Exosome Biogenesis in the Protozoa Parasite Giardia lamblia: A Model of Reduced Interorganellar Crosstalk

Extracellular vesicles (EVs) facilitate intercellular communication and are considered a promising therapeutic tool for the treatment of infectious diseases. These vesicles involve microvesicles (MVs) and exosomes and selectively transfer proteins, lipids, mRNAs, and microRNAs from one cell to another. While MVs are formed by extrusion of the plasma membrane, exosomes are a population of vesicles of endosomal origin that are stored inside the multivesicular bodies (MVBs) as intraluminal vesicles (ILVs) and are released when the MVBs fuse with the plasma membrane. Biogenesis of exosomes may be driven by the endosomal sorting complex required for transport (ESCRT) machinery or may be ESCRT independent, and it is still debated whether these are entirely separate pathways. In this manuscript, we report that the protozoan parasite, Giardia lamblia, although lacking a classical endo-lysosomal pathway, is able to produce and release exosome-like vesicles (ElV). By using a combination of biochemical and cell biology analyses, we found that the ElVs have the same size, shape, and protein and lipid composition as exosomes described for other eukaryotic cells. Moreover, we established that some endosome/lysosome peripheral vacuoles (PVs) contain ILV during the stationary phase. Our results indicate that ILV formation and ElV release depend on the ESCRT-associated AAA+-ATPase Vps4a, Rab11, and ceramide in this parasite. Interestingly, EIV biogenesis and release seems to occur in Giardia despite the fact that this parasite has lost most of the ESCRT machinery components during evolution and is unable to produce ceramide de novo. The differences in protozoa parasite EV composition, origin, and release may reveal functional and structural properties of EVs and, thus, may provide information on cell-to-cell communication and on survival mechanisms.

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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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Structure and mechanism of the ESCRT pathway AAA+ ATPase Vps4

Biochemical Society Transactions ◽

10.1042/bst20180260 ◽

2019 ◽

Vol 47 (1) ◽

pp. 37-45 ◽

Cited By ~ 11

Author(s):

Han Han ◽

Christopher P. Hill

Keyword(s):

Cell Biology ◽

Cellular Membrane ◽

Structural Features ◽

Transport Pathways ◽

Endosomal Sorting ◽

Aaa Atpase ◽

Protein Substrates ◽

Membrane Fission ◽

Aaa Atpases ◽

Equivalent Mechanism

Abstract The progression of ESCRT (Endosomal Sorting Complexes Required for Transport) pathways, which mediate numerous cellular membrane fission events, is driven by the enzyme Vps4. Understanding of Vps4 mechanism is, therefore, of fundamental importance in its own right and, moreover, it is highly relevant to the understanding of many related AAA+ ATPases that function in multiple facets of cell biology. Vps4 unfolds its ESCRT-III protein substrates by translocating them through its central hexameric pore, thereby driving membrane fission and recycling of ESCRT-III subunits. This mini-review focuses on recent advances in Vps4 structure and mechanism, including ideas about how Vps4 translocates and unfolds ESCRT-III subunits. Related AAA+ ATPases that share structural features with Vps4 and likely utilize an equivalent mechanism are also discussed.

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VPS37A directs ESCRT recruitment for phagophore closure

The Journal of Cell Biology ◽

10.1083/jcb.201902170 ◽

2019 ◽

Vol 218 (10) ◽

pp. 3336-3354 ◽

Cited By ~ 17

Author(s):

Yoshinori Takahashi ◽

Xinwen Liang ◽

Tatsuya Hattori ◽

Zhenyuan Tang ◽

Haiyan He ◽

...

Keyword(s):

Mammalian Cells ◽

Growth Factor Receptor ◽

Multivesicular Body ◽

Regulatory Mechanisms ◽

Endosomal Sorting ◽

Aaa Atpase ◽

Escrt Machinery ◽

Genome Wide ◽

A Genome ◽

Epidermal Growth

The process of phagophore closure requires the endosomal sorting complex required for transport III (ESCRT-III) subunit CHMP2A and the AAA ATPase VPS4, but their regulatory mechanisms remain unknown. Here, we establish a FACS-based HaloTag-LC3 autophagosome completion assay to screen a genome-wide CRISPR library and identify the ESCRT-I subunit VPS37A as a critical component for phagophore closure. VPS37A localizes on the phagophore through the N-terminal putative ubiquitin E2 variant domain, which is found to be required for autophagosome completion but dispensable for ESCRT-I complex formation and the degradation of epidermal growth factor receptor in the multivesicular body pathway. Notably, loss of VPS37A abrogates the phagophore recruitment of the ESCRT-I subunit VPS28 and CHMP2A, whereas inhibition of membrane closure by CHMP2A depletion or VPS4 inhibition accumulates VPS37A on the phagophore. These observations suggest that VPS37A coordinates the recruitment of a unique set of ESCRT machinery components for phagophore closure in mammalian cells.

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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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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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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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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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Arabidopsis SH3P2 is an ubiquitin-binding protein that functions together with ESCRT-I and the deubiquitylating enzyme AMSH3

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1710866114 ◽

2017 ◽

Vol 114 (34) ◽

pp. E7197-E7204 ◽

Cited By ~ 21

Author(s):

Marie-Kristin Nagel ◽

Kamila Kalinowska ◽

Karin Vogel ◽

Gregory D. Reynolds ◽

Zhixiang Wu ◽

...

Keyword(s):

Plasma Membrane ◽

Membrane Proteins ◽

Stress Responses ◽

Binding Protein ◽

In Planta ◽

Biotic And Abiotic Stress ◽

Plasma Membrane Proteins ◽

Endosomal Sorting ◽

Escrt Machinery ◽

Ubiquitin Binding

Clathrin-mediated endocytosis of plasma membrane proteins is an essential regulatory process that controls plasma membrane protein abundance and is therefore important for many signaling pathways, such as hormone signaling and biotic and abiotic stress responses. On endosomal sorting, plasma membrane proteins maybe recycled or targeted for vacuolar degradation, which is dependent on ubiquitin modification of the cargos and is driven by the endosomal sorting complexes required for transport (ESCRTs). Components of the ESCRT machinery are highly conserved among eukaryotes, but homologs of ESCRT-0 that are responsible for recognition and concentration of ubiquitylated proteins are absent in plants. Recently several ubiquitin-binding proteins have been identified that serve in place of ESCRT-0; however, their function in ubiquitin recognition and endosomal trafficking is not well understood yet. In this study, we identified Src homology-3 (SH3) domain-containing protein 2 (SH3P2) as a ubiquitin- and ESCRT-I–binding protein that functions in intracellular trafficking. SH3P2 colocalized with clathrin light chain-labeled punctate structures and interacted with clathrin heavy chain in planta, indicating a role for SH3P2 in clathrin-mediated endocytosis. Furthermore, SH3P2 cofractionates with clathrin-coated vesicles (CCVs), suggesting that it associates with CCVs in planta. Mutants of SH3P2 and VPS23 genetically interact, suggesting that they could function in the same pathway. Based on these results, we suggest a role of SH3P2 as an ubiquitin-binding protein that binds and transfers ubiquitylated proteins to the ESCRT machinery.

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T-cells play the classics with a different spin

Molecular Biology of the Cell ◽

10.1091/mbc.e13-11-0636 ◽

2014 ◽

Vol 25 (11) ◽

pp. 1699-1703 ◽

Cited By ~ 4

Author(s):

Michael L. Dustin

Keyword(s):

T Cells ◽

Plasma Membrane ◽

Immune Cells ◽

Cell Biology ◽

Immunological Synapse ◽

Cell Communication ◽

Intraflagellar Transport ◽

Cell Types ◽

Immune Synapse ◽

And Function

The immune system uses much of the classic machinery of cell biology, but in ways that put a different spin on organization and function. Striking recent examples include the demonstration of intraflagellar transport protein and hedgehog contributions to the immune synapse, even though immune cells lack a primary cilium that would be the typical setting for this machinery. In a second example, lymphocytes have their own subfamily of integrins, the β2 subfamily, and only integrins in this family form a stable adhesion ring using freely mobile ligands, a key feature of the immunological synapse. Finally, we showed recently that T-cells use endosomal sorting complexes required for transport (ESCRTs) at the plasma membrane to generate T-cell antigen receptor–enriched microvesicles. It is unusual for the ESCRT pathway to operate at the plasma membrane, but this may allow a novel form of cell–cell communication by providing a multivalent ligand for major histocompatibility complex–peptide complexes and perhaps other receptors on the partnering B-cell. Immune cells are thus an exciting system for novel cell biology even with classical pathways that have been studied extensively in other cell types.

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