Relationships between EEA1 binding partners and their role in endosome fusion

2001 ◽  
Vol 114 (10) ◽  
pp. 1959-1965 ◽  
Author(s):  
I.G. Mills ◽  
S. Urbe ◽  
M.J. Clague
Keyword(s):  
Protein Binding ◽  
Complex Network ◽  
Vital Role ◽  
Early Endosome ◽  
Stable Complex ◽  
Baby Hamster Kidney ◽  
Bhk Cells ◽  
Binding Partners ◽  
Early Endosomes

Homotypic fusion between early endosomes requires the phosphatidylinositol 3-phosphate (PI3P)-binding protein, Early Endosomal Autoantigen 1 (EEA1). We have investigated the role of other proteins that interact with EEA1 in the fusion of early endosomes derived from Baby Hamster Kidney (BHK) cells. We confirm a requirement for syntaxin 13, but additionally show that the assay is equally sensitive to reagents specifically targeted against syntaxin 6. Binding of EEA1 to immobilised GST-syntaxin 6 and 13 was directly compared; only syntaxin 6 formed a stable complex with EEA1. Early endosome fusion requires the release of intravesicular calcium, and calmodulin plays a vital role in the fusion pathway, as judged by sensitivity to antagonists. We demonstrate that both EEA1 and syntaxin 13 interact with calmodulin. In the case of EEA1, binding to calmodulin requires an IQ domain, which is adjacent to a C-terminal FYVE domain that specifically binds to PI3P. We have assessed the influence of protein binding partners on EEA1 interaction with PI3P and find that both calmodulin and rab5-GTP are antagonistic to PI3P binding, whilst syntaxins 6 and 13 have no effect. These studies reveal a complex network of interactions between the proteins required for endosome fusion.

scholarly journals Neurospora crassa NKIN2, a Kinesin-3 Motor, Transports Early Endosomes and Is Required for Polarized Growth

2013 ◽  
Vol 12 (7) ◽  
pp. 1020-1032 ◽  
Author(s):  
Constanze Seidel ◽  
Sergio David Moreno-Velásquez ◽  
Meritxell Riquelme ◽  
Reinhard Fischer
Keyword(s):  
Neurospora Crassa ◽  
Fluorescent Protein ◽  
Early Endosome ◽  
Mechanical Forces ◽  
Structural Components ◽  
Content Type ◽  
Early Endosomes ◽  
Polarized Cells ◽  
Green Fluorescent

ABSTRACT Biological motors are molecular nanomachines, which convert chemical energy into mechanical forces. The combination of mechanoenzymes with structural components, such as the cytoskeleton, enables eukaryotic cells to overcome entropy, generate molecular gradients, and establish polarity. Hyphae of filamentous fungi are among the most polarized cells, and polarity defects are most obvious. Here, we studied the role of the kinesin-3 motor, NKIN2, in Neurospora crassa . We found that NKIN2 localizes as fast-moving spots in the cytoplasm of mature hyphae. To test whether the spots represented early endosomes, the Rab5 GTPase YPT52 was used as an endosomal marker. NKIN2 colocalized with YPT52. Deletion of nkin2 caused strongly reduced endosomal movement. Combined, these results confirm the involvement of NKIN2 in early endosome transport. Introduction of a rigor mutation into NKIN2 labeled with green fluorescent protein (GFP) resulted in decoration of microtubules. Interestingly, NKIN2 rigor was associated with a subpopulation of microtubules, as had been shown earlier for the Aspergillus nidulans orthologue UncA. Other kinesins did not show this specificity.

The vital role of ditopic N–N bridging ligands with different lengths in the formation of new binuclear dioxomolybdenum(vi) complexes: synthesis, crystal structures, supramolecular framework and protein binding studies

2020 ◽  
Vol 44 (38) ◽  
pp. 16645-16664
Author(s):  
Debanjana Biswal ◽  
Malini Roy ◽  
Nikhil Ranjan Pramanik ◽  
Suvendu Paul ◽  
Michael G. B. Drew ◽  
...  
Keyword(s):  
Protein Binding ◽  
Crystal Structures ◽  
Vital Role ◽  
Supramolecular Framework ◽  
Binding Studies ◽  
Bridging Ligands ◽  
Supramolecular Architectures ◽  
Bsa Binding

Role of bis-(pyridyl) and bis-(imidazole) auxiliary ligands in the formation of supramolecular architectures and BSA binding with new binuclear dioxomolybdenum(vi) complexes.

The AGO proteins: an overview

2018 ◽  
Vol 399 (6) ◽  
pp. 525-547 ◽  
Author(s):  
Saife Niaz
Keyword(s):  
Protein Binding ◽  
Structure Function ◽  
Small Rnas ◽  
Biological Process ◽  
Biochemical Properties ◽  
The Core ◽  
Binding Partners

AbstractSmall RNAs govern almost every biological process in eukaryotes associating with the Argonaute (AGO) proteins to form the RNA-induced silencing complex (mRISC). AGO proteins constitute the core of RISCs with different members having variety of protein-binding partners and biochemical properties. This review focuses on the AGO subfamily of the AGOs that are ubiquitously expressed and are associated with small RNAs. The structure, function and role of the AGO proteins in the cell is discussed in detail.

scholarly journals Role of TSPAN9 in Alphavirus Entry and Early Endosomes

2016 ◽  
Vol 90 (9) ◽  
pp. 4289-4297 ◽  
Author(s):  
Katie M. Stiles ◽  
Margaret Kielian
Keyword(s):  
Membrane Fusion ◽  
Sindbis Virus ◽  
Semliki Forest Virus ◽  
Early Endosome ◽  
Late Endosome ◽  
Host Cells ◽  
Human Pathogens ◽  
Early Endosomes ◽  
Alphavirus Entry

ABSTRACTAlphaviruses are small enveloped RNA viruses that infect cells via clathrin-mediated endocytosis and low-pH-triggered fusion in the early endosome. Using a small interfering RNA (siRNA) screen in human cells, we previously identified TSPAN9 as a host factor that promotes infection by the alphaviruses Sindbis virus (SINV), Semliki Forest virus (SFV), and chikungunya virus (CHIKV). Depletion of TSPAN9 specifically decreases SFV membrane fusion in endosomes. TSPAN9 is a member of the tetraspanin family of multipass membrane proteins, but its cellular function is currently unknown. Here we used U-2 OS cells stably overexpressing TSPAN9 to show that TSPAN9 is localized at the plasma membrane and in early and late endosomes. Internalized SFV particles colocalized with TSPAN9 in vesicles early during infection. Depletion of TSPAN9 led to reductions in the amounts of the late endosomal proteins LAMP1 and CD63 and an increase in the amount of LAMP2. However, TSPAN9 depletion did not alter the delivery of SFV to early endosomes or change their pH or protease activity. Comparative studies showed that TSPAN9 depletion strongly inhibited infection by several viruses that fuse in early endosomes (SFV, SINV, CHIKV, and vesicular stomatitis virus [VSV]), while viruses that fuse in the late endosome (recombinant VSV-Lassa and VSV-Junin), including an SFV point mutant with a lower pH threshold for fusion (SFV E2 T12I), were relatively resistant. Our data suggest that TSPAN9 modulates the early endosome compartment to make it more permissive for membrane fusion of early-penetrating viruses.IMPORTANCEAlphaviruses are spread by mosquitoes and can cause serious human diseases such as arthritis and encephalitis. Recent outbreaks of CHIKV infection are responsible for millions of cases of acute illness and long-term complications. There are no vaccines or antiviral treatments for these important human pathogens. Alphaviruses infect host cells by utilizing the endocytic machinery of the cell and fusing their membrane with that of the endosome. Although the mechanism of virus-membrane fusion is well studied, we still know relatively little about the host cell proteins that are involved in alphavirus entry. Here we characterized the role of the host membrane protein TSPAN9 in alphavirus infection. TSPAN9 was localized to early endosomes containing internalized alphavirus, and depletion of TSPAN9 inhibited virus fusion with the early endosome membrane. In contrast, infection of viruses that enter through the late endosome was relatively resistant to TSPAN9 depletion, suggesting an important role for TSPAN9 in the early endosome.

scholarly journals Rings in the Extreme: PCNA Interactions and Adaptations in the Archaea

Archaea ◽  
2012 ◽  
Vol 2012 ◽  
pp. 1-8 ◽  
Author(s):  
Jody A. Winter ◽  
Karen A. Bunting
Keyword(s):  
Structural Analysis ◽  
Protein Binding ◽  
Protein Interactions ◽  
Extreme Environments ◽  
Binding Partners ◽  
Great Insight ◽  
Dna Replication And Repair ◽  
Evolution Of Proteins ◽  
Insight Into

Biochemical and structural analysis of archaeal proteins has enabled us to gain great insight into many eukaryotic processes, simultaneously offering fascinating glimpses into the adaptation and evolution of proteins at the extremes of life. The archaeal PCNAs, central to DNA replication and repair, are no exception. Characterisation of the proteins alone, and in complex with both peptides and protein binding partners, has demonstrated the diversity and subtlety in the regulatory role of these sliding clamps. Equally, studies have provided valuable detailed insight into the adaptation of protein interactions and mechanisms that are necessary for life in extreme environments.

scholarly journals Early endosome motility spatially organizes polysome distribution

2014 ◽  
Vol 204 (3) ◽  
pp. 343-357 ◽  
Author(s):  
Yujiro Higuchi ◽  
Peter Ashwin ◽  
Yvonne Roger ◽  
Gero Steinberg
Keyword(s):  
Rna Binding ◽  
Rna Binding Protein ◽  
Ribosomal Subunit ◽  
Protein Translation ◽  
Model System ◽  
Early Endosome ◽  
Long Distance ◽  
Translation Machinery ◽  
Early Endosomes

Early endosomes (EEs) mediate protein sorting, and their cytoskeleton-dependent motility supports long-distance signaling in neurons. Here, we report an unexpected role of EE motility in distributing the translation machinery in a fungal model system. We visualize ribosomal subunit proteins and show that the large subunits diffused slowly throughout the cytoplasm (Dc,60S = 0.311 µm2/s), whereas entire polysomes underwent long-range motility along microtubules. This movement was mediated by “hitchhiking” on kinesin-3 and dynein-driven EEs, where the polysomes appeared to translate EE-associated mRNA into proteins. Modeling indicates that this motor-driven transport is required for even cellular distribution of newly formed ribosomes. Indeed, impaired EE motility in motor mutants, or their inability to bind EEs in mutants lacking the RNA-binding protein Rrm4, reduced ribosome transport and induced ribosome aggregation near the nucleus. As a consequence, cell growth was severely restricted. Collectively, our results indicate that polysomes associate with moving EEs and that “off- and reloading” distributes the protein translation machinery.

scholarly journals Quantitative high-content imaging identifies novel regulators of Neo1 trafficking at endosomes

2017 ◽  
Vol 28 (11) ◽  
pp. 1539-1550 ◽  
Author(s):  
Lauren E. Dalton ◽  
Björn D. M. Bean ◽  
Michael Davey ◽  
Elizabeth Conibear
Keyword(s):  
Lipid Membrane ◽  
Membrane Asymmetry ◽  
High Content Imaging ◽  
Sorting Nexin ◽  
Binding Partners ◽  
Endosomal Recycling ◽  
Cargo Protein ◽  
Early Endosomes ◽  
Sorting Motif

P4-ATPases are a family of putative phospholipid flippases that regulate lipid membrane asymmetry, which is important for vesicle formation. Two yeast flippases, Drs2 and Neo1, have nonredundant functions in the recycling of the synaptobrevin-like v-SNARE Snc1 from early endosomes. Drs2 activity is needed to form vesicles and regulate its own trafficking, suggesting that flippase activity and localization are linked. However, the role of Neo1 in endosomal recycling is not well characterized. To identify novel regulators of Neo1 trafficking and activity at endosomes, we first identified mutants with impaired recycling of a Snc1-based reporter and subsequently used high-content microscopy to classify these mutants based on the localization of Neo1 or its binding partners, Mon2 and Dop1. This analysis identified a role for Arl1 in stabilizing the Mon2/Dop1 complex and uncovered a new function for Vps13 in early endosome recycling and Neo1 localization. We further showed that the cargo-selective sorting nexin Snx3 is required for Neo1 trafficking and identified an Snx3 sorting motif in the Neo1 N-terminus. Of importance, the Snx3-dependent sorting of Neo1 was required for the correct sorting of another Snx3 cargo protein, suggesting that the incorporation of Neo1 into recycling tubules may influence their formation.

Asiana Airlines Flight 214

2014 ◽  
Vol 4 (2) ◽  
pp. 113-121 ◽  
Author(s):  
Stephanie Chow ◽  
Stephen Yortsos ◽  
Najmedin Meshkati
Keyword(s):  
Human Factors ◽  
Aviation Safety ◽  
Cultural Factors ◽  
Vital Role ◽  
Cultural Issues ◽  
Flight Crew ◽  
Pilot Performance ◽  
Engineering Designs ◽  
Flight Deck

This article focuses on a major human factors–related issue that includes the undeniable role of cultural factors and cockpit automation and their serious impact on flight crew performance, communication, and aviation safety. The report concentrates on the flight crew performance of the Boeing 777–Asiana Airlines Flight 214 accident, by exploring issues concerning mode confusion and autothrottle systems. It also further reviews the vital role of cultural factors in aviation safety and provides a brief overview of past, related accidents. Automation progressions have been created in an attempt to design an error-free flight deck. However, to do that, the pilot must still thoroughly understand every component of the flight deck – most importantly, the automation. Otherwise, if pilots are not completely competent in terms of their automation, the slightest errors can lead to fatal accidents. As seen in the case of Asiana Flight 214, even though engineering designs and pilot training have greatly evolved over the years, there are many cultural, design, and communication factors that affect pilot performance. It is concluded that aviation systems designers, in cooperation with pilots and regulatory bodies, should lead the strategic effort of systematically addressing the serious issues of cockpit automation, human factors, and cultural issues, including their interactions, which will certainly lead to better solutions for safer flights.

Energy Efficiency Analysis in ERA using NFL-BA Algorithm

2020 ◽  
pp. 340-348
Author(s):  
Palky Mehta ◽  
H. L. Sharma
Keyword(s):  
Cluster Head ◽  
Vital Role ◽  
Wireless Sensor ◽  
Energy Usage ◽  
Cluster Heads ◽  
Comparison Results ◽  
Current Scenario ◽  
Energy Efficiency Analysis ◽  
Selection Of

In the current scenario of Wireless Sensor Network (WSN), power consumption is the major issue associated with nodes in WSN. LEACH technique plays a vital role of clustering in WSN and reduces the energy usage effectively. But LEACH has its own limitation in order to search cluster head nodes which are randomly distributed over the network. In this paper, ERA-NFL- BA algorithm is being proposed for selects the cluster heads in WSN. This algorithm help in selection of cluster heads can freely transform from global search to local search. At the end, a comparison has been done with earlier researcher using protocol ERA-NFL, which clearly shown that proposed Algorithm is best suited and from comparison results that ERA-NFL-BA has given better performance.

Tertiary English-majored Students’ Perceptions toward the Role of Pronunciation in English Language Learning and their Practicing Strategies

2020 ◽  
Vol 6 (2) ◽  
pp. 264-279
Keyword(s):  
Language Learning ◽  
English Language ◽  
Vital Role ◽  
English Language Learning ◽  
Structured Interviews ◽  
Efl Learners ◽  
The University

Accurate pronunciation has a vital role in English language learning as it can help learners to avoid misunderstanding in communication. However, EFL learners in many contexts, especially at the University of Phan Thiet, still encounter many difficulties in pronouncing English correctly. Therefore, this study endeavors to explore English-majored students’ perceptions towards the role of pronunciation in English language learning and examine their pronunciation practicing strategies (PPS). It involved 155 English-majored students at the University of Phan Thiet who answered closed-ended questionnaires and 18 English-majored students who participated in semi-structured interviews. The findings revealed that students strongly believed in the important role of pronunciation in English language learning; however, they sometimes employed PPS for their pronunciation improvement. Furthermore, the results showed that participants tended to use naturalistic practicing strategies and formal practicing strategies with sounds, but they overlooked strategies such as asking for help and cooperating with peers. Such findings could contribute further to the understanding of how students perceive the role of pronunciation and their PPS use in the research’s context and other similar ones. Received 10th June 2019; Revised 12th March 2020; Accepted 12th April 2020

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