Vps68 cooperates with ESCRT-III in intraluminal vesicle formation

Mapping Intimacies ◽

10.1101/2022.01.03.474785 ◽

2022 ◽

Author(s):

Sören Alsleben ◽

Ralf Kölling

Keyword(s):

Membrane Topology ◽

Endosomal Sorting ◽

Endosomal Membrane ◽

Amphipathic Helices ◽

Luminal Side ◽

Interaction Partners ◽

Associated Proteins ◽

Core Components ◽

Multivesicular Endosomes

The endosomal sorting complex required for transport (ESCRT)-III mediates budding and abscission of intraluminal vesicles (ILVs) into multivesicular endosomes. To further define the role of the ESCRT-III associated protein Mos10/Vps60 in ILV formation, we screened for new interaction partners by SILAC/MS. Here, we focused on the newly identified interaction partner Vps68. Our data suggest that Vps68 cooperates with ESCRT-III in ILV formation. The deletion of VPS68 caused a sorting defect similar to the SNF7 deletion, when the cargo load was high. The composition of ESCRT-III was altered, the level of core components was higher and the level of associated proteins was lower in the deletion strain. This suggests that a shift occurs from an active complex to a disassembly competent complex and that this shift is blocked in the Δvps68 strain. We present evidence that during this shift Snf7 is replaced by Mos10. Vps68 has an unusual membrane topology. Two of its potential membrane helices are amphipathic helices localized to the luminal side of the endosomal membrane. Based on this membrane topology we propose that Vps68 and ESCRT-III cooperate in the abscission step by weakening the luminal and cytosolic leaflets of the bilayer at the abscission site.

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Analysis of the dual function of the ESCRT-III protein Snf7 in endocytic trafficking and in gene expression

Biochemical Journal ◽

10.1042/bj20090957 ◽

2009 ◽

Vol 424 (1) ◽

pp. 89-97 ◽

Cited By ~ 20

Author(s):

Peter Weiss ◽

Stefanie Huppert ◽

Ralf Kölling

Keyword(s):

Endocytic Trafficking ◽

Dual Function ◽

Alkaline Ph ◽

Endosomal Sorting ◽

Endosomal Membrane ◽

Binding Partners ◽

Interaction Partners ◽

Pathway Function ◽

Multivesicular Endosomes ◽

Additional Role

ESCRT (endosomal sorting complex required for transport)-III mediates the budding and scission of intralumenal vesicles into multivesicular endosomes in yeast. For the main ESCRT-III subunit Snf7, an additional role in activation of the transcription factor Rim101 (the ‘Rim pathway’) is now also firmly established. In the present study, we investigate how these two Snf7 functions are related to each other. By generating SNF7 mutations that severely affect endocytic trafficking, but leave the Rim pathway function intact, we show that the two functions of SNF7 can be separated genetically. We analysed in detail how the SNF7 mutations affect the interaction of Snf7 with its various binding partners. Although the interactions with proteins Rim13 and Rim20, necessary for the Rim-pathway-related functions, were not altered by the mutations, there was a strong effect on interactions with components of the ESCRT pathway. The interactions, as measured by co-immunoprecipitation, with the ESCRT-III subunits Vps20 and Vps24 were strongly increased by the mutations, whereas the interactions with proteins Vps4 and Bro1, acting downstream of ESCRT-III, were reduced. As Vps4 is required for disassembly of ESCRT-III these results suggest that ESCRT-III is more stable in our SNF7 mutants. In line with this notion, a higher fraction of mutant Snf7 protein was detected at the membrane. Upon a shift to alkaline pH, a stronger binding signal for virtually all interaction partners, except Vps4, was observed. This indicates that the ESCRT network at the endosomal membrane is more extensive under these conditions.

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ESCRT-mediated sorting and intralumenal vesicle concatenation in plants

Biochemical Society Transactions ◽

10.1042/bst20170439 ◽

2018 ◽

Vol 46 (3) ◽

pp. 537-545 ◽

Cited By ~ 10

Author(s):

Marisa S. Otegui

Keyword(s):

Plasma Membrane ◽

Membrane Proteins ◽

Endocytic Pathway ◽

Endosomal Sorting ◽

Evolutionary Diversification ◽

Escrt Machinery ◽

Membrane Fission ◽

Associated Proteins

The degradation of plasma membrane and other membrane-associated proteins require their sorting at endosomes for delivery to the vacuole. Through the endocytic pathway, ubiquitinated membrane proteins (cargo) are delivered to endosomes where the ESCRT (endosomal sorting complex required for transport) machinery sorts them into intralumenal vesicles for degradation. Plants contain both conserved and plant-specific ESCRT subunits. In this review, I discuss the role of characterized plant ESCRT components, the evolutionary diversification of the plant ESCRT machinery, and a recent study showing that endosomal intralumenal vesicles form in clusters of concatenated vesicle buds by temporally uncoupling membrane constriction from membrane fission.

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Comprehensive analysis of yeast ESCRT-III composition in single ESCRT-III deletion mutants

Biochemical Journal ◽

10.1042/bcj20190141 ◽

2019 ◽

Vol 476 (14) ◽

pp. 2031-2046 ◽

Cited By ~ 2

Author(s):

Christian Heinzle ◽

Lara Mücke ◽

Thomas Brune ◽

Ralf Kölling

Keyword(s):

Family Members ◽

Cell Fractionation ◽

Endosomal Sorting ◽

Cellular Processes ◽

Associated Proteins ◽

Bona Fide ◽

Main Component ◽

The Impact

Abstract The endosomal sorting complex required for transport (ESCRT)-III is associated with a multitude of cellular processes involving membrane remodeling and abscission. The exact composition of ESCRT-III and the contribution of individual ESCRT-III family members to these diverse functions is unclear. Most of the currently available information about ESCRT-III was obtained with tagged, largely non-functional proteins, which may not correctly reflect the in vivo situation. Here, we performed a comprehensive biochemical analysis of ESCRT-III localization and composition in yeast under purely native conditions. Most of our findings are in line with the current concepts about ESCRT-III, but some findings are unexpected and call for adjustments to the model. In particular, our data suggest that the distinction between bona fide ESCRT-III components and ESCRT-III associated proteins is not justified. We detected a single complex containing all ESCRT-III members (except of Chm7) with Did2 as its main component. The classical core components were present in equimolar amounts. Our analysis of the impact of single deletions on the composition of ESCRT-III confirmed the central role of Snf7 for ESCRT-III assembly. For the other ESCRT-III family members predictions could be made about their role in ESCRT-III assembly. Furthermore, our cell fractionation points to a role of Vps20 at the endoplasmic reticulum.

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The role of ESCRT proteins in fusion events involving lysosomes, endosomes and autophagosomes

Biochemical Society Transactions ◽

10.1042/bst0381469 ◽

2010 ◽

Vol 38 (6) ◽

pp. 1469-1473 ◽

Cited By ~ 50

Author(s):

Daniel Metcalf ◽

Adrian M. Isaacs

Keyword(s):

Bacterial Pathogen ◽

Multivesicular Body ◽

Membrane Curvature ◽

Multivesicular Bodies ◽

Lysosomal Storage ◽

Endosomal Sorting ◽

Endosomal Membrane ◽

Pathogen Invasion ◽

Insight Into

ESCRT (endosomal sorting complex required for transport) proteins were originally identified for their role in delivering endocytosed proteins to the intraluminal vesicles of late-endosomal structures termed multivesicular bodies. Multivesicular bodies then fuse with lysosomes, leading to degradation of the internalized proteins. Four ESCRT complexes interact to concentrate cargo on the endosomal membrane, induce membrane curvature to form an intraluminal bud and finally pinch off the bud through a membrane-scission event to produce the intraluminal vesicle. Recent work suggests that ESCRT proteins are also required downstream of these events to enable fusion of multivesicular bodies with lysosomes. Autophagy is a related pathway required for the degradation of organelles, long-lived proteins and protein aggregates which also converges on lysosomes. The proteins or organelle to be degraded are encapsulated by an autophagosome that fuses either directly with a lysosome or with an endosome to form an amphisome, which then fuses with a lysosome. A common machinery is beginning to emerge that regulates fusion events in the multivesicular body and autophagy pathways, and we focus in the present paper on the role of ESCRT proteins. These fusion events have been implicated in diseases including frontotemporal dementia, Alzheimer's disease, lysosomal storage disorders, myopathies and bacterial pathogen invasion, and therefore further examination of the mechanisms involved may lead to new insight into disease pathogenesis and treatments.

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The role of ESCRT proteins in attenuation of cell signalling

Biochemical Society Transactions ◽

10.1042/bst0370137 ◽

2009 ◽

Vol 37 (1) ◽

pp. 137-142 ◽

Cited By ~ 20

Author(s):

Lina M. Rodahl ◽

Susanne Stuffers ◽

Viola H. Lobert ◽

Harald Stenmark

Keyword(s):

Cell Proliferation ◽

Growth Factor ◽

Membrane Proteins ◽

Protein Complexes ◽

Cell Signalling ◽

Control Cell ◽

Endosomal Sorting ◽

Tumour Suppression ◽

Multivesicular Endosomes

The ESCRT (endosomal sorting complex required for transport) machinery consists of four protein complexes that mediate sorting of ubiquitinated membrane proteins into the intraluminal vesicles of multivesicular endosomes, thereby targeting them for degradation in lysosomes. In the present paper, we review how ESCRT-mediated receptor down-regulation affects signalling downstream of Notch and growth factor receptors, and how ESCRTs may control cell proliferation, survival and cytoskeletal functions and contribute to tumour suppression.

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POPDC proteins and cardiac function

Biochemical Society Transactions ◽

10.1042/bst20190249 ◽

2019 ◽

Vol 47 (5) ◽

pp. 1393-1404 ◽

Cited By ~ 4

Author(s):

Thomas Brand

Keyword(s):

Potential Role ◽

Striated Muscle ◽

Short Review ◽

Effector Proteins ◽

Muscle Disease ◽

Disease Genes ◽

Protein Protein Interaction ◽

Interaction Partners ◽

And Function

Abstract The Popeye domain-containing gene family encodes a novel class of cAMP effector proteins in striated muscle tissue. In this short review, we first introduce the protein family and discuss their structure and function with an emphasis on their role in cyclic AMP signalling. Another focus of this review is the recently discovered role of POPDC genes as striated muscle disease genes, which have been associated with cardiac arrhythmia and muscular dystrophy. The pathological phenotypes observed in patients will be compared with phenotypes present in null and knockin mutations in zebrafish and mouse. A number of protein–protein interaction partners have been discovered and the potential role of POPDC proteins to control the subcellular localization and function of these interacting proteins will be discussed. Finally, we outline several areas, where research is urgently needed.

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Towards unravelling the role of the lipid droplet-associated proteins perilipin, adipophilin, and TIP47 in hepatocellular carcinoma

Zeitschrift für Gastroenterologie ◽

10.1055/s-0029-1191848 ◽

2009 ◽

Vol 47 (01) ◽

Author(s):

BK Straub ◽

S Singer ◽

J Lehmann-Koch ◽

H Heid ◽

E Specht ◽

...

Keyword(s):

Hepatocellular Carcinoma ◽

Lipid Droplet ◽

Associated Proteins

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A novel putative microtubule-associated protein is involved in arbuscule development during arbuscular mycorrhiza formation

Plant and Cell Physiology ◽

10.1093/pcp/pcaa159 ◽

2021 ◽

Author(s):

Tania Ho-Plágaro ◽

Raúl Huertas ◽

María I Tamayo-Navarrete ◽

Elison Blancaflor ◽

Nuria Gavara ◽

...

Keyword(s):

Plant Root ◽

Arbuscular Mycorrhizal ◽

Host Cells ◽

Root Cells ◽

Filament Dynamics ◽

Fungal Structure ◽

Genes Encoding ◽

Associated Proteins ◽

Mycorrhizal Development

Abstract The formation of arbuscular mycorrhizal (AM) symbiosis requires plant root host cells to undergo major structural and functional reprogramming in order to house the highly branched AM fungal structure for the reciprocal exchange of nutrients. These morphological modifications are associated with cytoskeleton remodelling. However, molecular bases and the role of microtubules (MTs) and actin filament dynamics during AM formation are largely unknown. In this study, the tomato tsb gene, belonging to a Solanaceae group of genes encoding MT-associated proteins for pollen development, was found to be highly expressed in root cells containing arbuscules. At earlier stages of mycorrhizal development, tsb overexpression enhanced the formation of highly developed and transcriptionally active arbuscules, while tsb silencing hampers the formation of mature arbuscules and represses arbuscule functionality. However, at later stages of mycorrhizal colonization, tsb OE roots accumulate fully developed transcriptionally inactive arbuscules, suggesting that the collapse and turnover of arbuscules might be impaired by TSB accumulation. Imaging analysis of the MT cytoskeleton in cortex root cells overexpressing tsb revealed that TSB is involved in MT-bundling. Taken together, our results provide unprecedented insights into the role of novel MT-associated protein in MT rearrangements throughout the different stages of the arbuscule life cycle.

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Membrane-associated phase separation: organization and function emerge from a two-dimensional milieu

Journal of Molecular Cell Biology ◽

10.1093/jmcb/mjab010 ◽

2021 ◽

Author(s):

Jonathon A Ditlev

Keyword(s):

Phase Separation ◽

Cell Biology ◽

Cellular Environment ◽

Condensate Formation ◽

Associated Proteins ◽

Available Technology ◽

And Function ◽

Surrounding Membrane

Abstract Liquid‒liquid phase separation (LLPS) of biomolecules has emerged as an important mechanism that contributes to cellular organization. Phase separated biomolecular condensates, or membrane-less organelles, are compartments composed of specific biomolecules without a surrounding membrane in the nucleus and cytoplasm. LLPS also occurs at membranes, where both lipids and membrane-associated proteins can de-mix to form phase separated compartments. Investigation of these membrane-associated condensates using in vitro biochemical reconstitution and cell biology has provided key insights into the role of phase separation in membrane domain formation and function. However, these studies have generally been limited by available technology to study LLPS on model membranes and the complex cellular environment that regulates condensate formation, composition, and function. Here, I briefly review our current understanding of membrane-associated condensates, establish why LLPS can be advantageous for certain membrane-associated condensates, and offer a perspective for how these condensates may be studied in the future.

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