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 Regulation of activity and localization of the WNK1 protein kinase by hyperosmotic stress

2006 ◽  
Vol 176 (1) ◽  
pp. 89-100 ◽  
Author(s):  
Anna Zagórska ◽  
Eulalia Pozo-Guisado ◽  
Jérôme Boudeau ◽  
Alberto C. Vitari ◽  
Fatema H. Rafiqi ◽  
...  
Keyword(s):  
Mutational Analysis ◽  
Adaptor Protein ◽  
Hyperosmotic Stress ◽  
Recycling Endosomes ◽  
Regulation Of Activity ◽  
Clathrin Adaptor ◽  
Golgi Network ◽  
Interfering Rna ◽  
Rna Depletion ◽  
And Oxidative Stress

Mutations within the WNK1 (with-no-K[Lys] kinase-1) gene cause Gordon's hypertension syndrome. Little is known about how WNK1 is regulated. We demonstrate that WNK1 is rapidly activated and phosphorylated at multiple residues after exposure of cells to hyperosmotic conditions and that activation is mediated by the phosphorylation of its T-loop Ser382 residue, possibly triggered by a transautophosphorylation reaction. Activation of WNK1 coincides with the phosphorylation and activation of two WNK1 substrates, namely, the protein kinases STE20/SPS1-related proline alanine–rich kinase (SPAK) and oxidative stress response kinase-1 (OSR1). Small interfering RNA depletion of WNK1 impairs SPAK/OSR1 activity and phosphorylation of residues targeted by WNK1. Hyperosmotic stress induces rapid redistribution of WNK1 from the cytosol to vesicular structures that may comprise trans-Golgi network (TGN)/recycling endosomes, as they display rapid movement, colocalize with clathrin, adaptor protein complex 1 (AP-1), and TGN46, but not the AP-2 plasma membrane–coated pit marker nor the endosomal markers EEA1, Hrs, and LAMP1. Mutational analysis suggests that the WNK1 C-terminal noncatalytic domain mediates vesicle localization. Our observations shed light on the mechanism by which WNK1 is regulated by hyperosmotic stress.

scholarly journals Ent3p and Ent5p Exhibit Cargo-specific Functions in Trafficking Proteins between the Trans-Golgi Network and the Endosomes in Yeast

2007 ◽  
Vol 18 (5) ◽  
pp. 1803-1815 ◽  
Author(s):  
Alenka Čopič ◽  
Trevor L. Starr ◽  
Randy Schekman
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Surface ◽  
Protein Transport ◽  
Adaptor Protein ◽  
Additional Function ◽  
Trans Golgi Network ◽  
Cargo Proteins ◽  
Clathrin Adaptor ◽  
Dependent Transport ◽  
Golgi Network

The phosphoinositide-binding proteins Ent3p and Ent5p are required for protein transport from the trans-Golgi network (TGN) to the vacuole in Saccharomyces cerevisiae. Both proteins interact with the monomeric clathrin adaptor Gga2p, but Ent5p also interacts with the clathrin adaptor protein 1 (AP-1) complex, which facilitates retention of proteins such as Chs3p at the TGN. When both ENT3 and ENT5 are mutated, Chs3p is diverted from an intracellular reservoir to the cell surface. However, Ent3p and Ent5p are not required for the function of AP-1, but rather they seem to act in parallel with AP-1 to retain proteins such as Chs3p at the TGN. They have all the properties of clathrin adaptors, because they can both bind to clathrin and to cargo proteins. Like AP-1, Ent5p binds to Chs3p, whereas Ent3p facilitates the interaction between Gga2p and the endosomal syntaxin Pep12p. Thus, Ent3p has an additional function in Gga-dependent transport to the late endosome. Ent3p also facilitates the association between Gga2p and clathrin; however, Ent5p can partially substitute for this function. We conclude that the clathrin adaptors AP-1, Ent3p, Ent5p, and the Ggas cooperate in different ways to sort proteins between the TGN and the endosomes.

scholarly journals The Yeast Adaptor Protein Complex, AP-3, Is Essential for the Efficient Delivery of Alkaline Phosphatase by the Alternate Pathway to the Vacuole

1997 ◽  
Vol 139 (7) ◽  
pp. 1761-1774 ◽  
Author(s):  
J. David Stepp ◽  
Kristen Huang ◽  
Sandra K. Lemmon
Keyword(s):  
Alkaline Phosphatase ◽  
Adaptor Protein ◽  
Carboxypeptidase Y ◽  
Loss Of Function ◽  
Synthetic Lethal ◽  
Efficient Delivery ◽  
Cargo Proteins ◽  
Clathrin Adaptor ◽  
Class E

A novel clathrin adaptor-like complex, adaptor protein (AP)-3, has recently been described in yeast and in animals. To gain insight into the role of yeast AP-3, a genetic strategy was devised to isolate gene products that are required in the absence of the AP-3 μ chain encoded by APM3. One gene identified by this synthetic lethal screen was VPS45. The Vps pathway defines the route that several proteins, including carboxypeptidase Y, take from the late Golgi to the vacuole. However, vacuolar alkaline phosphatase (ALP) is transported via an alternate, intracellular route. This suggested that the apm3-Δ vps45 synthetic phenotype could be caused by a block in both the alternate and the Vps pathways. Here we demonstrate that loss of function of the AP-3 complex results in slowed processing and missorting of ALP. ALP is no longer localized to the vacuole membrane by immunofluorescence, but is found in small punctate structures throughout the cell. This pattern is distinct from the Golgi marker Kex2p, which is unaffected in AP-3 mutants. We also show that in the apm3-Δ mutant some ALP is delivered to the vacuole by diversion into the Vps pathway. Class E vps mutants accumulate an exaggerated prevacuolar compartment containing membrane proteins on their way to the vacuole or destined for recycling to the Golgi. Surprisingly, in AP-3 class E vps double mutants these proteins reappear on the vacuole. We suggest that some AP-3–dependent cargo proteins that regulate late steps in Golgi to vacuole transport are diverted into the Vps pathway allowing completion of transfer to the vacuole in the class E vps mutant.

scholarly journals AP-4 mediated ATG9A sorting underlies axonal and autophagosome biogenesis defects in a mouse model of AP-4 deficiency syndrome

2017 ◽  
Author(s):  
Davor Ivankovic ◽  
Guillermo López-Doménech ◽  
James Drew ◽  
Sharon A. Tooze ◽  
Josef T. Kittler
Keyword(s):  
Mouse Model ◽  
Transmembrane Protein ◽  
Adaptor Protein ◽  
Deficiency Syndrome ◽  
Cellular Level ◽  
Loss Of Function ◽  
Golgi Network ◽  
Autophagosome Maturation

AbstractAdaptor protein (AP) complexes have critical roles in transmembrane protein sorting. AP-4 remains poorly understood in the brain despite its loss of function leading to a hereditary spastic paraplegia termed AP-4 deficiency syndrome. Here we demonstrate that knockout (KO) of AP-4 in a mouse model leads to thinning of the corpus callosum and ventricular enlargement, anatomical defects previously described in patients. At the cellular level, we find that AP-4 KO leads to defects in axonal extension and branching, in addition to aberrant distal swellings. Interestingly, we show that ATG9A, a key protein in autophagosome maturation, is critically dependent on AP-4 for its sorting from the trans-golgi network. Failure of AP-4 mediated ATG9A sorting results in its dramatic retention in the trans-golgi network in vitro and in vivo leading to a specific reduction of the axonal pool of ATG9A. As a result, autophagosome biogenesis is aberrant in the axon of AP-4 deficient neurons. The specific alteration to axonal integrity and axonal autophagosome maturation in AP-4 knockout neurons may underpin the pathology of AP-4 deficiency.

scholarly journals Ap2s1 mutation in mice causes familial hypocalciuric hypercalcemia type 3

2020 ◽  
Author(s):  
Fadil M. Hannan ◽  
Mark Stevenson ◽  
Asha L. Bayliss ◽  
Victoria J. Stokes ◽  
Michelle Stewart ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Fibroblast Growth Factor 23 ◽  
Plasma Concentrations ◽  
Adaptor Protein ◽  
Endosomal Trafficking ◽  
Familial Hypocalciuric Hypercalcemia ◽  
Loss Of Function ◽  
Dihydroxyvitamin D ◽  
Sigma Subunit ◽  
Type 3

AbstractMutations of the adaptor protein-2 sigma subunit (AP2S1) gene which encodes AP2σ2, a component of the ubiquitous AP2 heterotetrameric complex involved in endosomal trafficking of the calcium-sensing receptor (CaSR), cause familial hypocalciuric hypercalcemia type 3 (FHH3). FHH3 patients have heterozygous AP2S1 missense Arg15 mutations (p.Arg15Cys, p.Arg15His or p.Arg15Leu) with marked hypercalcemia and occasional hypophosphatemia and osteomalacia. To further characterise the phenotypic spectrum and calcitropic pathophysiology of FHH3, we used CRISPR/Cas9 genome editing to generate mice harboring the AP2S1 p.Arg15Leu mutation, which causes the most severe FHH3 phenotype. Heterozygous (Ap2s1+/L15) mice were viable, and had marked hypercalcemia, hypermagnesemia, hypophosphatemia, and increased plasma concentrations of parathyroid hormone, fibroblast growth factor 23 and alkaline phosphatase activity, but normal pro-collagen type 1 N-terminal pro-peptide and 1,25 dihydroxyvitamin D. Homozygous (Ap2s1L15/L15) mice invariably died perinatally. The AP2S1 p.Arg15Leu mutation impaired protein-protein interactions between AP2σ2 and the other AP2 subunits, and the CaSR. Cinacalcet, a CaSR allosteric activator, ameliorated the hypercalcemia and elevated PTH concentrations, but not the diminished AP2σ2-CaSR interaction. Thus, our studies have established a mouse model with a germline loss-of-function AP2S1 mutation that is representative for FHH3 in humans, and demonstrated that cinacalcet corrects the abnormalities of plasma calcium and PTH.

scholarly journals AP-1 and clathrin are essential for secretory granule biogenesis in Drosophila

2011 ◽  
Vol 22 (12) ◽  
pp. 2094-2105 ◽  
Author(s):  
Jason Burgess ◽  
Miluska Jauregui ◽  
Julie Tan ◽  
Janet Rollins ◽  
Sylvie Lallet ◽  
...  
Keyword(s):  
Secretory Granule ◽  
Secretory Granules ◽  
Adaptor Protein ◽  
Fruit Fly ◽  
Biologically Active ◽  
Granule Formation ◽  
The Third ◽  
Larval Salivary Gland ◽  
Clathrin Adaptor ◽  
Golgi Network

 Regulated secretion of hormones, digestive enzymes, and other biologically active molecules requires the formation of secretory granules. Clathrin and the clathrin adaptor protein complex 1 (AP-1) are necessary for maturation of exocrine, endocrine, and neuroendocrine secretory granules. However, the initial steps of secretory granule biogenesis are only minimally understood. Powerful genetic approaches available in the fruit fly Drosophila melanogaster were used to investigate the molecular pathway for biogenesis of the mucin-containing “glue granules” that form within epithelial cells of the third-instar larval salivary gland. Clathrin and AP-1 colocalize at the trans-Golgi network (TGN) and clathrin recruitment requires AP-1. Furthermore, clathrin and AP-1 colocalize with secretory cargo at the TGN and on immature granules. Finally, loss of clathrin or AP-1 leads to a profound block in secretory granule formation. These findings establish a novel role for AP-1– and clathrin-dependent trafficking in the biogenesis of mucin-containing secretory granules.

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 The clathrin adaptor complex 1 directly binds to a sorting signal in Ste13p to reduce the rate of its trafficking to the late endosome of yeast

2006 ◽  
Vol 173 (4) ◽  
pp. 615-626 ◽  
Author(s):  
Christopher Foote ◽  
Steven F. Nothwehr
Keyword(s):  
Alkaline Phosphatase ◽  
Steady State ◽  
Protein A ◽  
Adaptor Protein ◽  
Half Time ◽  
Sorting Signal ◽  
Early Endosomes ◽  
Direct Binding ◽  
Clathrin Adaptor ◽  
Golgi Network

Yeast trans-Golgi network (TGN) membrane proteins maintain steady-state localization by constantly cycling to and from endosomes. In this study, we examined the trafficking itinerary and molecular requirements for delivery of a model TGN protein A(F→A)–alkaline phosphatase (ALP) to the prevacuolar/endosomal compartment (PVC). A(F→A)-ALP was found to reach the PVC via early endosomes (EEs) with a half-time of ∼60 min. Delivery of A(F→A)-ALP to the PVC was not dependent on either the GGA or adaptor protein 1 (AP-1) type of clathrin adaptors, which are thought to function in TGN to PVC and TGN to EE transport, respectively. Surprisingly, in cells lacking the function of both GGA and AP-1 adaptors, A(F→A)-ALP transport to the PVC was dramatically accelerated. A 12-residue cytosolic domain motif of A(F→A)-ALP was found to mediate direct binding to AP-1 and was sufficient to slow TGN→EE→PVC trafficking. These results suggest a model in which this novel sorting signal targets A(F→A)-ALP into clathrin/AP-1 vesicles at the EE for retrieval back to the TGN.

scholarly journals Trafficking of the Menkes copper transporter ATP7A is regulated by clathrin-, AP-2–, AP-1–, and Rab22-dependent steps

2013 ◽  
Vol 24 (11) ◽  
pp. 1735-1748 ◽  
Author(s):  
Zoe G. Holloway ◽  
Antonio Velayos-Baeza ◽  
Gareth J. Howell ◽  
Clotilde Levecque ◽  
Sreenivasan Ponnambalam ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Adaptor Protein ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Copper Transporter ◽  
Caveolin 1 ◽  
Clathrin Adaptor ◽  
Partial Overlap ◽  
Golgi Network

The transporter ATP7A mediates systemic copper absorption and provides cuproenzymes in the trans-Golgi network (TGN) with copper. To regulate metal homeostasis, ATP7A constitutively cycles between the TGN and plasma membrane (PM). ATP7A trafficking to the PM is elevated in response to increased copper load and is reversed when copper concentrations are lowered. Molecular mechanisms underlying this trafficking are poorly understood. We assess the role of clathrin, adaptor complexes, lipid rafts, and Rab22a in an attempt to decipher the regulatory proteins involved in ATP7A cycling. While RNA interference (RNAi)–mediated depletion of caveolin 1/2 or flotillin had no effect on ATP7A localization, clathrin heavy chain depletion or expression of AP180 dominant-negative mutant not only disrupted clathrin-regulated pathways, but also blocked PM-to-TGN internalization of ATP7A. Depletion of the μ subunits of either adaptor protein-2 (AP-2) or AP-1 using RNAi further provides evidence that both clathrin adaptors are important for trafficking of ATP7A from the PM to the TGN. Expression of the GTP-locked Rab22aQ64L mutant caused fragmentation of TGN membrane domains enriched for ATP7A. These appear to be a subdomain of the mammalian TGN, showing only partial overlap with the TGN marker golgin-97. Of importance, ATP7A remained in the Rab22aQ64L-generated structures after copper treatment and washout, suggesting that forward trafficking out of this compartment was blocked. This study provides evidence that multiple membrane-associated factors, including clathrin, AP-2, AP-1, and Rab22, are regulators of ATP7A trafficking.

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.

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