RESUMEN
The RNA exosome complex associates with nuclear and cytoplasmic cofactors to mediate the decay, surveillance, or processing of a wide variety of transcripts. In the cytoplasm, the conserved core of the exosome (Exo10) functions together with the conserved Ski complex. The interaction of S. cerevisiae Exo10 and Ski is not direct but requires a bridging cofactor, Ski7. Here, we report the 2.65 Å resolution structure of S. cerevisiae Exo10 bound to the interacting domain of Ski7. Extensive hydrophobic interactions rationalize the high affinity and stability of this complex, pointing to Ski7 as a constitutive component of the cytosolic exosome. Despite the absence of sequence homology, cytoplasmic Ski7 and nuclear Rrp6 bind Exo10 using similar surfaces and recognition motifs. Knowledge of the interacting residues in the yeast complexes allowed us to identify a splice variant of human HBS1-Like as a Ski7-like exosome-binding protein, revealing the evolutionary conservation of this cytoplasmic cofactor.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Complejo Multienzimático de Ribonucleasas del Exosoma/metabolismo , Proteínas de Unión al GTP/metabolismo , Proteínas HSP70 de Choque Térmico/metabolismo , Factores de Elongación de Péptidos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas Adaptadoras Transductoras de Señales/química , Proteínas Adaptadoras Transductoras de Señales/genética , Secuencia de Aminoácidos , Sitios de Unión , Secuencia Conservada , Evolución Molecular , Complejo Multienzimático de Ribonucleasas del Exosoma/química , Complejo Multienzimático de Ribonucleasas del Exosoma/genética , Proteínas de Unión al GTP/genética , Proteínas HSP70 de Choque Térmico/genética , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Mutación , Proteínas Nucleares/metabolismo , Factores de Elongación de Péptidos/genética , Unión Proteica , Conformación Proteica , Dominios y Motivos de Interacción de Proteínas , Isoformas de Proteínas , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Relación Estructura-ActividadRESUMEN
The eukaryotic exosome is a conserved RNA-degrading complex that functions in RNA surveillance, turnover and processing. How the same machinery can either completely degrade or precisely trim RNA substrates has long remained unexplained. Here we report the crystal structures of a yeast nuclear exosome containing the 9-subunit core, the 3'-5' RNases Rrp44 and Rrp6, and the obligate Rrp6-binding partner Rrp47 in complex with different RNAs. The combined structural and biochemical data of this 12-subunit complex reveal how a single-stranded RNA can reach the Rrp44 or Rrp6 active sites directly or can bind Rrp6 and be threaded via the central channel towards the distal RNase Rrp44. When a bulky RNA is stalled at the entrance of the channel, Rrp6-Rrp47 swings open. The results suggest how the same molecular machine can coordinate processive degradation and partial trimming in an RNA-dependent manner by a concerted swinging mechanism of the two RNase subunits.
Asunto(s)
Complejo Multienzimático de Ribonucleasas del Exosoma/química , Complejo Multienzimático de Ribonucleasas del Exosoma/metabolismo , Estabilidad del ARN , Saccharomyces cerevisiae/enzimología , Cristalografía por Rayos X , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Modelos Moleculares , Movimiento , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Conformación de Ácido Nucleico , Conformación Proteica , Subunidades de Proteína/química , Subunidades de Proteína/metabolismo , ARN/química , ARN/metabolismo , ARN Ribosómico 5.8S/química , ARN Ribosómico 5.8S/genética , ARN Ribosómico 5.8S/metabolismo , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/metabolismo , Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Relación Estructura-ActividadRESUMEN
How granzymes gain entry into the cytosol of target cells during killer cell attack has been the subject of several studies in the past, but the effective delivery mechanism during target cell encounter has not been clarified. Here we show that granzyme B (GzmB) mutants lacking binding to negatively charged, essentially heparan-sulfate-containing membrane receptors are poorly endocytosed yet are delivered to the cytosol with efficacy similar to that of WT GzmB. In a cell-based system GzmB-deficient natural killer cells provided perforin (pfn) by natural polarized secretion and synergized with externally added GzmB. Whereas receptor (heparan sulfate)-dependent endocytosis was dispensable, delivery of larger cargo like that of GzmB fusion proteins and GzmB-antibody complexes was restricted by their size. Our data support the model in which granzymes are primarily translocated through repairable membrane pores of finite size and not by the disruption of endocytosed vesicles. We conclude that structurally related translocators, i.e., perforin and cholesterol-dependent cytolysins, deliver deathly cargo across host cell membranes in a similar manner.