RESUMEN
The recycling of SNARE proteins following complex formation and membrane fusion is an essential process in eukaryotic trafficking. A highly conserved AAA+ protein, NSF (N-ethylmaleimide sensitive factor) and an adaptor protein, SNAP (soluble NSF attachment protein), disassemble the SNARE complex. We report electron-cryomicroscopy structures of the complex of NSF, αSNAP, and the full-length soluble neuronal SNARE complex (composed of syntaxin-1A, synaptobrevin-2, SNAP-25A) in the presence of ATP under non-hydrolyzing conditions at ~3.9 Å resolution. These structures reveal electrostatic interactions by which two αSNAP molecules interface with a specific surface of the SNARE complex. This interaction positions the SNAREs such that the 15 N-terminal residues of SNAP-25A are loaded into the D1 ring pore of NSF via a spiral pattern of interactions between a conserved tyrosine NSF residue and SNAP-25A backbone atoms. This loading process likely precedes ATP hydrolysis. Subsequent ATP hydrolysis then drives complete disassembly.
Asunto(s)
Proteínas Sensibles a N-Etilmaleimida/metabolismo , Proteínas Solubles de Unión al Factor Sensible a la N-Etilmaleimida/química , Proteínas Solubles de Unión al Factor Sensible a la N-Etilmaleimida/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Cricetulus , Cinética , Modelos Moleculares , Proteínas Sensibles a N-Etilmaleimida/química , Proteínas Sensibles a N-Etilmaleimida/ultraestructura , Conformación Proteica , Subunidades de Proteína/química , Subunidades de Proteína/metabolismo , Proteínas Solubles de Unión al Factor Sensible a la N-Etilmaleimida/ultraestructura , Especificidad por SustratoRESUMEN
SNARE complex disassembly by the ATPase NSF is essential for neurotransmitter release and other membrane trafficking processes. We developed a single-molecule FRET assay to monitor repeated rounds of NSF-mediated disassembly and reassembly of individual SNARE complexes. For ternary neuronal SNARE complexes, disassembly proceeds in a single step within 100 msec. We observed short- (<0.32 s) and long-lived (≥0.32 s) disassembled states. The long-lived states represent fully disassembled SNARE complex, while the short-lived states correspond to failed disassembly or immediate reassembly. Either high ionic strength or decreased αSNAP concentration reduces the disassembly rate while increasing the frequency of short-lived states. NSF is also capable of disassembling anti-parallel ternary SNARE complexes, implicating it in quality control. Finally, complexin-1 competes with αSNAP binding to the SNARE complex; addition of complexin-1 has an effect similar to that of decreasing the αSNAP concentration, possibly differentially regulating cis and trans SNARE complexes disassembly.
Asunto(s)
Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Proteínas Sensibles a N-Etilmaleimida/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Proteínas SNARE/metabolismo , Animales , Cricetulus , Transferencia Resonante de Energía de Fluorescencia , Cinética , Ratones , Proteínas Mutantes/metabolismo , Mutación/genética , Proteínas Sensibles a N-Etilmaleimida/ultraestructura , Concentración Osmolar , Unión Proteica , Dominios Proteicos , Proteínas Qa-SNARE/química , Proteínas Qa-SNARE/metabolismo , Ratas , Imagen Individual de Molécula , Proteínas Solubles de Unión al Factor Sensible a la N-Etilmaleimida/metabolismo , Proteínas Solubles de Unión al Factor Sensible a la N-Etilmaleimida/ultraestructuraRESUMEN
Evolutionarily conserved SNARE (soluble N-ethylmaleimide sensitive factor attachment protein receptors) proteins form a complex that drives membrane fusion in eukaryotes. The ATPase NSF (N-ethylmaleimide sensitive factor), together with SNAPs (soluble NSF attachment protein), disassembles the SNARE complex into its protein components, making individual SNAREs available for subsequent rounds of fusion. Here we report structures of ATP- and ADP-bound NSF, and the NSF/SNAP/SNARE (20S) supercomplex determined by single-particle electron cryomicroscopy at near-atomic to sub-nanometre resolution without imposing symmetry. Large, potentially force-generating, conformational differences exist between ATP- and ADP-bound NSF. The 20S supercomplex exhibits broken symmetry, transitioning from six-fold symmetry of the NSF ATPase domains to pseudo four-fold symmetry of the SNARE complex. SNAPs interact with the SNARE complex with an opposite structural twist, suggesting an unwinding mechanism. The interfaces between NSF, SNAPs, and SNAREs exhibit characteristic electrostatic patterns, suggesting how one NSF/SNAP species can act on many different SNARE complexes.
Asunto(s)
Complejos Multiproteicos/química , Complejos Multiproteicos/metabolismo , Proteínas SNARE/química , Proteínas SNARE/metabolismo , Adenosina Difosfato/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Cricetulus , Microscopía por Crioelectrón , Modelos Moleculares , Complejos Multiproteicos/ultraestructura , Proteínas Sensibles a N-Etilmaleimida/química , Proteínas Sensibles a N-Etilmaleimida/metabolismo , Proteínas Sensibles a N-Etilmaleimida/ultraestructura , Unión Proteica , Estructura Terciaria de Proteína , Ratas , Proteínas SNARE/ultraestructura , Proteínas Solubles de Unión al Factor Sensible a la N-Etilmaleimida/química , Proteínas Solubles de Unión al Factor Sensible a la N-Etilmaleimida/metabolismo , Proteínas Solubles de Unión al Factor Sensible a la N-Etilmaleimida/ultraestructuraRESUMEN
Despite the progress in understanding nuclear envelope (NE) reformation after mitosis, it has remained unclear what drives the required membrane fusion and how exactly this is coordinated with nuclear pore complex (NPC) assembly. Here, we show that, like other intracellular fusion reactions, NE fusion in Xenopus laevis egg extracts is mediated by SNARE proteins that require activation by NSF. Antibodies against Xenopus NSF, depletion of NSF or the dominant-negative NSF(E329Q) variant specifically inhibited NE formation. Staging experiments further revealed that NSF was required until sealing of the envelope was completed. Moreover, excess exogenous alpha-SNAP that blocks SNARE function prevented membrane fusion and caused accumulation of non-flattened vesicles on the chromatin surface. Under these conditions, the nucleoporins Nup107 and gp210 were fully recruited, whereas assembly of FxFG-repeat-containing nucleoporins was blocked. Together, we define NSF- and SNARE-mediated membrane fusion events as essential steps during NE formation downstream of Nup107 recruitment, and upstream of membrane flattening and completion of NPC assembly.
Asunto(s)
Fusión de Membrana , Membrana Nuclear/metabolismo , Poro Nuclear/metabolismo , Óvulo/metabolismo , Proteínas SNARE/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus laevis/metabolismo , Animales , Extractos Celulares , Proteínas de la Membrana/metabolismo , Proteínas Mutantes/metabolismo , Membrana Nuclear/ultraestructura , Proteínas Nucleares/metabolismo , Óvulo/citología , Óvulo/ultraestructura , Proteínas Solubles de Unión al Factor Sensible a la N-Etilmaleimida/ultraestructura , Proteína de Unión al GTP ran/metabolismoRESUMEN
The AAA+ATPase p97/VCP, helped by adaptor proteins, exerts its essential role in cellular events such as endoplasmic reticulum-associated protein degradation or the reassembly of Golgi, ER and the nuclear envelope after mitosis. Here, we report the three-dimensional cryo-electron microscopy structures at approximately 20 Angstroms resolution in two nucleotide states of the endogenous hexameric p97 in complex with a recombinant p47 trimer, one of the major p97 adaptor proteins involved in membrane fusion. Depending on the nucleotide state, we observe the p47 trimer to be in two distinct arrangements on top of the p97 hexamer. By combining the EM data with NMR and other biophysical measurements, we propose a model of ATP-dependent p97(N) domain motions that lead to a rearrangement of p47 domains, which could result in the disassembly of target protein complexes.