A Bioinformatical Approach to Study the Endosomal Sorting Complex Required for Transport (ESCRT) Machinery in Protozoan Parasites: The Entamoeba histolytica Case

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.

Download Full-text

Entamoeba histolytica: identification of thioredoxin-targeted proteins and analysis of serine acetyltransferase-1 as a prototype example

Biochemical Journal ◽

10.1042/bj20121798 ◽

2013 ◽

Vol 451 (2) ◽

pp. 277-288 ◽

Cited By ~ 24

Author(s):

Sarah Schlosser ◽

David Leitsch ◽

Michael Duchêne

Keyword(s):

Entamoeba Histolytica ◽

Active Site ◽

Gel Electrophoresis ◽

Biosynthetic Pathway ◽

Interacting Proteins ◽

Protozoan Parasites ◽

Serine Acetyltransferase ◽

Two Dimensional Gel Electrophoresis ◽

Mixed Disulfide ◽

Far Western Blot

Entamoeba histolytica, the causative agent of amoebiasis, possesses the dithiol-containing redox proteins Trx (thioredoxin) and TrxR (Trx reductase). Both proteins were found to be covalently modified and inactivated by metronidazole, a 5-nitroimidazole drug that is commonly used to treat infections with microaerophilic protozoan parasites in humans. Currently, very little is known about enzymes and other proteins participating in the Trx-dependent redox network of the parasite that could be indirectly affected by metronidazole treatment. On the basis of the disulfide/dithiol-exchange mechanism we constructed an active-site mutant of Trx, capable of binding interacting proteins as a stable mixed disulfide intermediate to screen the target proteome of Trx in E. histolytica. By applying Trx affinity chromatography, two-dimensional gel electrophoresis and MS, peroxiredoxin and 15 further potentially redox-regulated proteins were identified. Among them, EhSat1 (E. histolytica serine acetyltransferase-1), an enzyme involved in the L-cysteine biosynthetic pathway, was selected for detailed analysis. Binding of Trx to EhSat1 was verified by Far-Western blot analysis. Trx was able to restore the activity of the oxidatively damaged EhSat1 suggesting that the TrxR/Trx system protects sensitive proteins against oxidative stress in E. histolytica. Furthermore, the activity of peroxiredoxin, which is dependent on a functioning TrxR/Trx system, was strongly reduced in metronidazole-treated parasites.

Download Full-text

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.

Download Full-text

Dynamics of endosomal sorting complex required for transport (ESCRT) machinery during cytokinesis and its role in abscission

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1102714108 ◽

2011 ◽

Vol 108 (12) ◽

pp. 4846-4851 ◽

Cited By ~ 255

Author(s):

N. Elia ◽

R. Sougrat ◽

T. A. Spurlin ◽

J. H. Hurley ◽

J. Lippincott-Schwartz

Keyword(s):

Endosomal Sorting ◽

Escrt Machinery

Download Full-text

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.

Download Full-text

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.

Download Full-text

Syntaxin 16 is a master recruitment factor for cytokinesis

Molecular Biology of the Cell ◽

10.1091/mbc.e13-06-0302 ◽

2013 ◽

Vol 24 (23) ◽

pp. 3663-3674 ◽

Cited By ~ 28

Author(s):

Hélia Neto ◽

Alexandra Kaupisch ◽

Louise L. Collins ◽

Gwyn W. Gould

Keyword(s):

Membrane Vesicles ◽

Attachment Protein ◽

Recycling Endosome ◽

Endosomal Sorting ◽

Escrt Machinery ◽

Protein Receptor ◽

Sensitive Factor ◽

Late Telophase ◽

Intracellular Organelles ◽

Factor Attachment Protein Receptor

Recently it was shown that both recycling endosome and endosomal sorting complex required for transport (ESCRT) components are required for cytokinesis, in which they are believed to act in a sequential manner to bring about secondary ingression and abscission, respectively. However, it is not clear how either of these complexes is targeted to the midbody and whether their delivery is coordinated. The trafficking of membrane vesicles between different intracellular organelles involves the formation of soluble N-ethylmaleimide–sensitive factor attachment protein receptor (SNARE) complexes. Although membrane traffic is known to play an important role in cytokinesis, the contribution and identity of intracellular SNAREs to cytokinesis remain unclear. Here we demonstrate that syntaxin 16 is a key regulator of cytokinesis, as it is required for recruitment of both recycling endosome–associated Exocyst and ESCRT machinery during late telophase, and therefore that these two distinct facets of cytokinesis are inextricably linked.

Download Full-text

Detection of the Endosomal Sorting Complex Required for Transport inEntamoeba histolyticaand Characterization of the EhVps4 Protein

Journal of Biomedicine and Biotechnology ◽

10.1155/2010/890674 ◽

2010 ◽

Vol 2010 ◽

pp. 1-15 ◽

Cited By ~ 12

Author(s):

Israel López-Reyes ◽

Guillermina García-Rivera ◽

Cecilia Bañuelos ◽

Silvia Herranz ◽

Olivier Vincent ◽

...

Keyword(s):

Plasma Membrane ◽

Entamoeba Histolytica ◽

Hepatic Damage ◽

Multivesicular Bodies ◽

Protein Overexpression ◽

Specific Antibodies ◽

Endosomal Sorting ◽

Biochemical Studies

Eukaryotic endocytosis involves multivesicular bodies formation, which is driven by endosomal sorting complexes required for transport (ESCRT). Here, we showed the presence and expression of homologous ESCRT genes inEntamoeba histolytica. We cloned and expressed theEhvps4gene, an ESCRT member, to obtain the recombinant EhVps4 and generate specific antibodies, which immunodetected EhVps4 in cytoplasm of trophozoites. Bioinformatics and biochemical studies evidenced that rEhVps4 is an ATPase, whose activity depends on the conserved E211 residue. Next, we generated trophozoites overexpressing EhVps4 and mutant EhVps4-E211Q FLAG-tagged proteins. The EhVps4-FLAG was located in cytosol and at plasma membrane, whereas the EhVps4-E211Q-FLAG was detected as abundant cytoplasmic dots in trophozoites. Erythrophagocytosis, cytopathic activity, and hepatic damage in hamsters were not improved in trophozoites overexpressing EhVps4-FLAG. In contrast, EhVps4-E211Q-FLAG protein overexpression impaired these properties. The localization of EhVps4-FLAG around ingested erythrocytes, together with our previous results, strengthens the role for EhVps4 inE. histolyticaphagocytosis and virulence.

Download Full-text

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.

Download Full-text