RESUMEN
The AAA+ ATPases are essential for various activities such as membrane trafficking, organelle biogenesis, DNA replication, intracellular locomotion, cytoskeletal remodelling, protein folding and proteolysis. The AAA ATPase Vps4, which is central to endosomal traffic to lysosomes, retroviral budding and cytokinesis, dissociates ESCRT complexes (the endosomal sorting complexes required for transport) from membranes. Here we show that, of the six ESCRT--related subunits in yeast, only Vps2 and Did2 bind the MIT (microtubule interacting and transport) domain of Vps4, and that the carboxy-terminal 30 residues of the subunits are both necessary and sufficient for interaction. We determined the crystal structure of the Vps2 C terminus in a complex with the Vps4 MIT domain, explaining the basis for selective ESCRT-III recognition. MIT helices alpha2 and alpha3 recognize a (D/E)xxLxxRLxxL(K/R) motif, and mutations within this motif cause sorting defects in yeast. Our crystal structure of the amino-terminal domain of an archaeal AAA ATPase of unknown function shows that it is closely related to the MIT domain of Vps4. The archaeal ATPase interacts with an archaeal ESCRT-III-like protein even though these organisms have no endomembrane system, suggesting that the Vps4/ESCRT-III partnership is a relic of a function that pre-dates the divergence of eukaryotes and Archaea.
Asunto(s)
Adenosina Trifosfatasas/química , Adenosina Trifosfatasas/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimología , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Secuencia Conservada , Cristalografía por Rayos X , Endocitosis , Complejos de Clasificación Endosomal Requeridos para el Transporte , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Terciaria de Proteína , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/metabolismo , Especificidad por Sustrato , Vacuolas/metabolismoRESUMEN
The ESCRT machinery mediates sorting of ubiquitinated transmembrane proteins to lysosomes via multivesicular bodies (MVBs) and also has roles in cytokinesis and viral budding. The ESCRT-III subunits are metastable monomers that transiently assemble on membranes. However, the nature of these assemblies is unknown. Among the core yeast ESCRT-III subunits, Snf7 and Vps24 spontaneously form ordered polymers in vitro. Single-particle EM reconstruction of helical Vps24 filaments shows both parallel and head-to-head subunit arrangements. Mutations of regions involved in intermolecular assembly in vitro result in cargo-sorting defects in vivo, suggesting that these homopolymers mimic interactions formed by ESCRT-III heteropolymers during MVB biogenesis. The C terminus of Vps24 is at the surface of the filaments and is not required for filament assembly. When this region is replaced by the MIT-interacting motif from the Vps2 subunit of ESCRT-III, the AAA-ATPase Vps4 can both bundle and disassemble the chimeric filaments in a nucleotide-dependent fashion.
Asunto(s)
Citoesqueleto/química , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Transporte Vesicular/química , Proteínas de Transporte Vesicular/metabolismo , Adenosina Trifosfatasas/metabolismo , Citoesqueleto/metabolismo , Dimerización , Complejos de Clasificación Endosomal Requeridos para el Transporte , Modelos Biológicos , Modelos Moleculares , Chaperonas Moleculares/química , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Mutación Missense/fisiología , Polímeros/metabolismo , Pliegue de Proteína , Estructura Cuaternaria de Proteína , Estructura Terciaria de Proteína/fisiología , Subunidades de Proteína/química , Transporte de Proteínas/genética , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Transporte Vesicular/genéticaRESUMEN
The AAA (ATPase associated with various cellular activities) proteins participate in membrane trafficking, organelle biogenesis, DNA replication, intracellular locomotion, cytoskeletal remodelling, protein folding and proteolysis. The AAA Vps (vacuolar protein sorting) 4 is central to traffic to lysosomes, retroviral budding and mammalian cell division. It dissociates ESCRTs (endosomal sorting complexes required for transport) from endosomal membranes, enabling their recycling to the cytosol, and plays a role in fission of intraluminal vesicles within MVBs (multivesicular bodies). The mechanism of Vps4-catalysed disassembly of ESCRT networks is unknown; however, it requires interaction between Vps4 and ESCRT-III subunits. The 30 C-terminal residues of Vps2 and Vps46 (Did2) subunits are both necessary and sufficient for interaction with the Vps4 N-terminal MIT (microtubule-interacting and transport) domain, and the crystal structure of the Vps2 C-terminus in a complex with the Vps4 MIT domain shows that MIT helices alpha2 and alpha3 recognize a (D/E)XXLXXRLXXL(K/R) MIM (MIT-interacting motif). These Vps2-MIT interactions are essential for vacuolar sorting and for Vps4-catalysed disassembly of ESCRT-III networks in vitro. Electron microscopy of ESCRT-III filaments assembled in vitro has enabled us to identify surfaces of the Vps24 subunit that are critical for protein sorting in vivo. The ESCRT-III-Vps4 interaction predates the divergence of Archaea and Eukarya. The Crenarchaea have three classes of ESCRT-III-like subunits, and one of these subunits interacts with an archaeal Vps4-like protein in a manner closely related to the human Vps4-human ESCRT-III subunit Vps20 interaction. This archaeal Vps4-ESCRT-III interaction appears to have a fundamental role in cell division in the Crenarchaea.