The endosomal sorting complexes required for transport (ESCRTs) are responsible for membrane remodeling in many cellular processes, such as multivesicular body biogenesis, viral budding, and ...cytokinetic abscission. ESCRT-III, the most abundant ESCRT subunit, assembles into flat spirals as the primed state, essential to initiate membrane invagination. However, the three-dimensional architecture of ESCRT-III flat spirals remained vague for decades due to highly curved filaments with a small diameter and a single preferred orientation on the membrane. Here, we unveiled that yeast Snf7, a component of ESCRT-III, forms flat spirals on the lipid monolayers using cryogenic electron microscopy. We developed a geometry-constrained Euler angle-assigned reconstruction strategy and obtained moderate-resolution structures of Snf7 flat spirals with varying curvatures. Our analyses showed that Snf7 subunits recline on the membrane with N-terminal motifs α0 as anchors, adopt an open state with fused α2/3 helices, and bend α2/3 gradually from the outer to inner parts of flat spirals. In all, we provide the orientation and conformations of ESCRT-III flat spirals on the membrane and unveil the underlying assembly mechanism, which will serve as the initial step in understanding how ESCRTs drive membrane abscission.
Range extended electric vehicles (REEVs), transition vehicles between combustion engine vehicles and electric vehicles, have been widely used due to their advantages of low fuel consumption and low ...emissions. By optimizing power management and after-treatment system control strategies, it is possible to achieve lower fuel consumption and emissions. This study proposes an integrated control method based on optimization strategies for the auxiliary power unit (APU) on/off system and energy management optimization for extended hybrid electric vehicles. The Elitist Nondominated Sorting Genetic Algorithm (NSGA-II) optimization method is used to determine the control parameters. In addition, considering the frequent start and stop characteristics of the APU caused by the optimization strategy, closed-loop control of the urea injection is established to solve the ammonia leakage problem. Due to their high computational efficiency, the proposed energy management and urea injection algorithms can be easily implemented in real time. The actual operating emissions of engines under the different strategies are tested on a semi-physical simulation platform, focusing on the analysis and comparison of NOx emissions. The conclusion has certain reference significance for vehicle production.
•A comprehensive optimization strategy of APU on/off + EMS + urea injection is developed.•Targeted urea injection optimization is carried out for the hybrid electric vehicles.•The NH3 leakage is significantly reduced after the SCR control improvement.•Emissions under four different EMSs are tested on the bench and analyzed.
Improving the efficiency of type-reduction algorithms continues to attract research interest. Recently, there has been some new type-reduction approaches claiming that they are more efficient than ...the well-known algorithms such as the enhanced Karnik-Mendel (EKM) and the enhanced iterative algorithm with stopping condition (EIASC). In a previous paper, we found that the computational efficiency of an algorithm is closely related to the platform, and how it is implemented. In computer science, the dependence on languages is usually avoided by focusing on the complexity of algorithms (using big O notation). In this article, the main contribution is the proposal of two novel type-reduction algorithms. Also, for the first time, a comprehensive study on both existing and new type-reduction approaches is made based on both algorithm complexity and practical computational time under a variety of programming languages. Based on the results, suggestions are given for the preferred algorithms in different scenarios depending on implementation platform and application context.
The AAA-ATPase Vps4 is critical for function of the multivesicular body sorting pathway, which impacts cellular phenomena ranging from receptor down-regulation to viral budding to cytokinesis. Vps4 ...activity is stimulated by the interaction between Vta1 and Vps60, but the structural basis for this interaction is unclear. The fragment Vps60(128–186) was reported to display the full activity of Vps60. Vta1 interacts with Vps60 using its N-terminal domain (Vta1NTD). In this work, the structure of Vps60(128–186) in complex with Vta1NTD was determined using NMR techniques, demonstrating a novel recognition mode of the microtubule-interacting and transport (MIT) domain in which Vps60(128–186) interacts with Vta1NTD through helices α4′ and α5′, extending over Vta1NTD MIT2 domain helices 1–3. The Vps60 binding does not result in Vta1 conformational changes, further revealing the fact that Vps4 ATPase is enhanced by the interaction between Vta1 and Vps60 in an unanticipated manner.
Vps4 ATPase is stimulated by the interaction between Vta1 and Vps60, but the structural basis for this interaction remains unclear.
The structure of the Vta1 N-terminal domain (Vta1NTD) in complex with Vps60(128–186) was determined.
Vps60(128–186) interacts with Vta1NTD through helices α4′ and α5′, extending over Vta1NTD MIT2 domain helices 1–3.
This is a novel MIT recognition mode.
Endosomal sorting complex required for transport (ESCRT)-III-mediated membrane invagination and scission are a critical step in multivesicular body (MVB) sorting of ubiquitinated membrane receptors, ...and generally thought to be required for degradation of these receptors in lysosomes. The adaptor protein Alix is critically involved in multiple ESCRT-III-mediated, membrane-remodelling processes in mammalian cells. However, Alix knockdown does not inhibit degradation of the activated epidermal growth factor receptor (EGFR) in mammalian cell lines, leading to a widely held notion that Alix is not critically involved in MVB sorting of ubiquitinated membrane receptors in mammalian cells. In the present study, we demonstrate that, despite its non-essential role in degradation of the activated EGFR, Alix plays a critical role in its MVB sorting and silencing Epidermal growth factor (EGF) stimulation of mammalian cell lines induces Alix's interaction with the ubiquitinated EGFR via the Alix V domain, and increases Alix's association with membrane-bound charged multivesicular body protein 4 (CHMP4) via the Alix Bro1 domain. Under both continuous and pulse-chase EGF stimulation conditions, inhibition of Alix's interaction with membrane-bound CHMP4, inhibition of Alix dimerization through the V domain or Alix knockdown dramatically inhibits MVB sorting of the activated EGFR and promotes sustained activation of extracellular-signal regulated kinase (ERK)1/2. Under the continuous EGF stimulation conditions, these cell treatments also retard degradation of the activated EGFR. These findings indicate that Alix is critically involved in MVB sorting of ubiquitinated membrane receptors in mammalian cells.
PspA is the main effector of the phage shock protein (Psp) system and preserves the bacterial inner membrane integrity and function. Here, we present the 3.6 Å resolution cryoelectron microscopy ...(cryo-EM) structure of PspA assembled in helical rods. PspA monomers adopt a canonical ESCRT-III fold in an extended open conformation. PspA rods are capable of enclosing lipids and generating positive membrane curvature. Using cryo-EM, we visualized how PspA remodels membrane vesicles into μm-sized structures and how it mediates the formation of internalized vesicular structures. Hotspots of these activities are zones derived from PspA assemblies, serving as lipid transfer platforms and linking previously separated lipid structures. These membrane fusion and fission activities are in line with the described functional properties of bacterial PspA/IM30/LiaH proteins. Our structural and functional analyses reveal that bacterial PspA belongs to the evolutionary ancestry of ESCRT-III proteins involved in membrane remodeling.
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•Cryo-EM structure of assembled helical PspA rods•PspA adopts a canonical ESCRT-III fold•PspA rods can enclose lipids generating positive membrane curvature•PspA remodels bacterial membranes following fusion and fission events
Structural and functional characterization of PspA reveals an unkown ESCRT-III family member capable of mediating membrane fission and fusion processes.
Cytokinesis requires the constriction of ESCRT-III filaments on the side of the midbody, where abscission occurs. After ESCRT recruitment at the midbody, it is not known how the ESCRT-III machinery ...localizes to the abscission site. To reveal actors involved in abscission, we obtained the proteome of intact, post-abscission midbodies (Flemmingsome) and identified 489 proteins enriched in this organelle. Among these proteins, we further characterized a plasma membrane-to-ESCRT module composed of the transmembrane proteoglycan syndecan-4, ALIX and syntenin, a protein that bridges ESCRT-III/ALIX to syndecans. The three proteins are highly recruited first at the midbody then at the abscission site, and their depletion delays abscission. Mechanistically, direct interactions between ALIX, syntenin and syndecan-4 are essential for proper enrichment of the ESCRT-III machinery at the abscission site, but not at the midbody. We propose that the ESCRT-III machinery must be physically coupled to a membrane protein at the cytokinetic abscission site for efficient scission, uncovering common requirements in cytokinesis, exosome formation and HIV budding.
The many functions of ESCRTs Vietri, Marina; Radulovic, Maja; Stenmark, Harald
Nature reviews. Molecular cell biology,
01/2020, Volume:
21, Issue:
1
Journal Article
Peer reviewed
Cellular membranes can form two principally different involutions, which either exclude or contain cytosol. The 'classical' budding reactions, such as those occurring during endocytosis or formation ...of exocytic vesicles, involve proteins that assemble on the cytosol-excluding face of the bud neck. Inverse membrane involution occurs in a wide range of cellular processes, supporting cytokinesis, endosome maturation, autophagy, membrane repair and many other processes. Such inverse membrane remodelling is mediated by a heteromultimeric protein machinery known as endosomal sorting complex required for transport (ESCRT). ESCRT proteins assemble on the cytosolic (or nucleoplasmic) face of the neck of the forming involution and cooperate with the ATPase VPS4 to drive membrane scission or sealing. Here, we review similarities and differences of various ESCRT-dependent processes, with special emphasis on mechanisms of ESCRT recruitment.
Mechanisms coordinating endosomal degradation and recycling are poorly understood, as are the cellular roles of microtubule (MT) severing. We show that cells lacking the MT-severing protein spastin ...had increased tubulation of and defective receptor sorting through endosomal tubular recycling compartments. Spastin required the ability to sever MTs and to interact with ESCRT-III (a complex controlling cargo degradation) proteins to regulate endosomal tubulation. Cells lacking IST1 (increased sodium tolerance 1), an endosomal sorting complex required for transport (ESCRT) component to which spastin binds, also had increased endosomal tubulation. Our results suggest that inclusion of IST1 into the ESCRT complex allows recruitment of spastin to promote fission of recycling tubules from the endosome. Thus, we reveal a novel cellular role for MT severing and identify a mechanism by which endosomal recycling can be coordinated with the degradative machinery. Spastin is mutated in the axonopathy hereditary spastic paraplegia. Zebrafish spinal motor axons depleted of spastin or IST1 also had abnormal endosomal tubulation, so we propose this phenotype is important for axonal degeneration.
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