Membrane manipulations by the ESCRT machinery

The endosomal sorting complexes required for transport (ESCRTs) collectively comprise a machinery that was first known for its function in the degradation of transmembrane proteins in the endocytic pathway of eukaryotic cells. Since their discovery, however, ESCRTs have been recognized as playing important roles at the plasma membrane, which appears to be the original site of function for the ESCRT machinery. This article reviews some of the major research findings that have shaped our current understanding of how the ESCRT machinery controls membrane dynamics and considers new roles for the ESCRT machinery that might be driven by these mechanisms.

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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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Tracking the Fate of Genetically Distinct Vesicular Stomatitis Virus Matrix Proteins Highlights the Role for Late Domains in Assembly

Journal of Virology ◽

10.1128/jvi.01371-15 ◽

2015 ◽

Vol 89 (23) ◽

pp. 11750-11760 ◽

Cited By ~ 10

Author(s):

Timothy K. Soh ◽

Sean P. J. Whelan

Keyword(s):

Plasma Membrane ◽

Vesicular Stomatitis Virus ◽

Matrix Protein ◽

Fluorescent Proteins ◽

Endosomal Sorting ◽

Escrt Machinery ◽

Viral Particles ◽

The Matrix ◽

Vesicular Stomatitis ◽

Late Domains

ABSTRACTVesicular stomatitis virus (VSV) assembly requires condensation of the viral ribonucleoprotein (RNP) core with the matrix protein (M) during budding from the plasma membrane. The RNP core comprises the negative-sense genomic RNA completely coated by the nucleocapsid protein (N) and associated by a phosphoprotein (P) with the large polymerase protein (L). To study the assembly of single viral particles, we tagged M and P with fluorescent proteins. We selected from a library of viruses with insertions in the M gene a replication-competent virus containing a fluorescent M and combined that with our previously described virus containing fluorescent P. Virus particles containing those fusions maintained the same bullet shape appearance as wild-type VSV but had a modest increase in particle length, reflecting the increased genome size. Imaging of the released particles revealed a variation in the amount of M and P assembled into the virions, consistent with a flexible packaging mechanism. We used the recombinants to further study the importance of the late domains in M, which serve to recruit the endosomal sorting complex required for transport (ESCRT) machinery during budding. Mutations in late domains resulted in the accumulation of virions that failed to pinch off from the plasma membrane. Imaging of single virions released from cells that were coinfected with M tagged with enhanced green fluorescent protein and M tagged with mCherry variants in which the late domains of one virus were inactivated by mutation showed a strong bias against the incorporation of the late-domain mutant into the released virions. In contrast, the intracellular expression and membrane association of the two variants were unaltered. These studies provide new tools for imaging particle assembly and enhance our resolution of existing models for assembly of VSV.IMPORTANCEAssembly of vesicular stomatitis virus (VSV) particles requires the separate trafficking of the viral replication machinery, a matrix protein (M) and a glycoprotein, to the plasma membrane. The matrix protein contains a motif termed a “late domain” that engages the host endosomal sorting complex required for transport (ESCRT) machinery to facilitate the release of viral particles. Inactivation of the late domains through mutation results in the accumulation of virions arrested at the point of release. In the study described here, we developed new tools to study VSV assembly by fusing fluorescent proteins to M and to a constituent of the replication machinery, the phosphoprotein (P). We used those tools to show that the late domains of M are required for efficient incorporation into viral particles and that the particles contain a variable quantity of M and P.

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ESCRT-dependent targeting of plasma membrane localized KCa3.1 to the lysosomes

AJP Cell Physiology ◽

10.1152/ajpcell.00120.2010 ◽

2010 ◽

Vol 299 (5) ◽

pp. C1015-C1027 ◽

Cited By ~ 19

Author(s):

Corina M. Balut ◽

Yajuan Gao ◽

Sandra A. Murray ◽

Patrick H. Thibodeau ◽

Daniel C. Devor

Keyword(s):

Plasma Membrane ◽

Molecular Mechanisms ◽

Close Association ◽

Significant Inhibition ◽

Dominant Negative ◽

Human Embryonic Kidney Cells ◽

Endosomal Sorting ◽

Escrt Machinery ◽

Interfering Rna

The number of intermediate-conductance, Ca2+-activated K+ channels (KCa3.1) present at the plasma membrane is deterministic in any physiological response. However, the mechanisms by which KCa3.1 channels are removed from the plasma membrane and targeted for degradation are poorly understood. Recently, we demonstrated that KCa3.1 is rapidly internalized from the plasma membrane, having a short half-life in both human embryonic kidney cells (HEK293) and human microvascular endothelial cells (HMEC-1). In this study, we investigate the molecular mechanisms controlling the degradation of KCa3.1 heterologously expressed in HEK and HMEC-1 cells. Using immunofluorescence and electron microscopy, as well as quantitative biochemical analysis, we demonstrate that membrane KCa3.1 is targeted to the lysosomes for degradation. Furthermore, we demonstrate that either overexpressing a dominant negative Rab7 or short interfering RNA-mediated knockdown of Rab7 results in a significant inhibition of channel degradation rate. Coimmunoprecipitation confirmed a close association between Rab7 and KCa3.1. On the basis of these findings, we assessed the role of the ESCRT machinery in the degradation of heterologously expressed KCa3.1, including TSG101 [endosomal sorting complex required for transport (ESCRT)-I] and CHMP4 (ESCRT-III) as well as VPS4, a protein involved in the disassembly of the ESCRT machinery. We demonstrate that TSG101 is closely associated with KCa3.1 via coimmunoprecipitation and that a dominant negative TSG101 inhibits KCa3.1 degradation. In addition, both dominant negative CHMP4 and VPS4 significantly decrease the rate of membrane KCa3.1 degradation, compared with wild-type controls. These results are the first to demonstrate that plasma membrane-associated KCa3.1 is targeted for lysosomal degradation via a Rab7 and ESCRT-dependent pathway.

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TOR complex 2 (TORC2) signaling and the ESCRT machinery cooperate in the protection of plasma membrane integrity in yeast

Journal of Biological Chemistry ◽

10.1074/jbc.ra120.013222 ◽

2020 ◽

Vol 295 (34) ◽

pp. 12028-12044

Author(s):

Oliver Schmidt ◽

Yannick Weyer ◽

Simon Sprenger ◽

Michael A. Widerin ◽

Sebastian Eising ◽

...

Keyword(s):

Plasma Membrane ◽

Membrane Integrity ◽

Homeostatic Regulation ◽

Membrane Tension ◽

Membrane Remodeling ◽

Plasma Membrane Integrity ◽

Endosomal Sorting ◽

Escrt Machinery ◽

Evolutionarily Conserved ◽

Calcineurin Activity

The endosomal sorting complexes required for transport (ESCRT) mediate evolutionarily conserved membrane remodeling processes. Here, we used budding yeast (Saccharomyces cerevisiae) to explore how the ESCRT machinery contributes to plasma membrane (PM) homeostasis. We found that in response to reduced membrane tension and inhibition of TOR complex 2 (TORC2), ESCRT-III/Vps4 assemblies form at the PM and help maintain membrane integrity. In turn, the growth of ESCRT mutants strongly depended on TORC2-mediated homeostatic regulation of sphingolipid (SL) metabolism. This was caused by calcineurin-dependent dephosphorylation of Orm2, a repressor of SL biosynthesis. Calcineurin activity impaired Orm2 export from the endoplasmic reticulum (ER) and thereby hampered its subsequent endosome and Golgi-associated degradation (EGAD). The ensuing accumulation of Orm2 at the ER in ESCRT mutants necessitated TORC2 signaling through its downstream kinase Ypk1, which repressed Orm2 and prevented a detrimental imbalance of SL metabolism. Our findings reveal compensatory cross-talk between the ESCRT machinery, calcineurin/TORC2 signaling, and the EGAD pathway important for the regulation of SL biosynthesis and the maintenance of PM homeostasis.

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Mvb12 Is a Novel Member of ESCRT-I Involved in Cargo Selection by the Multivesicular Body Pathway

Molecular Biology of the Cell ◽

10.1091/mbc.e06-07-0601 ◽

2007 ◽

Vol 18 (2) ◽

pp. 646-657 ◽

Cited By ~ 34

Author(s):

Andrea J. Oestreich ◽

Brian A. Davies ◽

Johanna A. Payne ◽

David J. Katzmann

Keyword(s):

Normal Cell ◽

Transmembrane Proteins ◽

Multivesicular Body ◽

Eukaryotic Cells ◽

Cell Physiology ◽

Cellular Functions ◽

Protein Loss ◽

Endosomal Sorting ◽

Vacuolar Protein ◽

Selection Of

The multivesicular body (MVB) sorting pathway impacts a variety of cellular functions in eukaryotic cells. Perhaps the best understood role for the MVB pathway is the degradation of transmembrane proteins within the lysosome. Regulation of cargo selection by this pathway is critically important for normal cell physiology, and recent advances in our understanding of this process have highlighted the endosomal sorting complexes required for transport (ESCRTs) as pivotal players in this reaction. To better understand the mechanisms of cargo selection during MVB sorting, we performed a genetic screen to identify novel factors required for cargo-specific selection by this pathway and identified the Mvb12 protein. Loss of Mvb12 function results in differential defects in the selection of MVB cargoes. A variety of analyses indicate that Mvb12 is a stable member of ESCRT-I, a heterologous complex involved in cargo selection by the MVB pathway. Phenotypes displayed upon loss of Mvb12 are distinct from those displayed by the previously described ESCRT-I subunits (vacuolar protein sorting 23, -28, and -37), suggesting a distinct function than these core subunits. These data support a model in which Mvb12 impacts the selection of MVB cargoes by modulating the cargo recognition capabilities of ESCRT-I.

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Multiple roles for the ESCRT machinery in maintaining plasma membrane homeostasis

10.1101/2020.02.25.964452 ◽

2020 ◽

Author(s):

Oliver Schmidt ◽

Yannick Weyer ◽

Simon Sprenger ◽

Michael A. Widerin ◽

Sebastian Eising ◽

...

Keyword(s):

Plasma Membrane ◽

Membrane Integrity ◽

Multiple Roles ◽

Membrane Tension ◽

Membrane Remodeling ◽

Membrane Stress ◽

Endosomal Sorting ◽

Protein Levels ◽

Escrt Machinery ◽

Calcineurin Phosphatase

ABSTRACTThe endosomal sorting complexes required for transport (ESCRT) execute evolutionary conserved membrane remodeling processes. Here we used budding yeast to explore how the ESCRT machinery contributes to plasma membrane (PM) homeostasis. In response to reduced membrane tension and inhibition of the target of rapamycin complex 2 (TORC2), ESCRT-III/Vps4 assemblies form at the PM and help to maintain membrane integrity. Conversely, the growth of ESCRT mutants strongly depends on TORC2-mediated homeostatic regulation of sphingolipid (SL) metabolism. This is caused by calcineurin phosphatase activity which causes Orm2 to accumulate at the endoplasmic reticulum (ER) in ESCRT mutants. Orm2 is a repressor of SL biosynthesis and its accumulation provokes increased membrane stress. This necessitates TORC2 signaling through its downstream kinase Ypk1 to control Orm2 protein levels and prevent a detrimental imbalance of SL metabolism. Our findings reveal new aspects of antagonistic calcineurin/TORC2 signaling for the regulation of SL biosynthesis and the maintenance of PM homeostasis, and suggest that the ESCRT machinery contributes directly and indirectly to these processes.

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Efficient Cargo Sorting by ESCRT-I and the Subsequent Release of ESCRT-I from Multivesicular Bodies Requires the Subunit Mvb12

Molecular Biology of the Cell ◽

10.1091/mbc.e06-07-0588 ◽

2007 ◽

Vol 18 (2) ◽

pp. 636-645 ◽

Cited By ~ 45

Author(s):

Matt Curtiss ◽

Charles Jones ◽

Markus Babst

Keyword(s):

Protein Complex ◽

Transmembrane Proteins ◽

Multivesicular Body ◽

Multivesicular Bodies ◽

Rapid Degradation ◽

Endosomal Sorting ◽

Escrt Machinery ◽

Complex Functions ◽

Vacuolar Protein ◽

Cargo Sorting

The endosomal sorting complex required for transport (ESCRT)-I protein complex functions in recognition and sorting of ubiquitinated transmembrane proteins into multivesicular body (MVB) vesicles. It has been shown that ESCRT-I contains the vacuolar protein sorting (Vps) proteins Vps23, Vps28, and Vps37. We identified an additional subunit of yeast ESCRT-I called Mvb12, which seems to associate with ESCRT-I by binding to Vps37. Transient recruitment of ESCRT-I to MVBs results in the rapid degradation of Mvb12. In contrast to mutations in other ESCRT-I subunits, which result in strong defects in MVB cargo sorting, deletion of MVB12 resulted in only a partial sorting phenotype. This trafficking defect was fully suppressed by overexpression of the ESCRT-II complex. Mutations in MVB12 did not affect recruitment of ESCRT-I to MVBs, but they did result in delivery of ESCRT-I to the vacuolar lumen via the MVB pathway. Together, these observations suggest that Mvb12 may function in regulating the interactions of ESCRT-I with cargo and other proteins of the ESCRT machinery to efficiently coordinate cargo sorting and release of ESCRT-I from the MVB.

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Evidence for Apical Endocytosis in Polarized Hepatic Cells: Phosphoinositide 3-Kinase Inhibitors Lead to the Lysosomal Accumulation of Resident Apical Plasma Membrane Proteins

The Journal of Cell Biology ◽

10.1083/jcb.145.5.1089 ◽

1999 ◽

Vol 145 (5) ◽

pp. 1089-1102 ◽

Cited By ~ 57

Author(s):

Pamela L. Tuma ◽

Catherine M. Finnegan ◽

Ji-Hyun Yi ◽

Ann L. Hubbard

Keyword(s):

Plasma Membrane ◽

Membrane Proteins ◽

Epithelial Cells ◽

Kinase Inhibitors ◽

Apical Membrane ◽

Membrane Dynamics ◽

Endocytic Pathway ◽

Apical Plasma Membrane ◽

Plasma Membrane Proteins ◽

Phosphoinositide 3 Kinase

The architectural complexity of the hepatocyte canalicular surface has prevented examination of apical membrane dynamics with methods used for other epithelial cells. By adopting a pharmacological approach, we have documented for the first time the internalization of membrane proteins from the hepatic apical surface. Treatment of hepatocytes or WIF-B cells with phosphoinositide 3-kinase inhibitors, wortmannin or LY294002, led to accumulation of the apical plasma membrane proteins, 5′-nucleotidase and aminopeptidase N in lysosomal vacuoles. By monitoring the trafficking of antibody-labeled molecules, we determined that the apical proteins in vacuoles came from the apical plasma membrane. Neither newly synthesized nor transcytosing apical proteins accumulated in vacuoles. In wortmannin-treated cells, transcytosing apical proteins traversed the subapical compartment (SAC), suggesting that this intermediate in the basolateral-to-apical transcytotic pathway remained functional. Ultrastructural analysis confirmed these results. However, apically internalized proteins did not travel through SAC en route to lysosomal vacuoles, indicating that SAC is not an intermediate in the apical endocytic pathway. Basolateral membrane protein distributions did not change in treated cells, uncovering another difference in endocytosis from the two domains. Similar effects were observed in polarized MDCK cells, suggesting conserved patterns of phosphoinositide 3-kinase regulation among epithelial cells. These results confirm a long-held but unproven assumption that lysosomes are the final destination of apical membrane proteins in hepatocytes. Significantly, they also confirm our hypothesis that SAC is not an apical endosome.

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Identification of an inhibitory budding signal that blocks the release of HIV particles and exosome/microvesicle proteins

Molecular Biology of the Cell ◽

10.1091/mbc.e10-07-0625 ◽

2011 ◽

Vol 22 (6) ◽

pp. 817-830 ◽

Cited By ~ 42

Author(s):

Xin Gan ◽

Stephen J. Gould

Keyword(s):

Plasma Membrane ◽

Plasma Membranes ◽

Animal Cells ◽

Virus Budding ◽

Gag Protein ◽

Endosomal Sorting ◽

Escrt Machinery ◽

Cargo Delivery ◽

Immunodeficiency Virus ◽

Cargo Proteins

Animal cells bud exosomes and microvesicles (EMVs) from endosome and plasma membranes. The combination of higher-order oligomerization and plasma membrane binding is a positive budding signal that targets diverse proteins into EMVs and retrovirus particles. Here we describe an inhibitory budding signal (IBS) from the human immunodeficiency virus (HIV) Gag protein. This IBS was identified in the spacer peptide 2 (SP2) domain of Gag, is activated by C-terminal exposure of SP2, and mediates the severe budding defect of p6-deficient and PTAP-deficient strains of HIV. This IBS also impairs the budding of CD63 and several other viral and nonviral EMV proteins. The IBS does not prevent cargo delivery to the plasma membrane, a major site of EMV and virus budding. However, the IBS does inhibit an interaction between EMV cargo proteins and VPS4B, a component of the endosomal sorting complexes required for transport (ESCRT) machinery. Taken together, these results demonstrate that inhibitory signals can block protein and virus budding, raise the possibility that the ESCRT machinery plays a role in EMV biogenesis, and shed new light on the role of the p6 domain and PTAP motif in the biogenesis of HIV particles.

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Retromer and sorting nexins in endosomal sorting

Biochemical Society Transactions ◽

10.1042/bst20140290 ◽

2015 ◽

Vol 43 (1) ◽

pp. 33-47 ◽

Cited By ~ 108

Author(s):

Matthew Gallon ◽

Peter J. Cullen

Keyword(s):

Plasma Membrane ◽

Present Article ◽

Transmembrane Proteins ◽

Molecular Architecture ◽

Sorting Nexins ◽

Endosomal Sorting ◽

Sorting Nexin ◽

Cargo Proteins ◽

Associated Proteins ◽

Lysosome Related Organelles

The evolutionarily conserved endosomal retromer complex rescues transmembrane proteins from the lysosomal degradative pathway and facilitates their recycling to other cellular compartments. Retromer functions in conjunction with numerous associated proteins, including select members of the sorting nexin (SNX) family. In the present article, we review the molecular architecture and cellular roles of retromer and its various functional partners. The endosomal network is a crucial hub in the trafficking of proteins through the cellular endomembrane system. Transmembrane proteins, here termed cargos, enter endosomes by endocytosis from the plasma membrane or by trafficking from the trans-Golgi network (TGN). Endosomal cargo proteins face one of the two fates: retention in the endosome, leading ultimately to lysosomal degradation or export from the endosome for reuse (‘recycling’). The balance of protein degradation and recycling is crucial to cellular homoeostasis; inappropriate sorting of proteins to either fate leads to cellular dysfunction. Retromer is an endosome-membrane-associated protein complex central to the recycling of many cargo proteins from endosomes, both to the TGN and the plasma membrane (and other specialized compartments, e.g. lysosome-related organelles). Retromer function is reliant on a number of proteins from the SNX family. In the present article, we discuss this inter-relationship and how defects in retromer function are increasingly being linked with human disease.

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