RESUMEN
Cohesin is a four-subunit ATPase in the family of structural maintenance of chromosomes (SMC). Cohesin promotes sister chromatid cohesion, chromosome condensation, DNA repair, and transcription regulation. Cohesin performs these functions as a DNA tether and potentially a DNA-based motor. At least one of its DNA binding activities involves entrapment of DNA within a lumen formed by its subunits. This activity can be reconstituted in vitro by incubating cohesin with DNA, ATP, and cohesin loader. Previously we showed that a mutant form of cohesin (DE-cohesin) possesses the ability to bind and tether DNA in vivo. Using in vitro reconstitution assays, we show that DE-cohesin can form stable complexes with DNA without ATP hydrolysis. We show that wild-type cohesin with ADP aluminum fluoride (cohesinADP/AlFx) can also form stable cohesin-DNA complexes. These results suggest that an intermediate nucleotide state of cohesin, likely cohesinADP-Pi, is capable of initially dissociating one interface between cohesin subunits to allow DNA entry into a cohesin lumen and subsequently interacting with the bound DNA to stabilize DNA entrapment. We also show that cohesinADP/AlFx binding to DNA is enhanced by cohesin loader, suggesting a function for loader other than stimulating the ATPase. Finally, we show that loader remains stably bound to cohesinADP/AlFx after DNA entrapment, potentially revealing a function for loader in tethering the second DNA substrate. These results provide important clues on how SMC complexes like cohesin can function as both DNA tethers and motors.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , ADN/metabolismo , Transportadoras de Casetes de Unión a ATP/metabolismo , Reparación del ADN , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Translocación Genética , CohesinasRESUMEN
Anhydrobiotes are rare microbes, plants and animals that tolerate severe water loss. Understanding the molecular basis for their desiccation tolerance may provide novel insights into stress biology and critical tools for engineering drought-tolerant crops. Using the anhydrobiote, budding yeast, we show that trehalose and Hsp12, a small intrinsically disordered protein (sIDP) of the hydrophilin family, synergize to mitigate completely the inviability caused by the lethal stresses of desiccation. We show that these two molecules help to stabilize the activity and prevent aggregation of model proteins both in vivo and in vitro. We also identify a novel in vitro role for Hsp12 as a membrane remodeler, a protective feature not shared by another yeast hydrophilin, suggesting that sIDPs have distinct biological functions.
Asunto(s)
Deshidratación , Proteínas de Choque Térmico/metabolismo , Proteínas Intrínsecamente Desordenadas/metabolismo , Viabilidad Microbiana , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/fisiología , Estrés Fisiológico , Trehalosa/metabolismo , Membrana Celular/metabolismo , Agregación Patológica de Proteínas/prevención & controlRESUMEN
Cohesin is essential for the hierarchical organization of the eukaryotic genome and plays key roles in many aspects of chromosome biology. The conformation of cohesin bound to DNA remains poorly defined, leaving crucial gaps in our understanding of how cohesin fulfills its biological functions. Here, we use single-molecule microscopy to directly observe the dynamic and functional characteristics of cohesin bound to DNA. We show that cohesin can undergo rapid one-dimensional (1D) diffusion along DNA, but individual nucleosomes, nucleosome arrays, and other protein obstacles significantly restrict its mobility. Furthermore, we demonstrate that DNA motor proteins can readily push cohesin along DNA, but they cannot pass through the interior of the cohesin ring. Together, our results reveal that DNA-bound cohesin has a central pore that is substantially smaller than anticipated. These findings have direct implications for understanding how cohesin and other SMC proteins interact with and distribute along chromatin.
Asunto(s)
Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Proteínas Cromosómicas no Histona/química , Proteínas Cromosómicas no Histona/metabolismo , ADN/metabolismo , Proteínas de Schizosaccharomyces pombe/química , Proteínas de Schizosaccharomyces pombe/metabolismo , Imagen Individual de Molécula/métodos , Secuencia Rica en At/genética , Secuencia de Bases , Difusión , Modelos Biológicos , Proteínas Motoras Moleculares/metabolismo , Nucleosomas/metabolismo , Porosidad , Unión Proteica , Conformación Proteica , Transporte de Proteínas , Schizosaccharomyces/metabolismo , CohesinasRESUMEN
Cohesin tethers together regions of DNA, thereby mediating higher order chromatin organization that is critical for sister chromatid cohesion, DNA repair and transcriptional regulation. Cohesin contains a heterodimeric ATP-binding Cassette (ABC) ATPase comprised of Smc1 and Smc3 ATPase active sites. These ATPases are required for cohesin to bind DNA. Cohesin's DNA binding activity is also promoted by the Eco1 acetyltransferase and inhibited by Wpl1. Recently we showed that after cohesin stably binds DNA, a second step is required for DNA tethering. This second step is also controlled by Eco1 acetylation. Here, we use genetic and biochemical analyses to show that this second DNA tethering step is regulated by cohesin ATPase. Furthermore, our results also suggest that Eco1 promotes cohesion by modulating the ATPase cycle of DNA-bound cohesin in a state that is permissive for DNA tethering and refractory to Wpl1 inhibition.
Asunto(s)
Acetiltransferasas/metabolismo , Adenosina Trifosfatasas/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , ADN/metabolismo , Proteínas Nucleares/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Ciclo Celular/genética , Cromatina/metabolismo , Proteínas Cromosómicas no Histona/genética , Proteínas de Saccharomyces cerevisiae/genética , CohesinasRESUMEN
Dynamin mediates various membrane fission events, including the scission of clathrin-coated vesicles. Here, we provide direct evidence for cooperative membrane recruitment of dynamin with the BIN/amphiphysin/Rvs (BAR) proteins, endophilin and amphiphysin. Surprisingly, endophilin and amphiphysin recruitment to membranes was also dependent on binding to dynamin due to auto-inhibition of BAR-membrane interactions. Consistent with reciprocal recruitment in vitro, dynamin recruitment to the plasma membrane in cells was strongly reduced by concomitant depletion of endophilin and amphiphysin, and conversely, depletion of dynamin dramatically reduced the recruitment of endophilin. In addition, amphiphysin depletion was observed to severely inhibit clathrin-mediated endocytosis. Furthermore, GTP-dependent membrane scission by dynamin was dramatically elevated by BAR domain proteins. Thus, BAR domain proteins and dynamin act in synergy in membrane recruitment and GTP-dependent vesicle scission.
Asunto(s)
Membrana Celular/metabolismo , Dinaminas/metabolismo , Guanosina Trifosfato/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Vesículas Secretoras/metabolismo , Línea Celular , Membrana Celular/genética , Dinaminas/genética , Guanosina Trifosfato/genética , Humanos , Proteínas del Tejido Nervioso/genética , Vesículas Secretoras/genéticaRESUMEN
Shallow hydrophobic insertions and crescent-shaped BAR scaffolds promote membrane curvature. Here, we investigate membrane fission by shallow hydrophobic insertions quantitatively and mechanistically. We provide evidence that membrane insertion of the ENTH domain of epsin leads to liposome vesiculation, and that epsin is required for clathrin-coated vesicle budding in cells. We also show that BAR-domain scaffolds from endophilin, amphiphysin, GRAF, and ß2-centaurin limit membrane fission driven by hydrophobic insertions. A quantitative assay for vesiculation reveals an antagonistic relationship between amphipathic helices and scaffolds of N-BAR domains in fission. The extent of vesiculation by these proteins and vesicle size depend on the number and length of amphipathic helices per BAR domain, in accord with theoretical considerations. This fission mechanism gives a new framework for understanding membrane scission in the absence of mechanoenzymes such as dynamin and suggests how Arf and Sar proteins work in vesicle scission.
Asunto(s)
Membranas Intracelulares/química , Membranas Intracelulares/metabolismo , Proteínas Adaptadoras del Transporte Vesicular/química , Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Animales , Línea Celular , Membrana Celular/química , Membrana Celular/metabolismo , Células HeLa , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Liposomas/química , Liposomas/metabolismo , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Modelos Moleculares , Estructura Terciaria de ProteínaRESUMEN
Compensatory endocytosis of exocytosed membrane and recycling of synaptic vesicle components is essential for sustained synaptic transmission at nerve terminals. At the ribbon-type synapse of retinal bipolar cells, manipulations expected to inhibit the interactions of the clathrin adaptor protein complex (AP2) affect only the slow phase of endocytosis (τ = 10-15 s), leading to the conclusion that fast endocytosis (τ = 1-2 s) occurs by a mechanism that differs from the classical pathway of clathrin-coated vesicle retrieval from the plasma membrane. Here we investigate the role of endophilin in endocytosis at this ribbon synapse. Endophilin A1 is a synaptically enriched N-BAR domain-containing protein, suggested to function in clathrin-mediated endocytosis. Internal dialysis of the synaptic terminal with dominant-negative endophilin A1 lacking its linker and Src homology 3 (SH3) domain inhibited the fast mode of endocytosis, while slow endocytosis continued. Dialysis of a peptide that binds endophilin SH3 domain also decreased fast retrieval. Electron microscopy indicated that fast endocytosis occurred by retrieval of small vesicles in most instances. These results indicate that endophilin is involved in fast retrieval of synaptic vesicles occurring by a mechanism that can be distinguished from the classical pathway involving clathrin-AP2 interactions.