scholarly journals Distinct sets of tethering complexes, SNARE complexes, and Rab GTPases mediate membrane fusion at the vacuole in Arabidopsis

2018 ◽  
Vol 115 (10) ◽  
pp. E2457-E2466 ◽  
Author(s):  
Kodai Takemoto ◽  
Kazuo Ebine ◽  
Jana Christin Askani ◽  
Falco Krüger ◽  
Zaida Andrés Gonzalez ◽  
...  
Keyword(s):  
Membrane Trafficking ◽  
Plant Evolution ◽  
Rab Gtpases ◽  
Core Complex ◽  
Transport Pathways ◽  
Protein Receptor ◽  
Evolutionarily Conserved ◽  
Vacuolar Transport ◽  
Higher Order Functions ◽  
Vacuolar Trafficking

Membrane trafficking plays pivotal roles in various cellular activities and higher-order functions of eukaryotes and requires tethering factors to mediate contact between transport intermediates and target membranes. Two evolutionarily conserved tethering complexes, homotypic fusion and protein sorting (HOPS) and class C core vacuole/endosome tethering (CORVET), are known to act in endosomal/vacuolar transport in yeast and animals. Both complexes share a core subcomplex consisting of Vps11, Vps18, Vps16, and Vps33, and in addition to this core, HOPS contains Vps39 and Vps41, whereas CORVET contains Vps3 and Vps8. HOPS and CORVET subunits are also conserved in the model plant Arabidopsis. However, vacuolar trafficking in plants occurs through multiple unique transport pathways, and how these conserved tethering complexes mediate endosomal/vacuolar transport in plants has remained elusive. In this study, we investigated the functions of VPS18, VPS3, and VPS39, which are core complex, CORVET-specific, and HOPS-specific subunits, respectively. Impairment of these tethering proteins resulted in embryonic lethality, distinctly altering vacuolar morphology and perturbing transport of a vacuolar membrane protein. CORVET interacted with canonical RAB5 and a plant-specific R-soluble NSF attachment protein receptor (SNARE), VAMP727, which mediates fusion between endosomes and the vacuole, whereas HOPS interacted with RAB7 and another R-SNARE, VAMP713, which likely mediates homotypic vacuolar fusion. These results indicate that CORVET and HOPS act in distinct vacuolar trafficking pathways in plant cells, unlike those of nonplant systems that involve sequential action of these tethering complexes during vacuolar/lysosomal trafficking. These results highlight a unique diversification of vacuolar/lysosomal transport that arose during plant evolution, using evolutionarily conserved tethering components.

scholarly journals Regulation of intracellular membrane trafficking and cell dynamics by syntaxin-6

2012 ◽  
Vol 32 (4) ◽  
pp. 383-391 ◽  
Author(s):  
Jae-Joon Jung ◽  
Shivangi M. Inamdar ◽  
Ajit Tiwari ◽  
Amit Choudhury
Keyword(s):  
Membrane Trafficking ◽  
Critical Role ◽  
Cell Types ◽  
Intracellular Membrane ◽  
Cellular Functions ◽  
Cell Dynamics ◽  
Transport Pathways ◽  
Protein Receptor ◽  
Secretory Transport ◽  
Target Membrane

Intracellular membrane trafficking along endocytic and secretory transport pathways plays a critical role in diverse cellular functions including both developmental and pathological processes. Briefly, proteins and lipids destined for transport to distinct locations are collectively assembled into vesicles and delivered to their target site by vesicular fusion. SNARE (soluble N-ethylmaleimide-sensitive factor-attachment protein receptor) proteins are required for these events, during which v-SNAREs (vesicle SNAREs) interact with t-SNAREs (target SNAREs) to allow transfer of cargo from donor vesicle to target membrane. Recently, the t-SNARE family member, syntaxin-6, has been shown to play an important role in the transport of proteins that are key to diverse cellular dynamic processes. In this paper, we briefly discuss the specific role of SNAREs in various mammalian cell types and comprehensively review the various roles of the Golgi- and endosome-localized t-SNARE, syntaxin-6, in membrane trafficking during physiological as well as pathological conditions.

scholarly journals Three Prevacuolar Compartment Rab GTPases Impact Candida albicans Hyphal Growth

2013 ◽  
Vol 12 (7) ◽  
pp. 1039-1050 ◽  
Author(s):  
Douglas A. Johnston ◽  
Arturo Luna Tapia ◽  
Karen E. Eberle ◽  
Glen E. Palmer
Keyword(s):  
Candida Albicans ◽  
Membrane Trafficking ◽  
Gene Deletion ◽  
Hyphal Growth ◽  
Rab Gtpases ◽  
Phenotypic Analysis ◽  
Pathogenic Yeast ◽  
Content Type ◽  
Growth Defects ◽  
Vacuolar Trafficking

ABSTRACTDisruption of vacuolar biogenesis in the pathogenic yeastCandida albicanscauses profound defects in polarized hyphal growth. However, the precise vacuolar pathways involved in yeast-hypha differentiation have not been determined. Previously we focused on Vps21p, a Rab GTPase involved in directing vacuolar trafficking through the late endosomalprevacuolarcompartment (PVC). Herein, we identify two additional Vps21p-related GTPases, Ypt52p and Ypt53p, that colocalize with Vps21p and can suppress the hyphal defects of thevps21Δ/Δ mutant. Phenotypic analysis of gene deletion strains revealed that loss of bothVPS21andYPT52causes synthetic defects in endocytic trafficking to the vacuole, as well as delivery of the virulence-associated vacuolar membrane protein Mlt1p from the Golgi compartment. Transcription of all three GTPase-encoding genes is increased under hyphal growth conditions, and overexpression of the transcription factor Ume6p is sufficient to increase the transcription of these genes. While only thevps21Δ/Δ single mutant has hyphal growth defects, these were greatly exacerbated in avps21Δ/Δypt52Δ/Δ double mutant. On the basis of relative expression levels and phenotypic analysis of gene deletion strains, Vps21p is the most important of the three GTPases, followed by Ypt52p, while Ypt53p has an only marginal impact onC. albicansphysiology. Finally, disruption of a nonendosomal AP-3-dependent vacuolar trafficking pathway in thevps21Δ/Δypt52Δ/Δ mutant, further exacerbated the stress and hyphal growth defects. These findings underscore the importance of membrane trafficking through the PVC in sustaining the invasive hyphal growth form ofC. albicans.

scholarly journals Cargo sorting zones in the trans-Golgi network visualized by super-resolution confocal live imaging microscopy in plants

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Yutaro Shimizu ◽  
Junpei Takagi ◽  
Emi Ito ◽  
Yoko Ito ◽  
Kazuo Ebine ◽  
...  
Keyword(s):  
Membrane Trafficking ◽  
High Speed ◽  
Super Resolution ◽  
Adaptor Protein ◽  
Transport Proteins ◽  
3D Localization ◽  
Golgi Network ◽  
Distinct Distribution ◽  
Vacuolar Trafficking ◽  
Cargo Sorting

AbstractThe trans-Golgi network (TGN) has been known as a key platform to sort and transport proteins to their final destinations in post-Golgi membrane trafficking. However, how the TGN sorts proteins with different destinies still remains elusive. Here, we examined 3D localization and 4D dynamics of TGN-localized proteins of Arabidopsis thaliana that are involved in either secretory or vacuolar trafficking from the TGN, by a multicolor high-speed and high-resolution spinning-disk confocal microscopy approach that we developed. We demonstrate that TGN-localized proteins exhibit spatially and temporally distinct distribution. VAMP721 (R-SNARE), AP (adaptor protein complex)−1, and clathrin which are involved in secretory trafficking compose an exclusive subregion, whereas VAMP727 (R-SNARE) and AP-4 involved in vacuolar trafficking compose another subregion on the same TGN. Based on these findings, we propose that the single TGN has at least two subregions, or “zones”, responsible for distinct cargo sorting: the secretory-trafficking zone and the vacuolar-trafficking zone.

scholarly journals Visualization of the exocyst complex dynamics at the plasma membrane of Arabidopsis thaliana

2013 ◽  
Vol 24 (4) ◽  
pp. 510-520 ◽  
Author(s):  
Matyáš Fendrych ◽  
Lukáš Synek ◽  
Tamara Pečenková ◽  
Edita Janková Drdová ◽  
Juraj Sekereš ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Fluorescent Protein ◽  
Complex Dynamics ◽  
Active Regions ◽  
Snare Complex ◽  
Epifluorescence Microscopy ◽  
Rab Gtpases ◽  
Exocyst Complex ◽  
Protein Receptor ◽  
Outer Domain

The exocyst complex, an effector of Rho and Rab GTPases, is believed to function as an exocytotic vesicle tether at the plasma membrane before soluble N-ethylmaleimide–sensitive factor attachment protein receptor (SNARE) complex formation. Exocyst subunits localize to secretory-active regions of the plasma membrane, exemplified by the outer domain of Arabidopsis root epidermal cells. Using variable-angle epifluorescence microscopy, we visualized the dynamics of exocyst subunits at this domain. The subunits colocalized in defined foci at the plasma membrane, distinct from endocytic sites. Exocyst foci were independent of cytoskeleton, although prolonged actin disruption led to changes in exocyst localization. Exocyst foci partially overlapped with vesicles visualized by VAMP721 v-SNARE, but the majority of the foci represent sites without vesicles, as indicated by electron microscopy and drug treatments, supporting the concept of the exocyst functioning as a dynamic particle. We observed a decrease of SEC6–green fluorescent protein foci in an exo70A1 exocyst mutant. Finally, we documented decreased VAMP721 trafficking to the plasma membrane in exo70A1 and exo84b mutants. Our data support the concept that the exocyst-complex subunits dynamically dock and undock at the plasma membrane to create sites primed for vesicle tethering.

scholarly journals Cleavage activates Dispatched for Sonic Hedgehog ligand release

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Daniel P Stewart ◽  
Suresh Marada ◽  
William J Bodeen ◽  
Ashley Truong ◽  
Sadie Miki Sakurada ◽  
...  
Keyword(s):  
Sonic Hedgehog ◽  
Membrane Trafficking ◽  
Developmental Disorders ◽  
Transmembrane Protein ◽  
Regulatory Mechanisms ◽  
Extracellular Loop ◽  
Site Mutation ◽  
Evolutionarily Conserved ◽  
Processing Site

Hedgehog ligands activate an evolutionarily conserved signaling pathway that provides instructional cues during tissue morphogenesis, and when corrupted, contributes to developmental disorders and cancer. The transmembrane protein Dispatched is an essential component of the machinery that deploys Hedgehog family ligands from producing cells, and is absolutely required for signaling to long-range targets. Despite this crucial role, regulatory mechanisms controlling Dispatched activity remain largely undefined. Herein, we reveal vertebrate Dispatched is activated by proprotein convertase-mediated cleavage at a conserved processing site in its first extracellular loop. Dispatched processing occurs at the cell surface to instruct its membrane re-localization in polarized epithelial cells. Cleavage site mutation alters Dispatched membrane trafficking and reduces ligand release, leading to compromised pathway activity in vivo. As such, convertase-mediated cleavage is required for Dispatched maturation and functional competency in Hedgehog ligand-producing cells.

LRRK2 and Rab GTPases

2018 ◽  
Vol 46 (6) ◽  
pp. 1707-1712 ◽  
Author(s):  
Suzanne R. Pfeffer
Keyword(s):  
Membrane Trafficking ◽  
Protein Function ◽  
Short Review ◽  
Rab Gtpase ◽  
Rab Gtpases ◽  
Rab Protein ◽  
Complex Interactions ◽  
Preferential Binding ◽  
Bona Fide ◽  
Mutant Lrrk2

Leucine-rich repeat kinase 2 (LRRK2) is mutated in familial Parkinson's disease, and pathogenic mutations activate the kinase activity. A tour de force screen by Mann and Alessi and co-workers identified a subset of Rab GTPases as bona fide LRRK2 substrates. Rab GTPases are master regulators of membrane trafficking and this short review will summarize what we know about the connection between LRRK2 and this family of regulatory proteins. While, in most cases, Rab GTPase phosphorylation is predicted to interfere with Rab protein function, the discovery of proteins that show preferential binding to phosphorylated Rabs suggests that more complex interactions may also contribute to mutant LRRK2-mediated pathology.

scholarly journals Components of the endocytic and recycling trafficking pathways interfere with the integrity of the Legionella-containing vacuole

2019 ◽  
Author(s):  
Ila S. Anand ◽  
Won Young Choi ◽  
Ralph R. Isberg
Keyword(s):  
Membrane Trafficking ◽  
Legionella Pneumophila ◽  
Host Cells ◽  
Rab Gtpases ◽  
Intracellular Growth ◽  
Mutant Proteins ◽  
Host Cytoplasm ◽  
Downstream Effectors ◽  
Cell Death Response ◽  
Host Membrane

SummaryLegionella pneumophila requires the Dot/Icm translocation system to replicate in a vacuolar compartment within host cells. Strains lacking the translocated substrate SdhA form a permeable vacuole during residence in the host cell, exposing bacteria to the host cytoplasm. In primary macrophages, mutants are defective for intracellular growth, with a pyroptotic cell death response mounted due to bacterial exposure to the cytosol. To understand how SdhA maintains vacuole integrity during intracellular growth, we performed high-throughput RNAi screens against host membrane trafficking genes to identify factors that antagonize vacuole integrity in the absence of SdhA. Depletion of host proteins involved in endocytic uptake and recycling resulted in enhanced intracellular growth and lower levels of permeable vacuoles surrounding the ΔsdhA mutant. Of interest were three different Rab GTPases involved in these processes: Rab11b, Rab8b and Rab5 isoforms, that when depleted resulted in enhanced vacuole integrity surrounding the sdhA mutant. Proteins regulated by these Rabs are responsible for interfering with proper vacuole membrane maintenance, as depletion of the downstream effectors EEA1, Rab11FIP1, or VAMP3 rescued vacuole integrity and intracellular growth of the sdhA mutant. To test the model that specific vesicular components associated with these effectors could act to destabilize the replication vacuole, EEA1 and Rab11FIP1 showed enhanced colocalization with the vacuole surrounding the sdhA mutant compared with the WT vacuole. Depletion of Rab5 isoforms or Rab11b reduced this aberrant colocalization. These findings are consistent with SdhA interfering with both endocytic and recycling membrane trafficking events that act to destabilize vacuole integrity during infection.

scholarly journals DRAM-3 modulates autophagy and promotes cell survival in the absence of glucose

2015 ◽  
Vol 22 (10) ◽  
pp. 1714-1726 ◽  
Author(s):  
M Mrschtik ◽  
J O'Prey ◽  
L Y Lao ◽  
J S Long ◽  
F Beaumatin ◽  
...  
Keyword(s):  
Cell Survival ◽  
Membrane Trafficking ◽  
Clonogenic Survival ◽  
Autophagic Flux ◽  
Normal Tissues ◽  
Atg Proteins ◽  
Dna Damaging Agents ◽  
Evolutionarily Conserved ◽  
Starvation Conditions

Abstract Macroautophagy is a membrane-trafficking process that delivers cytoplasmic constituents to lysosomes for degradation. The process operates under basal conditions as a mechanism to turnover damaged or misfolded proteins and organelles. As a result, it has a major role in preserving cellular integrity and viability. In addition to this basal function, macroautophagy can also be modulated in response to various forms of cellular stress, and the rate and cargoes of macroautophagy can be tailored to facilitate appropriate cellular responses in particular situations. The macroautophagy machinery is regulated by a group of evolutionarily conserved autophagy-related (ATG) proteins and by several other autophagy regulators, which either have tissue-restricted expression or operate in specific contexts. We report here the characterization of a novel autophagy regulator that we have termed DRAM-3 due to its significant homology to damage-regulated autophagy modulator (DRAM-1). DRAM-3 is expressed in a broad spectrum of normal tissues and tumor cells, but different from DRAM-1, DRAM-3 is not induced by p53 or DNA-damaging agents. Immunofluorescence studies revealed that DRAM-3 localizes to lysosomes/autolysosomes, endosomes and the plasma membrane, but not the endoplasmic reticulum, phagophores, autophagosomes or Golgi, indicating significant overlap with DRAM-1 localization and with organelles associated with macroautophagy. In this regard, we further proceed to show that DRAM-3 expression causes accumulation of autophagosomes under basal conditions and enhances autophagic flux. Reciprocally, CRISPR/Cas9-mediated disruption of DRAM-3 impairs autophagic flux confirming that DRAM-3 is a modulator of macroautophagy. As macroautophagy can be cytoprotective under starvation conditions, we also tested whether DRAM-3 could promote survival on nutrient deprivation. This revealed that DRAM-3 can repress cell death and promote long-term clonogenic survival of cells grown in the absence of glucose. Interestingly, however, this effect is macroautophagy-independent. In summary, these findings constitute the primary characterization of DRAM-3 as a modulator of both macroautophagy and cell survival under starvation conditions.

scholarly journals Local Secretory Trafficking Pathways in Neurons and the Role of Dendritic Golgi Outposts in Different Cell Models

2020 ◽  
Vol 13 ◽  
Author(s):  
Jingqi Wang ◽  
Lou Fourriere ◽  
Paul A. Gleeson
Keyword(s):  
Cell Body ◽  
Membrane Trafficking ◽  
Current Knowledge ◽  
Neurological Diseases ◽  
Model Organisms ◽  
Neuronal Structure ◽  
Anterograde Transport ◽  
Transport Pathways ◽  
Human Neurons

A fundamental characteristic of neurons is the relationship between the architecture of the polarized neuron and synaptic transmission between neurons. Intracellular membrane trafficking is paramount to establish and maintain neuronal structure; perturbation in trafficking results in defects in neurodevelopment and neurological disorders. Given the physical distance from the cell body to the distal sites of the axon and dendrites, transport of newly synthesized membrane proteins from the central cell body to their functional destination at remote, distal sites represents a conundrum. With the identification of secretory organelles in dendrites, including endoplasmic reticulum (ER) and Golgi outposts (GOs), recent studies have proposed local protein synthesis and trafficking distinct from the conventional anterograde transport pathways of the cell body. A variety of different model organisms, including Drosophila, zebrafish, and rodents, have been used to probe the organization and function of the local neuronal secretory network. Here, we review the evidence for local secretory trafficking pathways in dendrites in a variety of cell-based neuronal systems and discuss both the similarities and differences in the organization and role of the local secretory organelles, especially the GOs. In addition, we identify the gaps in the current knowledge and the potential advances using human induced pluripotent stem cells (iPSCs) in defining local membrane protein trafficking in human neurons and in understanding the molecular basis of neurological diseases.

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