Intralumenal vesicle formation of the multivesicular body is a critical step in the delivery of endocytic cargoes to the lysosome for degradation. Endosomal sorting complex required for transport III ...(ESCRT-III) subunits polymerize on endosomal membranes to facilitate membrane budding away from the cytoplasm to generate these intralumenal vesicles. The ATPase Vps4 remodels and disassembles ESCRT-III, but the manner in which Vps4 activity is coordinated with ESCRT-III function remains unclear. Ist1 is structurally homologous to ESCRT-III subunits and has been reported to inhibit Vps4 function despite the presence of a microtubule-interacting and trafficking domain-interacting motif (MIM) capable of stimulating Vps4 in the context of other ESCRT-III subunits. Here we report that Ist1 inhibition of Vps4 ATPase activity involves two elements in Ist1: the MIM itself and a surface containing a conserved ELYC sequence. In contrast, the MIM interaction, in concert with a more open conformation of the Ist1 core, resulted in stimulation of Vps4. Addition of the ESCRT-III subunit binding partner of Ist1, Did2, also converted Ist1 from an inhibitor to a stimulator of Vps4 ATPase activity. Finally, distinct regulation of Vps4 by Ist1 corresponded with altered ESCRT-III disassembly in vitro. Together, these data support a model in which Ist1-Did2 interactions during ESCRT-III polymerization coordinate Vps4 activity with the timing of ESCRT-III disassembly.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Microparticles (MPs) are cell-cell communication vesicles derived from the cell surface plasma membrane, although they are not known to originate from cardiac ventricular muscle. In ventricular ...cardiomyocytes, the membrane deformation protein cardiac bridging integrator 1 (cBIN1 or BIN1+13+17) creates transverse-tubule (t-tubule) membrane microfolds, which facilitate ion channel trafficking and modulate local ionic concentrations. The microfold-generated microdomains continuously reorganize, adapting in response to stress to modulate the calcium signaling apparatus. We explored the possibility that cBIN1-microfolds are externally released from cardiomyocytes. Using electron microscopy imaging with immunogold labeling, we found in mouse plasma that cBIN1 exists in membrane vesicles about 200 nm in size, which is consistent with the size of MPs. In mice with cardiac-specific heterozygous Bin1 deletion, flow cytometry identified 47% less cBIN1-MPs in plasma, supporting cardiac origin. Cardiac release was also evidenced by the detection of cBIN1-MPs in medium bathing a pure population of isolated adult mouse cardiomyocytes. In human plasma, osmotic shock increased cBIN1 detection by enzyme-linked immunosorbent assay (ELISA), and cBIN1 level decreased in humans with heart failure, a condition with reduced cardiac muscle cBIN1, both of which support cBIN1 release in MPs from human hearts. Exploring putative mechanisms of MP release, we found that the membrane fission complex endosomal sorting complexes required for transport (ESCRT)-III subunit charged multivesicular body protein 4B (CHMP4B) colocalizes and coimmunoprecipitates with cBIN1, an interaction enhanced by actin stabilization. In HeLa cells with cBIN1 overexpression, knockdown of CHMP4B reduced the release of cBIN1-MPs. Using truncation mutants, we identified that the N-terminal BAR (N-BAR) domain in cBIN1 is required for CHMP4B binding and MP release. This study links the BAR protein superfamily to the ESCRT pathway for MP biogenesis in mammalian cardiac ventricular cells, identifying elements of a pathway by which cytoplasmic cBIN1 is released into blood.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The endosomal sorting complex required for transport (ESCRT) machinery is an ancient system that deforms membrane and severs membrane necks from the inside. Extensive evidence has accumulated to ...demonstrate the conserved functions of plant ESCRTs in multivesicular body (MVB) biogenesis and MVB-mediated membrane protein sorting. In addition, recent exciting findings have uncovered unique plant ESCRT components and point to emerging roles for plant ESCRTs in non-endosomal sorting events such as autophagy, cytokinesis, and viral replication. Plant-specific processes, such as abscisic acid (ABA) signaling and chloroplast turnover, provide further evidence for divergences in the functions of plant ESCRTs during evolution. We summarize the multiple roles and current working models for plant ESCRT machinery and speculate on future ESCRT studies in the plant field.
ESCRT is an evolutionarily conserved machinery for membrane deformation and scission from the inner face of a membrane away from the cytoplasm.
Plants encode most ESCRT isoforms in their genome, including ESCRT-I, -II, -III, and VPS4/SKD1, with the exception of the canonical ESCRT-0. TOL (TOM1-like) proteins were identified as upstream ESCRT factors that partially fulfill ESCRT-0 function in plants.
Extensive evidence has accumulated to demonstrate the essential and conserved functions of ESCRTs in endosomal sorting in plants.
Plant-specific ESCRT components have been identified. In addition, ESCRTs in plants are also involved in a variety of non-endosomal sorting events such as autophagosome maturation, chloroplast turnover, cytokinesis, and viral replication.
Plant ESCRTs are also actively involved in hormone signaling and plant responses to biotic and abiotic stresses.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK, ZRSKP
Phosphatidylinositol 3-kinase Vps34 complexes regulate intracellular membrane trafficking in endocytic sorting, cytokinesis, and autophagy. We present the 4.4 angstrom crystal structure of the ...385-kilodalton endosomal complex II (PIK3C3-CII), consisting of Vps34, Vps15 (p150), Vps30/Atg6 (Beclin 1), and Vps38 (UVRAG). The subunits form a Y-shaped complex, centered on the Vps34 C2 domain. Vps34 and Vps15 intertwine in one arm, where the Vps15 kinase domain engages the Vps34 activation loop to regulate its activity. Vps30 and Vps38 form the other arm that brackets the Vps15/Vps34 heterodimer, suggesting a path for complex assembly. We used hydrogen-deuterium exchange mass spectrometry (HDX-MS) to reveal conformational changes accompanying membrane binding and identify a Vps30 loop that is critical for the ability of complex II to phosphorylate giant liposomes on which complex I is inactive.
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BFBNIB, NMLJ, NUK, PNG, SAZU, UL, UM, UPUK
Signal peptides (SPs) are short amino acid sequences in the amino terminus of many newly synthesized proteins that target proteins into, or across, membranes. Bioinformatic tools can predict SPs from ...amino acid sequences, but most cannot distinguish between various types of signal peptides. We present a deep neural network-based approach that improves SP prediction across all domains of life and distinguishes between three types of prokaryotic SPs.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
Exosomes transport a variety of macromolecules and modulate intercellular communication in physiology and disease. However, the regulation mechanisms that determine exosome contents during exosome ...biogenesis remain poorly understood. Here, we find that GPR143, an atypical GPCR, controls the endosomal sorting complex required for the transport (ESCRT)-dependent exosome biogenesis pathway. GPR143 interacts with HRS (an ESCRT-0 Subunit) and promotes its association to cargo proteins, such as EGFR, which subsequently enables selective protein sorting into intraluminal vesicles (ILVs) in multivesicular bodies (MVBs). GPR143 is elevated in multiple cancers, and quantitative proteomic and RNA profiling of exosomes in human cancer cell lines showed that the GPR143-ESCRT pathway promotes secretion of exosomes that carry unique cargo, including integrins signaling proteins. Through gain- and loss-of-function studies in mice, we show that GPR143 promotes metastasis by secreting exosomes and increasing cancer cell motility/invasion through the integrin/FAK/Src pathway. These findings provide a mechanism for regulating the exosomal proteome and demonstrate its ability to promote cancer cell motility.
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•GPR143 regulates ESCRT-dependent exosome production•GPR143 recruits HRS to endosomes to modulate interaction with cargo protein•GPR143 regulates protein sorting in ILVs and alters exosomal proteome composition•Exosomal integrins activate the cell motility signaling pathway to promote metastasis
Lee et al. find that GPR143, an atypical GPCR, controls the ESCRT-dependent exosome biogenesis pathway, which determines exosomal protein cargo composition. In cancer, GPR143 expression facilitates secretion of oncogenic exosomes, which contain integrins that promote cell motility and cancer metastasis.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
The current literature of evolutionary many-objective optimization is merely focused on the scalability to the number of objectives, while little work has considered the scalability to the number of ...decision variables. Nevertheless, many real-world problems can involve both many objectives and large-scale decision variables. To tackle such large-scale many-objective optimization problems (MaOPs), this paper proposes a specially tailored evolutionary algorithm based on a decision variable clustering method. To begin with, the decision variable clustering method divides the decision variables into two types: 1) convergence-related variables and 2) diversity-related variables. Afterward, to optimize the two types of decision variables, a convergence optimization strategy and a diversity optimization strategy are adopted. In addition, a fast nondominated sorting approach is developed to further improve the computational efficiency of the proposed algorithm. To assess the performance of the proposed algorithm, empirical experiments have been conducted on a variety of large-scale MaOPs with up to ten objectives and 5000 decision variables. Our experimental results demonstrate that the proposed algorithm has significant advantages over several state-of-the-art evolutionary algorithms in terms of the scalability to decision variables on MaOPs.
The ESCRT machinery Schmidt, Oliver; Teis, David
Current biology,
02/2012, Volume:
22, Issue:
4
Journal Article
Peer reviewed
Open access
The endosomal sorting complexes required for transport (ESCRT) assemble into a multisubunit machinery that performs a topologically unique membrane bending and scission reaction away from the ...cytoplasm. This evolutionarily highly conserved process is required for the multivesicular body (MVB) pathway, cytokinesis and HIV budding. The modular setup of the machinery with five distinct ESCRT complexes (ESCRT-0, -I, -II, -III and the Vps4 complex) that have a clear division of tasks — from interaction with ubiquitinated membrane proteins to membrane deformation and abscission — allows them to be flexibly integrated into these three very different biological processes (Figure 1).
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
HIV-1 infection causes AIDS, infecting millions worldwide. The virus can persist in a state of chronic infection due to its ability to become latent. We have previously shown a link between HIV-1 ...infection and exosome production. Specifically, we have reported that exosomes transport viral proteins and RNA from infected cells to neighboring uninfected cells. These viral products could then elicit an innate immune response, leading to activation of the Toll-like receptor and NF-κB pathways. In this study, we asked whether exosomes from uninfected cells could activate latent HIV-1 in infected cells. We observed that irrespective of combination antiretroviral therapy, both short- and long-length viral transcripts were increased in wild-type HIV-1–infected cells exposed to purified exosomes from uninfected cells. A search for a possible mechanism for this finding revealed that the exosomes increase RNA polymerase II loading onto the HIV-1 promoter in the infected cells. These viral transcripts, which include trans-activation response (TAR) RNA and a novel RNA that we termed TAR-gag, can then be packaged into exosomes and potentially be exported to neighboring uninfected cells, leading to increased cellular activation. To better decipher the exosome release pathways involved, we used siRNA to suppress expression of ESCRT (endosomal sorting complex required for transport) proteins and found that ESCRT II and IV significantly control exosome release. Collectively, these results imply that exosomes from uninfected cells activate latent HIV-1 in infected cells and that true transcriptional latency may not be possible in vivo, especially in the presence of combination antiretroviral therapy.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Metazoans require the sorting nexin (SNX) protein, SNX27, to recycle hundreds of important transmembrane protein receptors from endosomes to the plasma membrane. Cargo recycling by SNX27 requires its ...interaction with retromer, a heterotrimer known to assemble on membranes with multiple sorting nexins, including SNX-BAR proteins and SNX3. SNX27 has also been functionally linked to SNX-BARs, but the molecular basis of this interaction has been unknown. We identify a direct biochemical interaction between the conserved and flexible SNX1/SNX2 N-terminus and full-length SNX27 using purified proteins in pulldown experiments. Sequence alignments indicate both SNX1 and SNX2 contain two short and conserved stretches of acidic residues bearing a DxF motif in their flexible N-terminal regions. Biochemical pulldown and mapping experiments reveal forty residues in the N-terminus of either SNX1 or SNX2 can mediate binding to SNX27. SNX27 truncation analysis demonstrates the SNX27 FERM domain binds the SNX1 N-terminus. Calorimetry experiments quantified binding between the SNX1 N-terminus and SNX27 in the low micromolar affinity range (KD ∼10 μM) and suggest the second DxF motif may play a more prominent role in binding. Mutation of either DxF sequence in SNX1 abrogates measurable binding to SNX27 in the calorimeter. Modelling from both predicted and experimentally determined structures suggests the SNX27 FERM domain could accommodate both DxF motifs simultaneously. Together, these data suggest SNX27 is directly linked to specific SNX-BAR proteins through binding acidic motifs in the SNX1 or SNX2 N-terminus.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
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