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1.
The scission of biological membranes is facilitated by a variety of protein complexes that bind and manipulate lipid bilayers. ESCRT-III (endosomal sorting complex required for transport III) filaments mediate membrane scission during the ostensibly disparate processes of multivesicular endosome biogenesis, cytokinesis, and retroviral budding. However, mechanisms by which ESCRT-III subunits assemble into a polymer remain unknown. Using cryogenic electron microscopy (cryo-EM), we found that the full-length ESCRT-III subunit Vps32/CHMP4B spontaneously forms single-stranded spiral filaments. The resolution afforded by two-dimensional cryo-EM combined with molecular dynamics simulations revealed that individual Vps32/CHMP4B monomers within a filament are flexible and able to accommodate a range of bending angles. In contrast, the interface between monomers is stable and refractory to changes in conformation. We additionally found that the carboxyl terminus of Vps32/CHMP4B plays a key role in restricting the lateral association of filaments. Our findings highlight new mechanisms by which ESCRT-III filaments assemble to generate a unique polymer capable of membrane remodeling in multiple cellular contexts.  相似文献   

2.
ESCRT-I, -II, and -III protein complexes are sequentially recruited to endosomal membranes, where they orchestrate protein sorting and MVB biogenesis. In addition, they play a critical role in retrovirus budding. Structural understanding of ESCRT interaction networks is largely lacking. The 3.6 A structure of the yeast ESCRT-II core presented here reveals a trilobal complex containing two copies of Vps25, one copy of Vps22, and the C-terminal region of Vps36. Unexpectedly, the entire ESCRT-II core consists of eight repeats of a common building block, a "winged helix" domain. Two PPXY-motifs from Vps25 are involved in contacts with Vps22 and Vps36, and their mutation leads to ESCRT-II disruption. We show that purified ESCRT-II binds directly to the Vps20 component of ESCRT-III. Surprisingly, this binding does not require the protruding N-terminal coiled-coil of Vps22. Vps25 is the chief subunit responsible for Vps20 recruitment. This interaction dramatically increases binding of both components to lipid vesicles in vitro.  相似文献   

3.
Five endosomal sorting complexes required for transport (ESCRTs) mediate the degradation of ubiquitinated membrane proteins via multivesicular bodies (MVBs) in lysosomes. ESCRT-0, -I, and –II interact with cargo on endosomes. ESCRT-II also initiates the assembly of a ringlike ESCRT-III filament consisting of Vps20, Snf7, Vps24, and Vps2. The AAA–adenosine triphosphatase Vps4 disassembles and recycles the ESCRT-III complex, thereby terminating the ESCRT pathway. A mechanistic role for Vps4 in intraluminal vesicle (ILV) formation has been unclear. By combining yeast genetics, biochemistry, and electron tomography, we find that ESCRT-III assembly on endosomes is required to induce or stabilize the necks of growing MVB ILVs. Yet, ESCRT-III alone is not sufficient to complete ILV biogenesis. Rather, binding of Vps4 to ESCRT-III, coordinated by interactions with Vps2 and Snf7, is coupled to membrane neck constriction during ILV formation. Thus, Vps4 not only recycles ESCRT-III subunits but also cooperates with ESCRT-III to drive distinct membrane-remodeling steps, which lead to efficient membrane scission at the end of ILV biogenesis in vivo.  相似文献   

4.
Adell MA  Teis D 《FEBS letters》2011,585(20):3191-3196
The ESCRT (endosomal sorting complex required for transport) pathway promotes the final membrane scission step at the end of cytokinesis, assists viral budding and generates multivesicular bodies (MVBs). These seemingly unrelated processes require a topologically similar membrane deformation and scission event that buds membranes/vesicles out of the cytoplasm. The topology of this budding reaction is 'opposite' to reactions that bud endocytic and secretory vesicles into the cytoplasm. Here we summarize recent findings that help to understand how the ESCRT machinery, in particular the ESCRT-III complex, assembles on its target membranes, executes membrane scission and is disassembled by the AAA-ATPase Vps4.  相似文献   

5.
The sequential action of the Vps27/HRS complex, ESCRT-I, -II, and -III is required to sort ubiquitinated transmembrane proteins to the lumen of lysosomes via the multivesicular body (MVB) pathway. While Vps27/HRS, ESCRT-I, and -II are recruited to endosomes as preformed complexes, the ESCRT-III subunits Vps20, Snf7, Vps24, and Vps2 only assemble into a complex on endosomes. We have addressed the pathway and the regulation for ESCRT-III assembly. Our findings indicate the ordered assembly of a transient 450 kDa ESCRT-III complex on endosomes. Despite biochemical and structural similarity, each subunit contributes a specific function. Vps20 nucleates transient oligomerization of Snf7, which appears to sequester MVB cargo. Vps24 terminates Snf7 oligomerization by recruiting Vps2, which subsequently engages the AAA-ATPase Vps4 to dissociate ESCRT-III. We propose that the ordered assembly and disassembly of ESCRT-III delineates an MVB sorting domain to sequester cargo and complete the last steps of MVB sorting.  相似文献   

6.
Sorting of ubiquitinated endosomal membrane proteins into the MVB pathway is executed by the class E Vps protein complexes ESCRT-I, -II, and -III, and the AAA-type ATPase Vps4. This study characterizes ESCRT-II, a soluble approximately 155 kDa protein complex formed by the class E Vps proteins Vps22, Vps25, and Vps36. This protein complex transiently associates with the endosomal membrane and thereby initiates the formation of ESCRT-III, a membrane-associated protein complex that functions immediately downstream of ESCRT-II during sorting of MVB cargo. ESCRT-II in turn functions downstream of ESCRT-I, a protein complex that binds to ubiquitinated endosomal cargo. We propose that the ESCRT complexes perform a coordinated cascade of events to select and sort MVB cargoes for delivery to the lumen of the vacuole/lysosome.  相似文献   

7.
《Biophysical journal》2020,118(6):1333-1343
The endosomal sorting complex required for transport (ESCRT) machinery carries out the membrane scission reactions that are required for many biological processes throughout cells. How ESCRTs bind and deform cellular membranes and ultimately produce vesicles has been a matter of active research in recent years. In this study, we use fully atomistic molecular dynamics simulations to scrutinize the structural details of a filament composed of Vps32 protomers, a major component of ESCRT-III complexes. The simulations show that both hydrophobic and electrostatic interactions between monomers help maintain the structural stability of the filament, which exhibits an intrinsic bend and twist. Our findings suggest that the accumulation of bending and twisting stresses as the filament elongates on the membrane surface likely contributes to the driving force for membrane invagination. The filament exposes a large cationic surface that senses the negatively charged lipids in the membrane, and the N-terminal amphipathic helix of the monomers not only acts as a membrane anchor but also generates significant positive membrane curvature. Taking all results together, we discuss a plausible mechanism for membrane invagination driven by ESCRT-III.  相似文献   

8.
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.  相似文献   

9.
Endosomal sorting complexes required for transport (ESCRTs) promote the invagination of vesicles into the lumen of endosomes, the budding of enveloped viruses, and the separation of cells during cytokinesis. These processes share a topologically similar membrane scission event facilitated by ESCRT-III assembly at the cytosolic surface of the membrane. The Snf7 subunit of ESCRT-III in yeast binds directly to an auxiliary protein, Bro1. Like ESCRT-III, Bro1 is required for the formation of intralumenal vesicles at endosomes, but its role in membrane scission is unknown. We show that overexpression of Bro1 or its N-terminal Bro1 domain that binds Snf7 enhances the stability of ESCRT-III by inhibiting Vps4-mediated disassembly in vivo and in vitro. This stabilization effect correlates with a reduced frequency in the detachment of intralumenal vesicles as observed by electron tomography, implicating Bro1 as a regulator of ESCRT-III disassembly and membrane scission activity.  相似文献   

10.
Endosomal sorting complexes required for transport (ESCRT-0, ESCRT-I, ESCRT-II, and ESCRT-III) are selectively recruited to cellular membranes to exert their function in diverse processes, such as multivesicular body biogenesis, enveloped virus budding, and cytokinesis. ESCRT-III is composed of members of the charged multivesicular body protein (CHMP) family—cytosolic proteins that are targeted to membranes via yet unknown signals. Membrane targeting is thought to result in a membrane-associated protein network that presumably acts at a late budding step. Here we provide structural evidence based on small-angle X-ray scattering data that ESCRT-III CHMP3 can adopt two conformations in solution: a closed globular form that most likely represents the cytosolic conformation and an open extended conformation that might represent the activated form of CHMP3. Both the closed and open conformations of CHMP3 interact with AMSH with high affinity. Although the C-terminal region of CHMP3 is required for AMSH interaction, a peptide thereof reveals only weak binding to AMSH, suggesting that other regions of CHMP3 contribute to the high-affinity interaction. Thus, AMSH, including its MIT (microtubule interacting and transport) domain, interacts with ESCRT-III CHMP3 differently from reported Vps4 MIT domain-CHMP protein interactions.  相似文献   

11.
The sorting of transmembrane proteins (e.g., cell surface receptors) into the multivesicular body (MVB) pathway to the lysosomal/vacuolar lumen requires the function of the ESCRT protein complexes. The soluble coiled-coil-containing proteins Vps2, Vps20, Vps24, and Snf7 are recruited from the cytoplasm to endosomal membranes where they oligomerize into a protein complex, ESCRT-III. ESCRT-III contains two functionally distinct subcomplexes. The Vps20-Snf7 subcomplex binds to the endosomal membrane, in part via the myristoyl group of Vps20. The Vps2-Vps24 subcomplex binds to the Vps20-Snf7 complex and thereby serves to recruit additional cofactors to this site of protein sorting. We provide evidence for a role for ESCRT-III in sorting and/or concentration of MVB cargoes.  相似文献   

12.
The endosomal sorting complex required for transport (ESCRT)-III machinery contributes to membrane deformation and scission in cytokinesis, intraluminal vesicle formation, autophagy and virus budding. Recombinant ESCRT-III subunits polymerize in vitro into filaments, tubes, sheets or rings, and ESCRT-III-dependent filaments have been observed in cells at virus bud necks and at the cytokinetic abscission site. These observations have inspired speculation about how ESCRT-III could mediate constriction and fission of membrane necks. Based on the polymer structures observed in vitro and in vivo, we discuss models for ESCRT-III function and outline how emerging technologies could be used to test these models.  相似文献   

13.
The Saccharomyces cerevisiae ESCRT-III protein Snf7 is part of an intricate interaction network at the endosomal membrane. Interaction maps of Snf7 were established by measuring the degree of binding of individual binding partners to putative binding motifs along the Snf7 sequence by glutathione S-transferase (GST) pulldown. For each interaction partner, distinct binding profiles were obtained. The following observations were made. The ESCRT-III subunits Vps20 and Vps24 showed a complementary binding pattern, suggesting a model for the series of events in the ESCRT-III functional cycle. Vps4 bound to individual Snf7 motifs but not to full-length Snf7. This suggests that Vps4 does not bind to the closed conformation of Snf7. We also demonstrate for the first time that the ALIX/Bro1 homologue Rim20 binds to the α6 helix of Snf7. Analysis of a Snf7 α6 deletion mutant showed that the α6 helix is crucial for binding of Bro1 and Rim20 in vivo and is indispensable for the multivesicular body (MVB)-sorting and Rim-signaling functions of Snf7. The Snf7Δα6 protein still appeared to be incorporated into ESCRT-III complexes at the endosomal membrane, but disassembly of the complex seemed to be defective. In summary, our study argues against the view that the ESCRT cycle is governed by single one-to-one interactions between individual components and emphasizes the network character of the ESCRT interactions.  相似文献   

14.
Vps4 is a key enzyme that functions in endosomal protein trafficking, cytokinesis, and retroviral budding. Vps4 activity is regulated by its recruitment from the cytoplasm to ESCRT-III, where the protein oligomerizes into an active ATPase. The recruitment and oligomerization steps are mediated by a complex network of at least 12 distinct interactions between Vps4, ESCRT-III, Ist1, Vta1, and Did2. The order of events leading to active, ESCRT-III–associated Vps4 is poorly understood. In this study we present a systematic in vivo analysis of the Vps4 interaction network. The data demonstrated a high degree of redundancy in the network. Although no single interaction was found to be essential for the localization or activity of Vps4, certain interactions proved more important than others. The most significant among these were the binding of Vps4 to Vta1 and to the ESCRT-III subunits Vps2 and Snf7. In our model we propose the formation of a recruitment complex in the cytoplasm that is composed of Did2-Ist1-Vps4, which upon binding to ESCRT-III recruits Vta1. Vta1 in turn is predicted to cause a rearrangement of the Vps4 interactions that initiates the assembly of the active Vps4 oligomer.  相似文献   

15.
16.
Recruitment of endosomal sorting complexes required for transport (ESCRTs) to the cytosolic face of endosomes regulates selective inclusion of transmembrane proteins into the lumenal vesicles of multivesicular bodies (MVBs). ESCRT-0, -I, and -II bind directly to ubiquitinated transmembrane cargoes of the MVB pathway, whereas polymerization of ESCRT-III at endosomes is thought to bend the membrane and/or provide the energetic force that drives membrane scission and detachment of vesicles into the endosome lumen. Disassembly of the ESCRT-III polymer and dissociation of its subunits from endosomes requires the Vps4 ATPase, the activity of which is controlled in vivo by regulatory proteins. We identify distinct spatiotemporal roles for Vps4-regulating proteins through examinations of subcellular localization and endosome morphology. Did2 plays a unique role in the regulation of MVB lumenal vesicle size, whereas Vtal and Vps60 promote efficient membrane scission and delivery of membrane to the endosome lumen. These morphological effects probably result from Vps4-mediated manipulations of ESCRT-III, because we show dissociation of ESCRT-0, -I, and -II from endosomes is not directly dependent on Vps4 activity.  相似文献   

17.
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.  相似文献   

18.
The AAA-type ATPase Vps4 functions with components of the ESCRT (endosomal sorting complex required for transport) machinery in membrane fission events that are essential for endosomal maturation, cytokinesis, and the formation of retroviruses. A key step in these events is the assembly of monomeric Vps4 into the active ATPase complex, which is aided in part by binding of Vps4 via its N-terminal MIT (microtubule interacting and trafficking) domain to its substrate ESCRT-III. We found that the 40-amino acid linker region between the MIT and the ATPase domain of Vps4 is not required for proper function but plays a role in regulating Vps4 assembly and ATPase activity. Deletion of the linker is expected to bring the MIT domains into close proximity to the central pore of the Vps4 complex. We propose that this localization of the MIT domain in linker-deleted Vps4 mimics a repositioning of the MIT domain normally caused by binding of Vps4 to ESCRT-III. This structure would allow the Vps4 complex to engage ESCRT-III subunits with both the pore and the MIT domain simultaneously, which might be essential for the ATP-driven disassembly of ESCRT-III.  相似文献   

19.
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.  相似文献   

20.
The endosomal sorting complex required for transport (ESCRT) protein complexes function at the endosome in the formation of intraluminal vesicles (ILVs) containing cargo proteins destined for the vacuolar/lysosomal lumen. The early ESCRTs (ESCRT-0 and -I) are likely involved in cargo sorting, whereas ESCRT-III and Vps4 function to sever the neck of the forming ILVs. ESCRT-II links these functions by initiating ESCRT-III formation in an ESCRT-I–regulated manner. We identify a constitutively active mutant of ESCRT-II that partially suppresses the phenotype of an ESCRT-I or ESCRT-0 deletion strain, suggesting that these early ESCRTs are not essential and have redundant functions. However, the ESCRT-III/Vps4 system alone is not sufficient for ILV formation but requires cargo sorting mediated by one of the early ESCRTs.  相似文献   

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