RESUMEN
The study of conserved protein interaction networks seeks to better understand the evolution and regulation of protein interactions. Here, we present a quantitative proteomic analysis of 18 orthologous baits from three distinct chromatin-remodeling complexes in Saccharomyces cerevisiae and Homo sapiens. We demonstrate that abundance levels of orthologous proteins correlate strongly between the two organisms and both networks have highly similar topologies. We therefore used the protein abundances in one species to cross-predict missing protein abundance levels in the other species. Lastly, we identified a novel conserved low-abundance subnetwork further demonstrating the value of quantitative analysis of networks.
Asunto(s)
Ensamble y Desensamble de Cromatina/fisiología , Mapas de Interacción de Proteínas , Proteínas de Saccharomyces cerevisiae/metabolismo , ATPasas Asociadas con Actividades Celulares Diversas , Adenosina Trifosfatasas/metabolismo , ADN Helicasas/metabolismo , Proteínas de Unión al ADN , Histona Acetiltransferasas/metabolismo , Humanos , Lisina Acetiltransferasa 5 , Mapeo de Interacción de Proteínas/métodos , Proteómica/métodos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismoRESUMEN
Knowledge of structure and dynamics of proteins and protein complexes is important to unveil the molecular basis and mechanisms involved in most biological processes. Protein complex dynamics can be defined as the changes in the composition of a protein complex during a cellular process. Protein dynamics can be defined as conformational changes in a protein during enzyme activation, for example, when a protein binds to a ligand or when a protein binds to another protein. Mass spectrometry (MS) combined with affinity purification has become the analytical tool of choice for mapping protein-protein interaction networks and the recent developments in the quantitative proteomics field has made it possible to identify dynamically interacting proteins. Furthermore, hydrogen/deuterium exchange MS is emerging as a powerful technique to study structure and conformational dynamics of proteins or protein assemblies in solution. Methods have been developed and applied for the identification of transient and/or weak dynamic interaction partners and for the analysis of conformational dynamics of proteins or protein complexes. This review is an overview of existing and recent developments in studying the overall dynamics of in vivo protein interaction networks and protein complexes using MS-based methods.
Asunto(s)
Cromatografía de Afinidad/métodos , Medición de Intercambio de Deuterio/métodos , Espectrometría de Masas/métodos , Complejos Multiproteicos/análisis , Complejos Multiproteicos/aislamiento & purificación , Mapeo de Interacción de Proteínas/métodos , Proteínas/química , Proteínas/aislamiento & purificación , Deuterio , Hidrógeno , Ligandos , Estructura Molecular , Complejos Multiproteicos/química , Unión Proteica , Conformación Proteica , Proteómica/métodosRESUMEN
The C-terminal domain (CTD) of the largest subunit of RNA polymerase II (Pol II) serves an important role in coordinating stage-specific recruitment and release of cellular machines during transcription. Dynamic placement and removal of phosphorylation marks on different residues of a repeating heptapeptide (YSPTSPS) of the CTD underlies the engagement of relevant cellular machinery. Whereas sequential placement of phosphorylation marks is well explored, genome-wide engagement of phosphatases that remove these CTD marks is poorly understood. In particular, identifying the enzyme that erases phospho-Ser7 (Ser7-P) marks is especially important, because we find that substituting this residue with a glutamate, a phospho-mimic, is lethal. Our observations implicate Ssu72 as a Ser7-P phosphatase. We report that removal of all phospho-CTD marks during transcription termination is mechanistically coupled. An inability to remove these marks prevents Pol II from terminating efficiently and will likely impede subsequent assembly into the pre-initiation complex.
Asunto(s)
Fosfoproteínas Fosfatasas/metabolismo , ARN Polimerasa II/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Factores de Escisión y Poliadenilación de ARNm/metabolismo , Secuencias de Aminoácidos , Sustitución de Aminoácidos , Mutación Missense , Fosfoproteínas Fosfatasas/genética , Estructura Terciaria de Proteína , ARN Polimerasa II/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Serina/genética , Serina/metabolismo , Transcripción Genética , Factores de Escisión y Poliadenilación de ARNm/genéticaRESUMEN
Efficient determination of protein interactions and cellular localization remains a challenge in higher order eukaryotes and creates a need for robust technologies for functional proteomics studies. To address this, the HaloTag technology was developed for highly efficient and rapid isolation of intracellular complexes and correlative in vivo cellular imaging. Here we demonstrate the strength of this technology by simultaneous capture of human eukaryotic RNA polymerases (RNAP) I, II, and III using a shared subunit, POLR2H, fused to the HaloTag. Affinity purifications showed successful isolation, as determined using quantitative proteomics, of all RNAP core subunits, even at expression levels near endogenous. Transient known RNAP II interacting partners were identified as well as three previously uncharacterized interactors. These interactions were validated and further functionally characterized using cellular imaging. The multiple capabilities of the HaloTag technology demonstrate the ability to efficiently isolate highly challenging multiprotein complexes, discover new interactions, and characterize cellular localization.