scholarly journals The Arabidopsis R-SNARE VAMP714 is essential for polarization of PIN proteins in the establishment and maintenance of auxin gradients

2019 ◽  
Author(s):  
Xiaoyan Gu ◽  
Kumari Fonseka ◽  
Stuart A. Casson ◽  
Andrei Smertenko ◽  
Guangqin Guo ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Auxin Transport ◽  
Protein Localization ◽  
Adaptor Protein ◽  
Dominant Negative ◽  
Endocytic Pathway ◽  
Asymmetric Distribution ◽  
List Type ◽  
Loss Of Function ◽  
Regulatory Loop

SummaryThe plant hormone auxin and its directional intercellular transport plays a major role in diverse aspects of plant growth and development. The establishment of auxin gradients in plants requires asymmetric distribution of members of the auxin efflux carrier PIN-FORMED (PIN) protein family to the plasma membrane. An endocytic pathway regulates the recycling of PIN proteins between the plasma membrane and endosomes, providing a mechanism for dynamic localization.N-ethylmaleimide-sensitive factor adaptor protein receptors (SNAP receptors, SNAREs) mediate fusion between vesicles and target membranes and are classed as Q- or R-SNAREs based on their sequence. We analysed gain- and loss-of-function mutants, dominant negative transgenics and protein localization of the Arabidopsis R-SNARE VAMP714 to understand its function.We demonstrate that VAMP714 is essential for the insertion of PINs into the plasmamembrane, for polar auxin transport, and for root gravitropism and morphogenesis. VAMP714 gene expression is upregulated by auxin, and the VAMP714 protein co-localizes with ER and Golgi vesicles and with PIN proteins at the plasma membrane.It is proposed that VAMP714 mediates the delivery of PIN-carrying vesicles to the plasma membrane, and that this forms part of a positive regulatory loop in which auxin activates a VAMP714-dependent PIN/auxin transport system to control development.

scholarly journals Auxin methylation is required for differential growth inArabidopsis

2018 ◽  
Vol 115 (26) ◽  
pp. 6864-6869 ◽  
Author(s):  
Mohamad Abbas ◽  
Jorge Hernández-García ◽  
Stephan Pollmann ◽  
Sophia L. Samodelov ◽  
Martina Kolb ◽  
...  
Keyword(s):  
Gene Expression ◽  
Auxin Transport ◽  
Polar Auxin Transport ◽  
Asymmetric Distribution ◽  
Loss Of Function ◽  
Differential Growth ◽  
Specific Expression ◽  
Auxin Homeostasis ◽  
Auxin Distribution ◽  
Gene Expression Levels

Asymmetric auxin distribution is instrumental for the differential growth that causes organ bending on tropic stimuli and curvatures during plant development. Local differences in auxin concentrations are achieved mainly by polarized cellular distribution of PIN auxin transporters, but whether other mechanisms involving auxin homeostasis are also relevant for the formation of auxin gradients is not clear. Here we show that auxin methylation is required for asymmetric auxin distribution across the hypocotyl, particularly during its response to gravity. We found that loss-of-function mutants inArabidopsis IAA CARBOXYL METHYLTRANSFERASE1(IAMT1) prematurely unfold the apical hook, and that their hypocotyls are impaired in gravitropic reorientation. This defect is linked to an auxin-dependent increase inPINgene expression, leading to an increased polar auxin transport and lack of asymmetric distribution of PIN3 in theiamt1mutant. Gravitropic reorientation in theiamt1mutant could be restored with either endodermis-specific expression ofIAMT1or partial inhibition of polar auxin transport, which also results in normalPINgene expression levels. We propose that IAA methylation is necessary in gravity-sensing cells to restrict polar auxin transport within the range of auxin levels that allow for differential responses.

scholarly journals Clathrin Hub Expression Affects Early Endosome Distribution with Minimal Impact on Receptor Sorting and Recycling

2001 ◽  
Vol 12 (9) ◽  
pp. 2790-2799 ◽  
Author(s):  
Elizabeth M. Bennett ◽  
Sharron X. Lin ◽  
Mhairi C. Towler ◽  
Frederick R. Maxfield ◽  
Frances M. Brodsky
Keyword(s):  
Plasma Membrane ◽  
Early Endosome ◽  
Dominant Negative ◽  
Endocytic Pathway ◽  
Endocytic Trafficking ◽  
Minimal Impact ◽  
Recycling Endosomes ◽  
Receptor Mediated Endocytosis ◽  
Early Endosomes ◽  
Endosomal Membranes

Clathrin-coated vesicles execute receptor-mediated endocytosis at the plasma membrane. However, a role for clathrin in later endocytic trafficking processes, such as receptor sorting and recycling or maintaining the organization of the endocytic pathway, has not been thoroughly characterized. The existence of clathrin-coated buds on endosomes suggests that clathrin might mediate later endocytic trafficking events. To investigate the function of clathrin-coated buds on endosomal membranes, endosome function and distribution were analyzed in a HeLa cell line that expresses the dominant-negative clathrin inhibitor Hub in an inducible manner. As expected, Hub expression reduced receptor-mediated endocytosis at the plasma membrane. Hub expression also induced a perinuclear aggregation of early endosome antigen 1-positive early endosomes, such that sorting and recycling endosomes were found tightly concentrated in the perinuclear region. Despite the dramatic redistribution of endosomes, Hub expression did not affect the overall kinetics of receptor sorting or recycling. These data show that clathrin function is necessary to maintain proper cellular distribution of early endosomes but does not play a prominent role in sorting and recycling events. Thus, clathrin's role on endosomal membranes is to influence organelle localization and is distinct from its role in trafficking pathways at the plasma membrane and trans-Golgi network.

scholarly journals Eps15 Is Recruited to the Plasma Membrane upon Epidermal Growth Factor Receptor Activation and Localizes to Components of the Endocytic Pathway during Receptor Internalization

1999 ◽  
Vol 10 (2) ◽  
pp. 417-434 ◽  
Author(s):  
Maria Rosaria Torrisi ◽  
Lavinia Vittoria Lotti ◽  
Francesca Belleudi ◽  
Roberto Gradini ◽  
Anna Elisabetta Salcini ◽  
...  
Keyword(s):  
Epidermal Growth Factor Receptor ◽  
Plasma Membrane ◽  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Growth Factor Receptor ◽  
Adaptor Protein ◽  
Receptor Activation ◽  
Endocytic Pathway ◽  
Receptor Internalization ◽  
Epidermal Growth

Eps15 is a substrate for the tyrosine kinase of the epidermal growth factor receptor (EGFR) and is characterized by the presence of a novel protein:protein interaction domain, the EH domain. Eps15 also stably binds the clathrin adaptor protein complex AP-2. Previous work demonstrated an essential role for eps15 in receptor-mediated endocytosis. In this study we show that, upon activation of the EGFR kinase, eps15 undergoes dramatic relocalization consisting of 1) initial relocalization to the plasma membrane and 2) subsequent colocalization with the EGFR in various intracellular compartments of the endocytic pathway, with the notable exclusion of coated vesicles. Relocalization of eps15 is independent of its binding to the EGFR or of binding of the receptor to AP-2. Furthermore, eps15 appears to undergo tyrosine phosphorylation both at the plasma membrane and in a nocodazole-sensitive compartment, suggesting sustained phosphorylation in endocytic compartments. Our results are consistent with a model in which eps15 undergoes cycles of association:dissociation with membranes and suggest multiple roles for this protein in the endocytic pathway.

scholarly journals ATANN3 is involved in extracellular ATP-regulated auxin transport and distribution in Arabidopsis thaliana seedlings

2021 ◽  
Author(s):  
Lijuan Han ◽  
Shuyan Xia ◽  
Jiawei Xu ◽  
Ruojia Zhu ◽  
Zhonglin Shang ◽  
...  
Keyword(s):  
Arabidopsis Thaliana ◽  
Stress Responses ◽  
Auxin Transport ◽  
Inhibitory Effect ◽  
Extracellular Atp ◽  
Root Tip ◽  
Asymmetric Distribution ◽  
Loss Of Function ◽  
Hypocotyl Growth

Abstract Background Extracellular ATP (eATP) exists in the apoplast of plants and plays multiple roles in growth, development, and stress responses. It has been reported that eATP stimulation suppresses growth rate and alters growth orientation of root and hypocotyls of Arabidopsis thaliana seedlings by affecting auxin accumulation and transport in these organs. However, the mechanism of eATP-stimulated vegetative organ growth remains unclear. Annexins are involved in multiple aspects of plant cellular metabolism, while the role of annexins in response to apoplast signal remains unclear. Here, by using loss-of-function mutants, we investigated the role of several annexins in eATP-regulated root and hypocotyl growth. Since mutant of AtANN3 did not respond to eATP sensitively, the role of AtANN3 in eATP regulated auxin transport was intensively investigated. Results First, the inhibitory effect of eATP on root or hypocotyl elongation was weakened or impaired in AtANN3 null mutants (atann3). Meanwhile, single-, double- or triple-null mutant of AtANN1, AtANN2 or AtANN4 responded to eATP stimulation in same manner and degree with Col-0. The abundance and distribution of Dr5-GUS and Dr5-GFP indicated that eATP-induced accumulation and asymmetric distribution of auxin in root tip or hypocotyl cells, which appeared in wild type controls, were lacking in atann3 seedlings. Further, eATP-induced accumulation and asymmetric distribution of PIN2-GFP in root tip cells or PIN3-GFP in hypocotyl cells were reduced in atann3 seedlings. Conclusions AtANN3 may be involved in eATP-regulated seedling growth through regulating auxin transport and accumulation in vegetative organs.

scholarly journals Mechanism for Removal of Tumor Necrosis Factor Receptor 1 from the Cell Surface by the Adenovirus RIDα/β Complex

2005 ◽  
Vol 79 (21) ◽  
pp. 13606-13617 ◽  
Author(s):  
Y. Rebecca Chin ◽  
Marshall S. Horwitz
Keyword(s):  
Plasma Membrane ◽  
Tumor Necrosis Factor ◽  
Tumor Necrosis ◽  
Tumor Necrosis Factor Receptor ◽  
Adaptor Protein ◽  
Dominant Negative ◽  
Sorting Motif ◽  
Interfering Rna ◽  
Necrosis Factor

ABSTRACT Proteins encoded in adenovirus early region 3 have important immunoregulatory properties. We have recently shown that the E3-10.4K/14.5K (RIDα/β) complex downregulates tumor necrosis factor receptor 1 (TNFR1) expression at the plasma membrane. To study the role of the RIDβ tyrosine sorting motif in the removal of surface TNFR1, tyrosine 122 on RIDβ was mutated to alanine or phenylalanine. Both RIDβ mutations not only abolished the downregulation of surface TNFR1 but paradoxically increased surface TNFR1 levels. RID also downregulates other death receptors, such as FAS; however, surface FAS expression was not increased by RIDβ mutants, suggesting that regulation of TNFR1 and that of FAS by RID are mechanistically different. In the mixing experiments, the wild-type (WT) RID-mediated TNFR1 downregulation was partially inhibited in the presence of RIDβ mutants, indicating that the mutants compete for TNFR1 access. Indeed, an association between RIDβ and TNFR1 was shown by coimmunoprecipitation. In contrast, the mutants did not affect the WT RID-induced downregulation of FAS. These differential effects support a model in which RID associates with TNFR1 on the plasma membrane, whereas RID probably associates with FAS in a cytoplasmic compartment. By using small interfering RNA against the μ2 subunit of adaptor protein 2, dominant negative dynamin construct K44A, and the lysosomotropic agents bafilomycin A1 and ammonium chloride, we also demonstrated that surface TNFR1 was internalized by RID by a clathrin-dependent process involving μ2 and dynamin, followed by degradation of TNFR1 via an endosomal/lysosomal pathway.

scholarly journals Mutations in Auxilin cause parkinsonism via impaired clathrin-mediated trafficking at the Golgi apparatus and synapse

2019 ◽  
Author(s):  
Dorien A. Roosen ◽  
Natalie Landeck ◽  
Melissa Conti ◽  
Nathan Smith ◽  
Sara Saez-Atienzar ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Adaptor Protein ◽  
Cellular Function ◽  
Motor Disorder ◽  
List Type ◽  
Nigrostriatal Pathway ◽  
Loss Of Function ◽  
Motor Impairments ◽  
Clathrin Adaptor ◽  
Golgi Network

AbstractParkinson’s disease (PD) is a common neurodegenerative motor disorder characterized in part by neuropathological lesions in the nigrostriatal pathway. While most cases of PD are sporadic in nature, several inherited monogenic syndromes exist that overlap clinically and pathologically with sporadic PD. Of these, loss of function mutations in DNAJC6, which encodes the protein Auxilin, cause an aggressive form of juvenile onset PD. Auxilin and its homologues are known to play a role in clathrin-mediated trafficking, which is crucial for cellular function in all eukaryotes and plays a specialized role in synaptic transmission in higher organisms. Auxilin is the major neuronal uncoating protein for clathrin-coated vesicles required for delivery of cargo from the plasma membrane and trans-Golgi network to intracellular destinations. However, how mutations in Auxilin cause PD is currently not understood. To address this problem, we generated a novel mouse model carrying an endogenous pathogenic Auxilin mutation. When bred to homozygosity, this mutation induced neurological phenotypes that phenocopy clinical features observed in patients, including motor impairments reminiscent of bradykinesia and gait problems. Mapping the interactome of Auxilin confirmed clathrin and synaptic clathrin adaptor protein interactions and also identified novel Golgi-resident interactors. Critically, all tested pathogenic mutations in Auxilin retained clathrin adaptor protein binding but lost interaction with clathrin itself. These observations describe a mechanism by which impaired clathrin-mediated trafficking in R857G Auxilin mice, both at the Golgi and the synapse, results in neuropathological lesions in the nigrostriatal pathway. Collectively, these results provide novel insights for PD pathogenesis in Auxilin mutation carriers, reinforcing a key role for clathrin-mediated trafficking in PD, and expand our understanding of the cellular function of Auxilin.HighlightsAuxilin interacts with Golgi-resident clathrin adaptor protein GGA2Auxilin is involved with uncoating of CCVs at the Golgi and synapseImpaired clathrin-mediated trafficking underlies PD-like phenotypes in R857G Auxilin mice

scholarly journals Role of AP1 and Gadkin in the traffic of secretory endo-lysosomes

2011 ◽  
Vol 22 (12) ◽  
pp. 2068-2082 ◽  
Author(s):  
Karine Laulagnier ◽  
Nicole L. Schieber ◽  
Tanja Maritzen ◽  
Volker Haucke ◽  
Robert G. Parton ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Secretory Pathway ◽  
Protein Complexes ◽  
Adaptor Protein ◽  
Cytosolic Calcium ◽  
Endocytic Pathway ◽  
Secretory Process ◽  
Microtubule Depolymerization ◽  
Lysosome Related Organelles ◽  
Endocytic Compartments

Whereas lysosome-related organelles (LRO) of specialized cells display both exocytic and endocytic features, lysosomes in nonspecialized cells can also acquire the property to fuse with the plasma membrane upon an acute rise in cytosolic calcium. Here, we characterize this unconventional secretory pathway in fibroblast-like cells, by monitoring the appearance of Lamp1 on the plasma membrane and the release of lysosomal enzymes into the medium. After sequential ablation of endocytic compartments in living cells, we find that donor membranes primarily derive from a late compartment, but that an early compartment is also involved. Strikingly, this endo-secretory process is not affected by treatments that inhibit endosome dynamics (microtubule depolymerization, cholesterol accumulation, overexpression of Rab7 or its effector Rab-interacting lysosomal protein [RILP], overexpression of Rab5 mutants), but depends on Rab27a, a GTPase involved in LRO secretion, and is controlled by F-actin. Moreover, we find that this unconventional endo-secretory pathway requires the adaptor protein complexes AP1, Gadkin (which recruits AP1 by binding to the γ1 subunit), and AP2, but not AP3. We conclude that a specific fraction of the AP2-derived endocytic pathway is dedicated to secretory purposes under the control of AP1 and Gadkin.

Abstract 2257: KCNJ2 Mutations in Patients Referred for Catecholaminergic Polymorphic Ventricular Tachycardia Gene Screening

Circulation ◽  
2007 ◽  
Vol 116 (suppl_16) ◽  
Author(s):  
Yanfei Ruan ◽  
Juliane Theilade ◽  
Mirella Memmi ◽  
Luciana D Giuli ◽  
Nicoletta Rizzi ◽  
...  
Keyword(s):  
Ventricular Tachycardia ◽  
Mutational Analysis ◽  
Dominant Negative ◽  
Catecholaminergic Polymorphic Ventricular Tachycardia ◽  
Polymorphic Ventricular Tachycardia ◽  
Dominant Negative Effect ◽  
Confocal Laser ◽  
List Type ◽  
Missense Mutations ◽  
Loss Of Function

Background : Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an inherited arrhythmogenic disease characterized by adrenergically mediated polymorphic or bidirectional ventricular tachycardia (BVT). Andersen-Tawil syndrome (ATS1), which are mainly caused by KCNJ2 mutations, phenocopies CPVT and may manifest the typical adrenergically mediated BVT. The purpose of this study was assess whether patients (pts) lacking periodic paralysis typical of ATS1 and diagnosed as CPVT because of BVT carry KCNJ2 mutations. Methods and Results : Mutational analysis of KCNJ2 was performed in 23 RyR2 and CASQ2 genotype-negative CPVT pts with normal QT interval. Two novel missense mutations (S220I and T305I) were identified. Mutations were absent in >400 reference alleles. Both of the pts present exercise or isoproterenol induced BVT, baseline ECG with prominent U wave and mild facial abnormalities. In vitro characterization showed that no current is detectable when S220I and T305I mutants clones are expressed; on the contrary co-expression of WT and mutant KCNJ2 to mimic heterozygosis present in patients, caused significant dominant negative effect (see figure ). Confocal laser microscopy revealed normal sarcolemmal localization of the mutant channels and of the heterozygous channels. Conclusions : KCNJ2 mutation with loss of function are present in a minority of pts with clinical diagnosis of CPVT. Given the limited number of CPVT pts with KCNJ2 mutations it is impossible to determine whether their prognosis is identical to that of RyR2 and CASQ2 related CPVT. Screening of KCNJ2 should be considered in CPVT pts without mutations in RyR2 and CASQ2 genes.

scholarly journals Phosphoinositide–Ap-2 Interactions Required for Targeting to Plasma Membrane Clathrin-Coated Pits

1999 ◽  
Vol 146 (4) ◽  
pp. 755-764 ◽  
Author(s):  
Ibragim Gaidarov ◽  
James H. Keen
Keyword(s):  
Plasma Membrane ◽  
Mammalian Cells ◽  
Limited Proteolysis ◽  
Adaptor Protein ◽  
Dominant Negative ◽  
Coated Pits ◽  
Intact Cells ◽  
Α Subunit ◽  
General Role ◽  
Receptor Complexes

The clathrin-associated AP-2 adaptor protein is a major polyphosphoinositide-binding protein in mammalian cells. A high affinity binding site has previously been localized to the NH2-terminal region of the AP-2 α subunit (Gaidarov et al. 1996. J. Biol. Chem. 271:20922–20929). Here we used deletion and site- directed mutagenesis to determine that α residues 21–80 comprise a discrete folding and inositide-binding domain. Further, positively charged residues located within this region are involved in binding, with a lysine triad at positions 55–57 particularly critical. Mutant peptides and protein in which these residues were changed to glutamine retained wild-type structural and functional characteristics by several criteria including circular dichroism spectra, resistance to limited proteolysis, and clathrin binding activity. When expressed in intact cells, mutated α subunit showed defective localization to clathrin-coated pits; at high expression levels, the appearance of endogenous AP-2 in coated pits was also blocked consistent with a dominant-negative phenotype. These results, together with recent work indicating that phosphoinositides are also critical to ligand-dependent recruitment of arrestin-receptor complexes to coated pits (Gaidarov et al. 1999. EMBO (Eur. Mol. Biol. Organ.) J. 18:871–881), suggest that phosphoinositides play a critical and general role in adaptor incorporation into plasma membrane clathrin-coated pits.

scholarly journals Clathrin Assembly Lymphoid Myeloid Leukemia (CALM) Protein: Localization in Endocytic-coated Pits, Interactions with Clathrin, and the Impact of Overexpression on Clathrin-mediated Traffic

1999 ◽  
Vol 10 (8) ◽  
pp. 2687-2702 ◽  
Author(s):  
Francesc Tebar ◽  
Stefan K. Bohlander ◽  
Alexander Sorkin
Keyword(s):  
Plasma Membrane ◽  
Heavy Chain ◽  
Myeloid Leukemia ◽  
Protein Localization ◽  
Adaptor Protein ◽  
Coated Pits ◽  
Trans Golgi Network ◽  
Clathrin Heavy Chain ◽  
Golgi Network ◽  
The Impact

The clathrin assembly lymphoid myeloid leukemia (CALM) gene encodes a putative homologue of the clathrin assembly synaptic protein AP180. Hence the biochemical properties, the subcellular localization, and the role in endocytosis of a CALM protein were studied. In vitro binding and coimmunoprecipitation demonstrated that the clathrin heavy chain is the major binding partner of CALM. The bulk of cellular CALM was associated with the membrane fractions of the cell and localized to clathrin-coated areas of the plasma membrane. In the membrane fraction, CALM was present at near stoichiometric amounts relative to clathrin. To perform structure–function analysis of CALM, we engineered chimeric fusion proteins of CALM and its fragments with the green fluorescent protein (GFP). GFP–CALM was targeted to the plasma membrane–coated pits and also found colocalized with clathrin in the Golgi area. High levels of expression of GFP–CALM or its fragments with clathrin-binding activity inhibited the endocytosis of transferrin and epidermal growth factor receptors and altered the steady-state distribution of the mannose-6-phosphate receptor in the cell. In addition, GFP–CALM overexpression caused the loss of clathrin accumulation in the trans-Golgi network area, whereas the localization of the clathrin adaptor protein complex 1 in the trans-Golgi network remained unaffected. The ability of the GFP-tagged fragments of CALM to affect clathrin-mediated processes correlated with the targeting of the fragments to clathrin-coated areas and their clathrin-binding capacities. Clathrin–CALM interaction seems to be regulated by multiple contact interfaces. The C-terminal part of CALM binds clathrin heavy chain, although the full-length protein exhibited maximal ability for interaction. Altogether, the data suggest that CALM is an important component of coated pit internalization machinery, possibly involved in the regulation of clathrin recruitment to the membrane and/or the formation of the coated pit.

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