 additional interactions between pairs of complexes. The data (all of which are available online) will help future studies on individual proteins as well as functional genomics and systems biology.
Asunto(s)
Proteoma/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Evolución Biológica , Secuencia Conservada , Espectrometría de Masas , Complejos Multiproteicos/química , Complejos Multiproteicos/metabolismo , Unión Proteica , Proteoma/química , Proteómica , Proteínas de Saccharomyces cerevisiae/químicaRESUMEN
The yeast histone deacetylase Rpd3 can be recruited to promoters to repress transcription initiation. Biochemical, genetic, and gene-expression analyses show that Rpd3 exists in two distinct complexes. The smaller complex, Rpd3C(S), shares Sin3 and Ume1 with Rpd3C(L) but contains the unique subunits Rco1 and Eaf3. Rpd3C(S) mutants exhibit phenotypes remarkably similar to those of Set2, a histone methyltransferase associated with elongating RNA polymerase II. Chromatin immunoprecipitation and biochemical experiments indicate that the chromodomain of Eaf3 recruits Rpd3C(S) to nucleosomes methylated by Set2 on histone H3 lysine 36, leading to deacetylation of transcribed regions. This pathway apparently acts to negatively regulate transcription because deleting the genes for Set2 or Rpd3C(S) bypasses the requirement for the positive elongation factor Bur1/Bur2.
Asunto(s)
Histona Desacetilasas/metabolismo , Histonas/metabolismo , Lisina/metabolismo , Metiltransferasas/metabolismo , Proteínas Represoras/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Factores de Transcripción/metabolismo , Acetilación , Acetiltransferasas/genética , Acetiltransferasas/metabolismo , Secuencia de Aminoácidos , Análisis por Conglomerados , Quinasas Ciclina-Dependientes/genética , Regulación hacia Abajo/genética , Epistasis Genética , Eliminación de Gen , Regulación Fúngica de la Expresión Génica , Histona Desacetilasas/genética , Metilación , Metiltransferasas/genética , Datos de Secuencia Molecular , Mutación/genética , Análisis de Secuencia por Matrices de Oligonucleótidos , Unión Proteica , ARN Polimerasa II/metabolismo , Proteínas Represoras/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Homología de Secuencia de Aminoácido , Proteínas Reguladoras de Información Silente de Saccharomyces cerevisiae/genética , Proteínas Reguladoras de Información Silente de Saccharomyces cerevisiae/metabolismo , Sirtuina 2 , Sirtuinas/genética , Sirtuinas/metabolismo , Supresión Genética , Factores de Transcripción/genéticaRESUMEN
We present a strategy for generating and analyzing comprehensive genetic-interaction maps, termed E-MAPs (epistatic miniarray profiles), comprising quantitative measures of aggravating or alleviating interactions between gene pairs. Crucial to the interpretation of E-MAPs is their high-density nature made possible by focusing on logically connected gene subsets and including essential genes. Described here is the analysis of an E-MAP of genes acting in the yeast early secretory pathway. Hierarchical clustering, together with novel analytical strategies and experimental verification, revealed or clarified the role of many proteins involved in extensively studied processes such as sphingolipid metabolism and retention of HDEL proteins. At a broader level, analysis of the E-MAP delineated pathway organization and components of physical complexes and illustrated the interconnection between the various secretory processes. Extension of this strategy to other logically connected gene subsets in yeast and higher eukaryotes should provide critical insights into the functional/organizational principles of biological systems.