RESUMEN
Fueled by ATP hydrolysis in N-ethylmaleimide sensitive factor (NSF), the 20S complex disassembles rigid SNARE (soluble NSF attachment protein receptor) complexes in single unraveling step. This global disassembly distinguishes NSF from other molecular motors that make incremental and processive motions, but the molecular underpinnings of its remarkable energy efficiency remain largely unknown. Using multiple single-molecule methods, we found remarkable cooperativity in mechanical connection between NSF and the SNARE complex, which prevents dysfunctional 20S complexes that consume ATP without productive disassembly. We also constructed ATP hydrolysis cycle of the 20S complex, in which NSF largely shows randomness in ATP binding but switches to perfect ATP hydrolysis synchronization to induce global SNARE disassembly, minimizing ATP hydrolysis by non-20S complex-forming NSF molecules. These two mechanisms work in concert to concentrate ATP consumption into functional 20S complexes, suggesting evolutionary adaptations by the 20S complex to the energetically expensive mechanical task of SNARE complex disassembly.
Asunto(s)
Adenosina Trifosfato/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteínas SNARE/metabolismo , Animales , Bovinos , Cricetulus , Hidrólisis , Modelos Moleculares , Proteínas Sensibles a N-Etilmaleimida/aislamiento & purificación , Proteínas Sensibles a N-Etilmaleimida/metabolismo , Complejo de la Endopetidasa Proteasomal/genética , Complejo de la Endopetidasa Proteasomal/aislamiento & purificación , Unión Proteica , Multimerización de Proteína , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Proteínas SNARE/genética , Proteínas SNARE/aislamiento & purificación , Imagen Individual de Molécula , Proteínas Solubles de Unión al Factor Sensible a la N-Etilmaleimida/genética , Proteínas Solubles de Unión al Factor Sensible a la N-Etilmaleimida/aislamiento & purificación , Proteínas Solubles de Unión al Factor Sensible a la N-Etilmaleimida/metabolismoRESUMEN
Endothelial exocytosis of granules is a rapid response to vascular injury. However, the molecular machinery that regulates exocytosis in endothelial cells is not well understood. Recently developed techniques have defined the endothelial proteins that control vesicle and granule trafficking in endothelial cells. These techniques have revealed that syntaxin 4, synaptobrevin 3, and N-ethylmaleimide-sensitive factor (NSF) play a critical role in endothelial granule exocytosis. Additional studies have shown that nitric oxide regulates exocytosis by chemically modifying NSF. Further characterization of the factors that regulate exocytosis will lead to novel treatments for vascular diseases such as myocardial infarction and stroke.
