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Systematic analysis of ESCRT mutants reveals an essential function for the MVB pathway in amino acid recycling.

ABSTRACT: The ESCRT machinery drives the multivesicular body (MVB) pathway in eukaryotic cells and thus is required for the degradation of ubiquitinated membrane proteins in lysosomes. To systematically characterize how cells respond to loss of ESCRT function, we used quantitative proteomics and gene expression profiling in yeast. We find that ESCRT mutants display severe defects in amino acid homeostasis, resulting in lower levels of free intracellular amino acids. This deficit renders the growth of ESCRT mutants highly sensitive to nutrient limitation. Further proteomic analysis revealed that the MVB pathway essentially contributes to proteome remodeling under nutrient limitation. The rapid decline of protein synthesis upon starvation no longer enables ESCRT mutants to complete their cell cycle and properly enter a quiescent state, which strongly affects their long-term survival. Thus, the MVB pathway functions to selectively down-regulate integral membrane proteins and, together with autophagy and proteasomal degradation, considerably contributes to amino acid recycling. We used microarrays to identify gene expression changes between delta VPS4 knock out and wild type Saccharomyces cerevisiae strains

ORGANISM(S): Saccharomyces cerevisiae Schizosaccharomyces pombe

PROVIDER: GSE53774 | GEO | 2018/01/01

SECONDARY ACCESSION(S): PRJNA232853

REPOSITORIES: GEO

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ESCRT-I controls lysosomal membrane protein homeostasis and restricts MCOLN1-dependent TFEB/TFE3 signaling.

Project description:Within the endolysosomal pathway in mammalian cells, ESCRT complexes facilitate degradation of membrane proteins from limiting membranes of late endosomes. Recent studies revealed that yeast ESCRT proteins also sort for degradation, ubiquitinated proteins from vacuolar membrane. However, whether mammalian ESCRTs perform similar function at lysosomes remained unknown. Here, we studied the involvement of mammalian ESCRT-I in maintaining lysosomal membrane homeostasis and its implication in lysosome-related signaling. We show that ESCRT-I restricts the size of lysosomes and promotes degradation of lysosomal Ca2+ channel, MCOLN1 protein. ESCRT-I depletion induced transcriptional response related to MiT-TFE signaling and cholesterol biosynthesis, pointing to lysosomal dysfunction. The lack of ESCRT-I promoted abnormal cholesterol accumulation on lysosomes and activated TFEB/TFE3 transcription factors in Ca2+-MCOLN1-dependent, but lipid-independent manner. Hence, our study provides evidence that ESCRT-I maintains lysosomal homeostasis and elucidates MiT-TFE regulatory mechanism activated in response to ESCRT-I deprivation

2022-03-28 | GSE178665 | GEO

Components of the ESCRT machinery act downstream of MLKL to regulate necroptotic cell death and its consequences

Project description:The activation of Mixed Lineage Kinase-Like (MLKL) by Receptor Interacting Protein Kinase-3 (RIPK3) results in plasma membrane (PM) disruption and a form of regulated necrosis, called necroptosis. Here we show that during necroptosis, MLKL-dependent calcium (Ca++) influx and phosphatidylserine (PS) exposure on the outer leaflet of the plasma membrane preceded loss of PM integrity. Activation of MLKL results in the generation of broken, PM “bubbles” with exposed PS that are released from the surface of the otherwise intact cell. Components of the ESCRT machinery are required for formation of these bubbles, and act to sustain survival of the cell when MLKL activation is limited or reversed. Under conditions of necroptotic cell death, ESCRT controls the duration of plasma membrane integrity. As a consequence of the action of ESCRT, cells undergoing necroptosis can express chemokines and other regulatory molecules, and promote antigenic cross-priming of CD8+ T cells.

2017-06-16 | GSE85660 | GEO

GPR143 regulates ESCRT-dependent exosome biogenesis and promotes cancer metastasis

Project description: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 identify GPR143, an atypical GPCR, as a regulator of the endosomal sorting complex required for 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 intralumenal vesicles (ILVs) in multivesicular bodies (MVBs). GPR143 is elevated in multiple cancers and quantitative proteomic and RNA profiling of exosomes revealed the GPR143-ESCRT pathway promotes secretion of exosomes that carry unique cargo, including integrins signaling proteins. By gain- and loss-of-function studies, we reveal GPR143 promotes metastasis by secreting exosomes and increasing cell motility/invasion through the integrin/FAK/Src pathway. These finding uncover a mechanism that regulates the exosomal proteome and demonstrate its ability to promote cancer metastasis.

2022-12-20 | PXD031515 | Pride

The ESCRT-III protein CHMP1A mediates secretion of sonic hedgehog on a novel class of extracellular vesicles

Project description:Extracellular vesicles (EVs) enable cell-to-cell communication in the nervous system essential for development and adult function. Endosomal Sorting Complex Required for Transport (ESCRT) complex proteins regulate EV formation and release. Recent work shows loss of function (LOF) mutations in, CHMP1A, which encodes one ESCRT-III member, cause autosomal recessive microcephaly with pontocerebellar hypoplasia in humans (Mochida et al., 2012). Here we show CHMP1A is required for maintenance of progenitors in human cerebral organoids and that mouse Chmp1a is required for progenitor proliferation in cortex and cerebellum and specifically for sonic hedgehog (SHH) mediated proliferation through SHH secretion. CHMP1A mutation reduces intraluminal vesicle (ILV) formation in multivesicular bodies (MVBs), and EV release. SHH protein is present on a subset of EVs marked by a unique set of proteins we call ART-EVs. CHMP1A’s requirement in formation of ART-EVs and other EVs provides a model to elucidate EV functions in multiple brain processes.

2018-06-16 | GSE115867 | GEO

Selective microautophagy of proteasomes is initiated by ESCRT-0 and is promoted by proteasome ubiquitylation

Project description:The proteasome is central to proteolysis by the ubiquitin-proteasome system under normal growth conditions but is itself degraded through macroautophagy under nutrient stress. A recently described AMPactivated protein kinase (AMPK)-regulated endosomal sorting complex required for transport (ESCRT)-dependent microautophagy pathway also regulates proteasome trafficking and degradation in low-glucose conditions in yeast. Aberrant proteasomes are more prone to microautophagy, suggesting the ESCRT system fine-tunes proteasome quality control under low-glucose stress. Here, we uncover additional features of the selective microautophagy of proteasomes in budding yeast. Genetic or pharmacological induction of aberrant proteasomes is associated with increased mono- or oligo-ubiquitylation of proteasome components, which appears to be recognized by ESCRT-0. AMPK controls this pathway in part by regulating the trafficking of ESCRT-0 to the vacuole surface, which also leads to degradation of the Vps27 subunit of ESCRT-0. The Rsp5 ubiquitin ligase contributes to proteasome subunit ubiquitylation, and multiple ubiquitin-binding elements in Vps27 are involved in their recognition.We propose that ESCRT-0 at the vacuole surface recognizes ubiquitylated proteasomes and initiates their microautophagic elimination during glucose depletion.

2022-02-06 | PXD031469 | iProX

The Plant Unique ESCRT Component FREE1 Regulates Autophagosome Closure

Project description:The energy sensor AMP-activated protein kinase (AMPK) can activate autophagy when cellular energy production becomes compromised. However, the degree to which nutrient sensing impinges on the autophagosome closure remains unknown. Here, we provide the mechanism underlying a plant unique protein FREE1, upon autophagy-induced SnRK1α1-mediated phosphorylation, functions as a linkage between ATG conjugation system and ESCRT machinery to regulate the autophagosome closure upon nutrient deprivation. Using high-resolution microscopy, 3D-electron tomography, and protease protection assay, we showed that unclosed autophagosomes accumulated in free1 mutants. Proteomic, cellular and biochemical analysis revealed the mechanistic connection between FREE1 and the ATG conjugation system/ESCRT-III complex in regulating autophagosome closure. Mass spectrometry analysis showed that the evolutionary conserved plant energy sensor SnRK1α1 phosphorylates FREE1 and recruits it to the autophagosomes to promote closure. Mutagenesis of the phosphorylation site on FREE1 caused the autophagosome closure failure. Our findings unveil how cellular energy sensing pathways regulate autophagosome closure to maintain cellular homeostasis.

2023-03-30 | PXD040435 | Pride

Concurrent depletion of Vps37 proteins evokes inflammatory response and cell growth inhibition via ESCRT-I destabilization.

Project description:Ample evidence points to the contribution of endocytosis to oncogenesis at the cellular level but molecular details still remain obscure. Our analysis of The Cancer Genome Atlas data involving cohorts of colorectal cancer (CRC) patients revealed stage-dependent alterations in the expression of 113 genes whose function is related to endocytic transport. Among differentially expressed genes we observed decreased transcription of VPS37B and to a lesser extent VPS37A in the advanced stages of CRC. One member of the Vps37 protein family (Vps37A, Vps37B, Vps37C, Vps37D) together with Tsg101 and Vps28 forms the core of the Endosomal Sorting Complex Required for Transport (ESCRT)-I mediating membrane-remodeling processes. Our analysis of an independent cohort of CRC patients corroborated that both VPS37B and VPS37A genes are downregulated at mRNA and protein levels. Depletion of both Vps37A and Vps37B proteins elicits multifaceted transcriptional alterations, including cell growth arrest and induction of sterile inflammatory response, which are further potentiated by co-silencing of VPS37C. Among analyzed silencing condition, concurrent knockdown of VPS37A, VPS37B and VPS37C evokes the greatest magnitude of p21-mediated inhibition of cell proliferation and activation of Nuclear Factor-κB and mitogen-activated protein kinases signaling. These processes are independently induced after co-silencing of VPS37 paralogs. We further show that the type and magnitude of transcriptional responses correlate with the ESCRT-I stability, which is differentially affected upon individual and concurrent Vps37 depletion. Our study provides novel insights into cancer cell biology by describing a cellular stress response that is associated with ESCRT-I destabilization, which might occur in CRC patients

2020-12-17 | GSE152195 | GEO

UMAD1 contributes to ESCRT-III dynamic subunit turnover during cytokinetic abscission

Project description:Abscission is the final stage of cytokinesis whereby the midbody, a thin intercellular bridge, is resolved to separate the daughter cells. Cytokinetic abscission is mediated by the Endosomal Sorting Complex Required for Transport (ESCRT), a conserved membrane remodelling machinery. The midbody organiser CEP55 recruits early acting ESCRT factors such as ESCRT-I and ALIX, which subsequently initiate the formation of ESCRT-III polymers that sever the midbody. We now identify UMAD1 as an ESCRT-I subunit that facilitates abscission. UMAD1 selectively associates with VPS37C and VPS37B, supporting the formation of cytokinesis-specific ESCRT-I assemblies. TSG101 recruits UMAD1 to the site of midbody abscission, to stabilise the CEP55/ESCRT-I interaction. We further demonstrate that the UMAD1/ESCRT-I interaction facilitates the final step of cytokinesis. Paradoxically, UMAD1 and ALIX co-depletion has synergistic effects on abscission whilst ESCRT-III recruitment to the midbody is not inhibited. Importantly, we find that both UMAD1 and ALIX are required for the dynamic exchange of ESCRT-III subunits at the midbody. Therefore, UMAD1 reveals a key functional connection between ESCRT-I and ESCRT-III that is required for cytokinesis.

2023-08-04 | PXD043940 | Pride

The Flemmingsome reveals an ESCRT-to-membrane coupling required for cytokinesis

Project description:Cytokinesis requires the constriction of ESCRT-III filaments on the midbody side, where abscission occurs. After ESCRT recruitment at the midbody, it is not known how the ESCRT-III machinery localizes to the abscission site. We obtained the proteome of intact, post-abscission midbodies (Flemmingsome) and revealed enriched proteins in this organelle. We propose that the ESCRT-III machinery must be physically coupled to a membrane protein at the cytokinetic abscission site for productive scission, revealing novel common requirements in cytokinesis, exosome formation and retroviral budding.

2020-05-22 | PXD013219 | Pride

ESCRTIII-associated protein AtALIX mediates high affinity phosphate transporter trafficking to maintain phosphate homeostasis in Arabidopsis

Project description:Prior to the release of their cargoes into the vacuolar lumen, sorting endosomes mature into multivesicular bodies (MVB) through the action of ENDOSOMAL COMPLEX REQUIRED FOR TRANSPORT (ESCRT) protein complexes. MVB-mediated sorting of high affinity phosphate transporters (PHT1) to the vacuole limits their plasma membrane levels under phosphate sufficient conditions, a process that allows plants to maintain phosphate homeostasis. We describe here AtALIX, a cytosolic protein that associates to MVB by interacting with ESCRT-III subunit SNF7 and enables PHT1;1 trafficking to the vacuole. Thus, we show that the partial loss of function mutant Atalix-1 displays reduced vacuolar degradation of PHT1;1. AtALIX versions containing the Atalix-1 mutation showed reduced interaction with SNF7, providing a simple molecular explanation for impaired cargo trafficking in Atalix-1 mutants. Indeed, Atalix-1 mutation also hampered vacuolar sorting of brassinosteroid receptor BRI1. In line with a presumed broad target spectrum, Atalix-1 mutation is pleiotropic, provoking reduced plant growth, late flowering and altered vacuole morphogenesis, whereas null mutants are lethal, indicating that AtALIX controls diverse processes in plants being essential for their life.

2015-09-09 | PXD002075 | Pride

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