RESUMO
In eukaryotes, DNA replication initiates from multiple origins of replication for timely genome duplication. These sites are selected by origin licensing, during which the core enzyme of the eukaryotic DNA replicative helicase, the Mcm2-7 (minichromosome maintenance) complex, is loaded at each origin. This origin licensing requires loading two Mcm2-7 helicases around origin DNA in a head-to-head orientation. Current models suggest that the origin-recognition complex (ORC) and cell-division cycle 6 (Cdc6) proteins recognize and encircle origin DNA and assemble an Mcm2-7 double-hexamer around adjacent double-stranded DNA. To test this model and assess the location of Mcm2-7 initial loading, we placed DNA-protein roadblocks at defined positions adjacent to the essential ORC-binding site within Saccharomyces cerevisiae origin DNA. Roadblocks were made either by covalent cross-linking of the HpaII methyltransferase to DNA or through binding of a transcription activator-like effector (TALE) protein. Contrary to the sites of Mcm2-7 recruitment being precisely defined, only single roadblocks that inhibited ORC-DNA binding showed helicase loading defects. We observed inhibition of helicase loading without inhibition of ORC-DNA binding only when roadblocks were placed on both sides of the origin to restrict sliding of a helicase-loading intermediate. Consistent with a sliding helicase-loading intermediate, when either one of the flanking roadblocks was eliminated, the remaining roadblock had no effect on helicase loading. Interestingly, either origin-flanking nucleosomes or roadblocks resulted in helicase loading being dependent on an additional origin sequence known to be a weaker ORC-DNA-binding site. Together, our findings support a model in which sliding helicase-loading intermediates increase the flexibility of the DNA sequence requirements for origin licensing.
Assuntos
Proteínas de Arabidopsis/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas de Manutenção de Minicromossomo/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Replicação do DNA/genética , Replicação do DNA/fisiologia , Componente 7 do Complexo de Manutenção de Minicromossomo/metabolismo , Proteínas de Manutenção de Minicromossomo/fisiologia , Complexo de Reconhecimento de Origem/genética , Ligação Proteica , Domínios Proteicos , Origem de Replicação/genética , Origem de Replicação/fisiologia , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
The endosomal sorting complexes required for transport (ESCRTs) impact multiple cellular processes including multivesicular body sorting, abscission, and viral budding. The AAA-ATPase Vps4 is required for ESCRT function, and its full activity is dependent upon the co-factor Vta1. The Vta1 carboxyl-terminal Vta1 SBP1 Lip5 (VSL) domain stimulates Vps4 function by facilitating oligomerization of Vps4 into its active state. Here we report the identification of the Vps4 stimulatory element (VSE) within Vta1 that is required for additional stimulation of Vps4 activity in vitro and in vivo. VSE activity is autoinhibited in a manner dependent upon the unstructured linker region joining the amino-terminal microtubule interacting and trafficking domains and the carboxyl-terminal VSL domain. The VSE is also required for Vta1-mediated Vps4 stimulation by ESCRT-III subunits Vps60 and Did2. These results suggest that ESCRT-III binding to the Vta1 microtubule interacting and trafficking domains relieves linker region autoinhibition of the VSE to produce maximal activation of Vps4 during ESCRT function.
Assuntos
Adenosina Trifosfatases/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Multimerização Proteica/fisiologia , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Adenosina Trifosfatases/genética , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Ativação Enzimática/fisiologia , Estrutura Terciária de Proteína , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
The MVB pathway plays essential roles in several eukaryotic cellular processes. Proper function of the MVB pathway requires reversible membrane association of the ESCRTs, a process catalyzed by Vps4 ATPase. Vta1 regulates the Vps4 activity, but its mechanism of action was poorly understood. We report the high-resolution crystal structures of the Did2- and Vps60-binding N-terminal domain and the Vps4-binding C-terminal domain of S. cerevisiae Vta1. The C-terminal domain also mediates Vta1 dimerization and both subunits are required for its function as a Vps4 regulator. Emerging from our analysis is a mechanism of regulation by Vta1 in which the C-terminal domain stabilizes the ATP-dependent double ring assembly of Vps4. In addition, the MIT motif-containing N-terminal domain, projected by a long disordered linker, allows contact between the Vps4 disassembly machinery and the accessory ESCRT-III proteins. This provides an additional level of regulation and coordination for ESCRT-III assembly and disassembly.
Assuntos
Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/química , Proteínas de Transporte Vesicular/química , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Animais , Sequência Conservada , Cristalografia por Raios X , Complexos Endossomais de Distribuição Requeridos para Transporte , Humanos , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Conformação Proteica , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Especificidade da Espécie , Proteínas de Transporte Vesicular/metabolismoRESUMO
The AAA-ATPase Vps4 is critical for function of the MVB sorting pathway, which in turn impacts cellular phenomena ranging from receptor downregulation to viral budding to cytokinesis. Vps4 dissociates ESCRTs from endosomal membranes during MVB sorting, but it is unclear how Vps4 ATPase activity is synchronized with ESCRT release. Vta1 potentiates Vps4 activity and interacts with ESCRT-III family members. We have investigated the impact of Vta1 and ESCRT-III family members on Vps4 ATPase activity. Two distinct mechanisms of Vps4 stimulation are described: Vps2 can directly stimulate Vps4 via its MIT domain, whereas Vps60 stimulates via Vta1. Moreover, Did2 can stimulate Vps4 by both mechanisms in distinct contexts. Recent structural determination of the ESCRT-III-binding region of Vta1 unexpectedly revealed a MIT-like region. These data support a model wherein a network of MIT and MIT-like domain interactions with ESCRT-III subunits contributes to the regulation of Vps4 activity during MVB sorting.
Assuntos
Adenosina Trifosfatases/metabolismo , Biomarcadores Tumorais/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Proteínas de Transporte Vesicular/metabolismo , Adenosina Trifosfatases/química , Adenosina Trifosfatases/genética , Sítios de Ligação , Biomarcadores Tumorais/química , Complexos Endossomais de Distribuição Requeridos para Transporte , Cinética , Modelos Moleculares , Proteínas do Tecido Nervoso/química , Plasmídeos , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Transporte Vesicular/química , Proteínas de Transporte Vesicular/genéticaRESUMO
Endosomal sorting complexes required for transport (ESCRT-0, -I, -II, -III) execute cargo sorting and intralumenal vesicle (ILV) formation during conversion of endosomes to multivesicular bodies (MVBs). The AAA-ATPase Vps4 regulates the ESCRT-III polymer to facilitate membrane remodeling and ILV scission during MVB biogenesis. Here, we show that the conserved V domain of ESCRT-associated protein Bro1 (the yeast homologue of mammalian proteins ALIX and HD-PTP) directly stimulates Vps4. This activity is required for MVB cargo sorting. Furthermore, the Bro1 V domain alone supports Vps4/ESCRT-driven ILV formation in vivo without efficient MVB cargo sorting. These results reveal a novel activity of the V domains of Bro1 homologues in licensing ESCRT-III-dependent ILV formation and suggest a role in coordinating cargo sorting with membrane remodeling during MVB sorting. Moreover, ubiquitin binding enhances V domain stimulation of Vps4 to promote ILV formation via the Bro1-Vps4-ESCRT-III axis, uncovering a novel role for ubiquitin during MVB biogenesis in addition to facilitating cargo recognition.
Assuntos
Adenosina Trifosfatases/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Corpos Multivesiculares/enzimologia , Biogênese de Organelas , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Adenosina Trifosfatases/genética , Sítios de Ligação , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Ativação Enzimática , Microscopia de Fluorescência , Modelos Moleculares , Corpos Multivesiculares/genética , Mutação , Domínios Proteicos , Transporte Proteico , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Ubiquitina/metabolismo , UbiquitinaçãoRESUMO
MVB (multivesicular body) formation occurs when the limiting membrane of an endosome invaginates into the intraluminal space and buds into the lumen, bringing with it a subset of transmembrane cargoes. Exvagination of the endosomal membrane from the cytosol is topologically similar to the budding of retroviral particles and cytokinesis, wherein membranes bud away from the cytoplasm, and the machinery responsible for MVB sorting has been implicated in these phenomena. The AAA (ATPase associated with various cellular activities) Vps4 (vacuolar protein sorting 4) performs a critical function in the MVB sorting pathway. Vps4 appears to dissociate the ESCRTs (endosomal sorting complexes required for transport) from endosomal membranes during the course of MVB sorting, but it is unclear how Vps4 ATPase activity is synchronized with ESCRT release. We have investigated the mechanisms by which ESCRT components stimulate the ATPase activity of Vps4. These studies support a model wherein Vps4 activity is subject to spatial and temporal regulation via distinct mechanisms during MVB sorting.
Assuntos
Adenosina Trifosfatases/metabolismo , Endossomos/metabolismo , Complexos Multiproteicos/metabolismo , Adenosina Trifosfatases/química , Animais , Ativação Enzimática , Estrutura Quaternária de Proteína , Transporte ProteicoRESUMO
Eukaryotic replication origin licensing, activation and timing are influenced by chromatin but a mechanistic understanding is lacking. Using reconstituted nucleosomal DNA replication assays, we assessed the impact of nucleosomes on replication initiation. To generate distinct nucleosomal landscapes, different chromatin-remodeling enzymes (CREs) were used to remodel nucleosomes on origin-DNA templates. Nucleosomal organization influenced two steps of replication initiation: origin licensing and helicase activation. Origin licensing assays showed that local nucleosome positioning enhanced origin specificity and modulated helicase loading by influencing ORC DNA binding. Interestingly, SWI/SNF- and RSC-remodeled nucleosomes were permissive for origin licensing but showed reduced helicase activation. Specific CREs rescued replication of these templates if added prior to helicase activation, indicating a permissive chromatin state must be established during origin licensing to allow efficient origin activation. Our studies show nucleosomes directly modulate origin licensing and activation through distinct mechanisms and provide insights into the regulation of replication initiation by chromatin.
Assuntos
Replicação do DNA , Nucleossomos/metabolismo , Origem de Replicação , DNA/metabolismo , DNA Helicases/metabolismo , Ligação Proteica , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/fisiologiaRESUMO
ESCRT-III undergoes dynamic assembly and disassembly to facilitate membrane exvagination processes including multivesicular body (MVB) formation, enveloped virus budding, and membrane abscission during cytokinesis. The AAA-ATPase Vps4 is required for ESCRT-III disassembly, however the coordination of Vps4 ATP hydrolysis with ESCRT-III binding and disassembly is not understood. Vps4 ATP hydrolysis has been proposed to execute ESCRT-III disassembly as either a stable oligomer or an unstable oligomer whose dissociation drives ESCRT-III disassembly. An in vitro ESCRT-III disassembly assay was developed to analyze Vps4 function during this process. The studies presented here support a model in which Vps4 acts as a stable oligomer during ATP hydrolysis and ESCRT-III disassembly. Moreover, Vps4 oligomer binding to ESCRT-III induces coordination of ATP hydrolysis at the level of individual Vps4 subunits. These results suggest that Vps4 functions as a stable oligomer that acts upon individual ESCRT-III subunits to facilitate ESCRT-III disassembly.