scholarly journals Ear1p and Ssh4p Are New Adaptors of the Ubiquitin Ligase Rsp5p for Cargo Ubiquitylation and Sorting at Multivesicular Bodies

2008 ◽  
Vol 19 (6) ◽  
pp. 2379-2388 ◽  
Author(s):  
Sébastien Léon ◽  
Zoi Erpapazoglou ◽  
Rosine Haguenauer-Tsapis
Keyword(s):  
Plasma Membrane ◽  
Membrane Proteins ◽  
Ubiquitin Ligase ◽  
Multivesicular Bodies ◽  
Endosomal Sorting ◽  
Frame Fusion ◽  
Vacuolar Targeting

The ubiquitylation of membrane proteins destined for the vacuole/lysosome is essential for their recognition by the endosomal sorting machinery and their internalization into vesicles of multivesicular bodies (MVBs). In yeast, this process requires Rsp5p, an essential ubiquitin ligase of the Nedd4 family. We describe here two redundant proteins, Ear1p and Ssh4p, required for the vacuolar targeting of several cargoes originating from the Golgi or the plasma membrane. Ear1p is an endosomal protein that interacts with Rsp5p through its PPxY motifs, and it is required for the ubiquitylation of selected cargoes before their MVB sorting. In-frame fusion of cargo to ubiquitin overcomes the need for Ear1p/Ssh4p, confirming a role for these proteins in cargo ubiquitylation. Interestingly, Ear1p is itself ubiquitylated by Rsp5p and targeted to the vacuole. Finally, Ear1p overexpression leads to Rsp5p accumulation at endosomes, interfering with some of its functions in trafficking. Therefore, Ear1p/Ssh4p recruit Rsp5p and assist it in its function at MVBs by directing the ubiquitylation of specific cargoes.

scholarly journals Plasma membrane deformation by circular arrays of ESCRT-III protein filaments

2008 ◽  
Vol 180 (2) ◽  
pp. 389-402 ◽  
Author(s):  
Phyllis I. Hanson ◽  
Robyn Roth ◽  
Yuan Lin ◽  
John E. Heuser
Keyword(s):  
Electron Microscopy ◽  
Plasma Membrane ◽  
Negative Curvature ◽  
Multivesicular Body ◽  
Multivesicular Bodies ◽  
Deficient Mutant ◽  
Membrane Deformation ◽  
Endosomal Sorting ◽  
Viral Budding ◽  
Circular Arrays

Endosomal sorting complex required for transport III (ESCRT-III) proteins function in multivesicular body biogenesis and viral budding. They are recruited from the cytoplasm to the membrane, where they assemble into large complexes. We used “deep-etch” electron microscopy to examine polymers formed by the ESCRT-III proteins hSnf7-1 (CHMP4A) and hSnf7-2 (CHMP4B). When overexpressed, these proteins target to endosomes and the plasma membrane. Both hSnf7 proteins assemble into regular approximately 5-nm filaments that curve and self-associate to create circular arrays. Binding to a coexpressed adenosine triphosphate hydrolysis–deficient mutant of VPS4B draws these filaments together into tight circular scaffolds that bend the membrane away from the cytoplasm to form buds and tubules protruding from the cell surface. Similar buds develop in the absence of mutant VPS4B when hSnf7-1 is expressed without its regulatory C-terminal domain. We demonstrate that hSnf7 proteins form novel membrane-attached filaments that can promote or stabilize negative curvature and outward budding. We suggest that ESCRT-III polymers delineate and help generate the luminal vesicles of multivesicular bodies.

scholarly journals Revisiting caveolin trafficking: the end of the caveosome

2010 ◽  
Vol 191 (3) ◽  
pp. 439-441 ◽  
Author(s):  
Robert G. Parton ◽  
Mark T. Howes
Keyword(s):  
Membrane Proteins ◽  
Multivesicular Bodies ◽  
Endosomal Sorting ◽  
Cell Biol ◽  
New Findings

In this issue, a study by Hayer et al. (2010. J. Cell Biol. doi: 10.1083/jcb.201003086) provides insights into the trafficking of caveolins, the major membrane proteins of caveolae. As well as providing evidence for ubiquitin-mediated endosomal sorting and degradation of caveolin in multivesicular bodies (MVBs), the new findings question the existence of a unique organelle proposed nine years ago, the caveosome.

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

2010 ◽  
Vol 2010 ◽  
pp. 1-15 ◽  
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.

Maturation of reticulocytes: formation of exosomes as a mechanism for shedding membrane proteins

1992 ◽  
Vol 70 (3-4) ◽  
pp. 179-190 ◽  
Author(s):  
R. M. Johnstone
Keyword(s):  
Plasma Membrane ◽  
Membrane Proteins ◽  
Transferrin Receptor ◽  
Structural Changes ◽  
Surface Proteins ◽  
Multivesicular Bodies ◽  
Red Cell ◽  
Nucleoside Transporter ◽  
Selective Loss ◽  
Anion Transporter

The transferrin receptor is a member of a group of reticulocyte surface proteins that disappear from the membranes of reticulocytes as the cells mature to the erythrocyte stage. The selective loss of membrane proteins appears to be preceded by the formation of multivesicular bodies (MVBs). At the reticulocyte stage, many species of mammalian red cells including man, and one nucleated avian species (chicken), contain these intracellular structures in both natural and induced anemias. Also characteristic of blood containing reticulocytes is the presence of circulating vesicles (exosomes), which contain proteins and lipids characteristic of the plasma membrane. These exosomes appear to arise from the contents of the MVBs, after the fusion of MVBs with the plasma membrane. The proteins in the exosomes are those frequently lost during red cell maturation (e.g., transferrin receptor). The major transmembrane proteins (such as the anion transporter) are fully retained into the mature red cell, indicating a highly selective mechanism of recognition of a specific group of proteins. The exosomes are largely devoid of soluble proteins and proteins associated with lysozomes or mitochondria. A speculative model is proposed which addresses the questions of the maturation-induced structural changes in a class of membrane proteins, their recognition and selective loss involving exosome formation, and the release of exosomes to the circulation.Key words: transferrin receptor, nucleoside transporter, reticulocyte maturation, multivesicular bodies, 70-kilodalton protein.

scholarly journals Differential degradation of PIN2 auxin efflux carrier by retromer-dependent vacuolar targeting

2008 ◽  
Vol 105 (46) ◽  
pp. 17812-17817 ◽  
Author(s):  
Jürgen Kleine-Vehn ◽  
Johannes Leitner ◽  
Marta Zwiewka ◽  
Michael Sauer ◽  
Lindy Abas ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Membrane Proteins ◽  
Brefeldin A ◽  
Fine Tuning ◽  
Environmental Signals ◽  
Basal Plasma ◽  
Vacuolar Protein ◽  
Differential Degradation ◽  
Vacuolar Targeting

All eukaryotic cells present at the cell surface a specific set of plasma membrane proteins that modulate responses to internal and external cues and whose activity is also regulated by protein degradation. We characterized the lytic vacuole-dependent degradation of membrane proteins in Arabidopsis thaliana by means of in vivo visualization of vacuolar targeting combined with quantitative protein analysis. We show that the vacuolar targeting pathway is used by multiple cargos including PIN-FORMED (PIN) efflux carriers for the phytohormone auxin. In vivo visualization of PIN2 vacuolar targeting revealed its differential degradation in response to environmental signals, such as gravity. In contrast to polar PIN delivery to the basal plasma membrane, which depends on the vesicle trafficking regulator ARF-GEF GNOM, PIN sorting to the lytic vacuolar pathway requires additional brefeldin A-sensitive ARF-GEF activity. Furthermore, we identified putative retromer components SORTING NEXIN1 (SNX1) and VACUOLAR PROTEIN SORTING29 (VPS29) as important factors in this pathway and propose that the retromer complex acts to retrieve PIN proteins from a late/pre-vacuolar compartment back to the recycling pathways. Our data suggest that ARF GEF- and retromer-dependent processes regulate PIN sorting to the vacuole in an antagonistic manner and illustrate instrumentalization of this mechanism for fine-tuning the auxin fluxes during gravitropic response.

ESCRT-mediated sorting and intralumenal vesicle concatenation in plants

2018 ◽  
Vol 46 (3) ◽  
pp. 537-545 ◽  
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.

scholarly journals ESCRT ubiquitin-binding domains function cooperatively during MVB cargo sorting

2009 ◽  
Vol 185 (2) ◽  
pp. 213-224 ◽  
Author(s):  
S. Brookhart Shields ◽  
Andrea J. Oestreich ◽  
Stanley Winistorfer ◽  
Doris Nguyen ◽  
Johanna A. Payne ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Structural Information ◽  
Multivesicular Bodies ◽  
Endosomal Sorting ◽  
Binding Domains ◽  
Ubiquitin Binding ◽  
Cargo Sorting

Ubiquitin (Ub) sorting receptors facilitate the targeting of ubiquitinated membrane proteins into multivesicular bodies (MVBs). Ub-binding domains (UBDs) have been described in several endosomal sorting complexes required for transport (ESCRT). Using available structural information, we have investigated the role of the multiple UBDs within ESCRTs during MVB cargo selection. We found a novel UBD within ESCRT-I and show that it contributes to MVB sorting in concert with the known UBDs within the ESCRT complexes. These experiments reveal an unexpected level of coordination among the ESCRT UBDs, suggesting that they collectively recognize a diverse set of cargo rather than act sequentially at discrete steps.

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

2017 ◽  
Vol 114 (34) ◽  
pp. E7197-E7204 ◽  
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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