scholarly journals EpsinR

2003 ◽  
Vol 160 (2) ◽  
pp. 213-222 ◽  
Author(s):  
Ian G. Mills ◽  
Gerrit J.K. Praefcke ◽  
Yvonne Vallis ◽  
Brian J. Peter ◽  
Lene E. Olesen ◽  
...  
Keyword(s):  
Cathepsin D ◽  
Adaptor Protein ◽  
Biological Data ◽  
Coated Vesicle ◽  
Binding Motif ◽  
Binding Partner ◽  
High Affinity ◽  
Ef Hand ◽  
Phosphatidylinositol 4 ◽  
Golgi Network

EpsinR is a clathrin-coated vesicle (CCV) enriched 70-kD protein that binds to phosphatidylinositol-4-phosphate, clathrin, and the gamma appendage domain of the adaptor protein complex 1 (AP1). In cells, its distribution overlaps with the perinuclear pool of clathrin and AP1 adaptors. Overexpression disrupts the CCV-dependent trafficking of cathepsin D from the trans-Golgi network to lysosomes and the incorporation of mannose-6-phosphate receptors into CCVs. These biochemical and cell biological data point to a role for epsinR in AP1/clathrin budding events in the cell, just as epsin1 is involved in the budding of AP2 CCVs. Furthermore, we show that two gamma appendage domains can simultaneously bind to epsinR with affinities of 0.7 and 45 μM, respectively. Thus, potentially, two AP1 complexes can bind to one epsinR. This high affinity binding allowed us to identify a consensus binding motif of the form DFxDF, which we also find in γ-synergin and use to predict that an uncharacterized EF-hand–containing protein will be a new gamma binding partner.

scholarly journals High-Affinity Binding Of The AP-1 Adaptor Complex to Trans-Golgi Network Membranes Devoid Of Mannose 6-Phosphate Receptors

1999 ◽  
Vol 10 (3) ◽  
pp. 537-549 ◽  
Author(s):  
Yunxiang Zhu ◽  
Linton M. Traub ◽  
Stuart Kornfeld
Keyword(s):  
Coated Vesicle ◽  
Chemical Extraction ◽  
Normal Cells ◽  
High Affinity ◽  
Affinity Membrane ◽  
Docking Proteins ◽  
Mannose 6 Phosphate ◽  
Golgi Network

The GTP-binding protein ADP-ribosylation factor (ARF) initiates clathrin-coat assembly at the trans-Goli network (TGN) by generating high-affinity membrane-binding sites for the AP-1 adaptor complex. Both transmembrane proteins, which are sorted into the assembling coated bud, and novel docking proteins have been suggested to be partners with GTP-bound ARF in generating the AP-1-docking sites. The best characterized, and probably the major transmembrane molecules sorted into the clathrin-coated vesicles that form on the TGN, are the mannose 6-phosphate receptors (MPRs). Here, we have examined the role of the MPRs in the AP-1 recruitment process by comparing fibroblasts derived from embryos of either normal or MPR-negative animals. Despite major alterations to the lysosome compartment in the MPR-deficient cells, the steady-state distribution of AP-1 at the TGN is comparable to that of normal cells. Golgi-enriched membranes prepared from the receptor-negative cells also display an apparently normal capacity to recruit AP-1 in vitro in the presence of ARF and either GTP or GTPγS. The AP-1 adaptor is recruited specifically onto the TGN and not onto the numerous abnormal membrane elements that accumulate within the MPR-negative fibroblasts. AP-1 bound to TGN membranes from either normal or MPR-negative fibroblasts is fully resistant to chemical extraction with 1 M Tris-HCl, pH 7, indicating that the adaptor binds to both membrane types with high affinity. The only difference we do note between the Golgi prepared from the MPR-deficient cells and the normal cells is that AP-1 recruited onto the receptor-lacking membranes in the presence of ARF1·GTP is consistently more resistant to extraction with Tris. Because sensitivity to Tris extraction correlates well with nucleotide hydrolysis, this finding might suggest a possible link between MPR sorting and ARF GAP regulation. We conclude that the MPRs are not essential determinants in the initial steps of AP-1 binding to the TGN but, instead, they may play a regulatory role in clathrin-coated vesicle formation by affecting ARF·GTP hydrolysis.

FAPP2 is required for aquaporin-2 apical sorting at trans-Golgi network in polarized MDCK cells

2009 ◽  
Vol 297 (6) ◽  
pp. C1389-C1396 ◽  
Author(s):  
Naofumi Yui ◽  
Rie Okutsu ◽  
Eisei Sohara ◽  
Tatemitsu Rai ◽  
Akihito Ohta ◽  
...  
Keyword(s):  
Cell Lines ◽  
Plasma Membranes ◽  
Mdck Cells ◽  
Adaptor Protein ◽  
Trans Golgi Network ◽  
Aquaporin 2 ◽  
Phosphatidylinositol 4 ◽  
Apical Membranes ◽  
Apical Sorting ◽  
Golgi Network

FAPP2 is an adaptor protein of phosphatidylinositol-4-phosphate and is involved in the transport of some apical cargos from the trans-Golgi network (TGN). To investigate whether the regulated apical transport of aquaporin-2 (AQP2) is involved in the FAPP2-dependent apical protein-sorting machinery, we measured apical sorting of AQP2 in Madin-Darby canine kidney (MDCK) cells with or without FAPP2 knockdown. We established MDCK cell lines that stably express rat AQP2 without any tag sequence. Then, FAPP2-deficient stable cell lines were established from the AQP2-expressing cell lines by a retrovirus-mediated RNA interference system. In the established cell lines, AQP2 was detected in both apical and basolateral membranes. Forskolin increased only the apical localization of AQP2, which was not affected by basolateral treatment with 0.5% tannic acid, indicating that the forskolin-induced apical transport of AQP2 did not include the transcytotic pathway from basolateral to apical membranes but is a direct transport from TGN to the apical membranes. Using these cell lines, we tested the effect of FAPP2 knockdown on the polarized AQP2 transport to plasma membranes and found that the forskolin-induced apical transport of AQP2 was completely abolished by FAPP2 knockdown. By contrast, the basolateral localization of AQP2 was not affected by FAPP2 knockdown. AQP2 phosphorylation by forskolin was also impaired in FAPP2 knockdown MDCK cells. These results suggest that FAPP2 is necessary to generate AQP2-bearing vesicles at trans-Golgi that will undergo phosphorylation by PKA in subapical regions.

scholarly journals Canonical Interaction of Cyclin G–associated Kinase with Adaptor Protein 1 Regulates Lysosomal Enzyme Sorting

2007 ◽  
Vol 18 (8) ◽  
pp. 2991-3001 ◽  
Author(s):  
Satoshi Kametaka ◽  
Kengo Moriyama ◽  
Patricia V. Burgos ◽  
Evan Eisenberg ◽  
Lois E. Greene ◽  
...  
Keyword(s):  
Lysosomal Enzyme ◽  
Cathepsin D ◽  
Adaptor Protein ◽  
Physiological Relevance ◽  
Lysosomal Sorting ◽  
Canonical Motif ◽  
Cyclin G ◽  
Golgi Network

The adaptor protein 1 (AP1) complex is a heterotetramer that participates in cargo sorting into clathrin-coated vesicles at the trans-Golgi network (TGN) and endosomes. The γ subunit of AP1 possesses a C-terminal “ear” domain that recruits a cohort of accessory proteins through recognition of a shared canonical motif, ΨG[PDE][ΨLM] (where Ψ is an aromatic residue). The physiological relevance of these ear-motif interactions, however, remains to be demonstrated. Here we report that the cyclin G–associated kinase (GAK) has two sequences fitting this motif, FGPL and FGEF, which mediate binding to the AP1-γ-ear domain in vitro. Mutation of both γ-ear–binding sequences or depletion of AP1-γ by RNA interference (RNAi) decreases the association of GAK with the TGN in vivo. Depletion of GAK by RNAi impairs the sorting of the acid hydrolase, cathepsin D, to lysosomes. Importantly, expression of RNAi-resistant GAK restores the lysosomal sorting of cathepsin D in cells depleted of endogenous GAK, whereas expression of a similar construct bearing mutations in both γ-ear–binding sequences fails to correct the sorting defect. Thus, interactions between the ΨG[PDE][ΨLM]-motif sequences in GAK and the AP1-γ-ear domain are critical for the recruitment of GAK to the TGN and the function of GAK in lysosomal enzyme sorting.

scholarly journals Clathrin-dependent Association of CVAK104 with Endosomes and the Trans-Golgi Network

2006 ◽  
Vol 17 (10) ◽  
pp. 4513-4525 ◽  
Author(s):  
Michael Düwel ◽  
Ernst J. Ungewickell
Keyword(s):  
Fluorescent Protein ◽  
Brefeldin A ◽  
Adaptor Protein ◽  
Terminal Segment ◽  
Coated Vesicle ◽  
Membrane Association ◽  
Permeabilized Cells ◽  
Trans Golgi Network ◽  
Golgi Network

CVAK104 is a novel coated vesicle-associated protein with a serine/threonine kinase homology domain that was recently shown to phosphorylate the β2-subunit of the adaptor protein (AP) complex AP2 in vitro. Here, we demonstrate that a C-terminal segment of CVAK104 interacts with the N-terminal domain of clathrin and with the α-appendage of AP2. CVAK104 localizes predominantly to the perinuclear region of HeLa and COS-7 cells, but it is also present on peripheral vesicular structures that are accessible to endocytosed transferrin. The distribution of CVAK104 overlaps extensively with that of AP1, AP3, the mannose 6-phosphate receptor, and clathrin but not at all with its putative phosphorylation target AP2. RNA interference-mediated clathrin knockdown reduced the membrane association of CVAK104. Recruitment of CVAK104 to perinuclear membranes of permeabilized cells is enhanced by guanosine 5′-O-(3-thio)triphosphate, and brefeldin A redistributes CVAK104 in cells. Both observations suggest a direct or indirect requirement for GTP-binding proteins in the membrane association of CVAK104. Live-cell imaging showed colocalization of green fluorescent protein-CVAK104 with endocytosed transferrin and with red fluorescent protein-clathrin on rapidly moving endosomes. Like AP1-depleted COS-7 cells, CVAK104-depleted cells missort the lysosomal hydrolase cathepsin D. Together, our data suggest a function for CVAK104 in clathrin-dependent pathways between the trans-Golgi network and the endosomal system.

scholarly journals γ Subunit of the AP-1 Adaptor Complex Binds Clathrin: Implications for Cooperative Binding in Coated Vesicle Assembly

2001 ◽  
Vol 12 (7) ◽  
pp. 1925-1935 ◽  
Author(s):  
Balraj Doray ◽  
Stuart Kornfeld
Keyword(s):  
Coated Vesicle ◽  
Glutathione S Transferase ◽  
High Affinity ◽  
Terminal Domain ◽  
Γ Subunit ◽  
New Variant ◽  
Golgi Network ◽  
Binding Sequence ◽  
Β Chain ◽  
Human And Mouse

The heterotetrameric AP-1 adaptor complex is involved in the assembly of clathrin-coated vesicles originating from thetrans-Golgi network (TGN). The β1 subunit of AP-1 is known to contain a consensus clathrin binding sequence, LLNLD (the so-called clathrin box motif), in its hinge segment through which the β chain interacts with the N-terminal domains of clathrin trimers. Here, we report that the hinge region of the γ subunit of human and mouse AP-1 contains two copies of a new variant, LLDLL, of the clathrin box motif that also bind to the terminal domain of the clathrin heavy chain. High-affinity binding of the γ hinge to clathrin trimers requires both LLDLL sequences to be present and the spacing between them to be maintained. We also identify an independent clathrin-binding site within the appendage domain of the γ subunit that interacts with a region of clathrin other than the N-terminal domain. Clathrin polymerization is promoted by glutathione S-transferase (GST)-γ hinge, but not by GST-γ appendage. However, the hinge and appendage domains of γ function in a cooperative manner to recruit and polymerize clathrin, suggesting that clathrin lattice assembly at the TGN involves multivalent binding of clathrin by the γ and β1 subunits of AP-1.

scholarly journals FAPP2 is involved in the transport of apical cargo in polarized MDCK cells

2005 ◽  
Vol 170 (4) ◽  
pp. 521-526 ◽  
Author(s):  
Otilia V. Vieira ◽  
Paul Verkade ◽  
Aki Manninen ◽  
Kai Simons
Keyword(s):  
Fluorescent Protein ◽  
Mdck Cells ◽  
Basolateral Membrane ◽  
Adaptor Protein ◽  
Pleckstrin Homology Domain ◽  
Transport Pathways ◽  
Protein Encoding ◽  
Phosphatidylinositol 4 ◽  
Golgi Network ◽  
Darby Canine Kidney

Phosphatidylinositol-4-phosphate (PI(4)P) is the main phosphoinositide in the Golgi complex and has been reported to play a pleiotropic role in transport of cargo from the trans-Golgi network to the plasma membrane (PM) in polarized Madin–Darby canine kidney (MDCK) cells. Overexpression of the chimeric fluorescent protein encoding the pleckstrin homology domain, which is specific for PI(4)P, inhibited both apical and basolateral transport pathways. The transport of apical cargo from the Golgi was shown to be specifically decreased by adenovirus-mediated RNA interference directed against PI(4)P adaptor protein (FAPP) 2. FAPP1 depletion had no effect on transport. On the other hand, FAPP2 was not involved in the Golgi-to-PM transport of cargo that was targeted to the basolateral membrane domain. Thus, we conclude that FAPP2 plays a specific role in apical transport in MDCK cells.

SAC3B is a target of CML19, the centrin 2 of Arabidopsis thaliana

2020 ◽  
Vol 477 (1) ◽  
pp. 173-189 ◽  
Author(s):  
Marco Pedretti ◽  
Carolina Conter ◽  
Paola Dominici ◽  
Alessandra Astegno
Keyword(s):  
Excision Repair ◽  
Complex Structure ◽  
Binding Motif ◽  
C Terminus ◽  
Repair Protein ◽  
Nucleotide Excision ◽  
Ef Hand ◽  
Molecular Determinants ◽  
Structure In Solution

Arabidopsis centrin 2, also known as calmodulin-like protein 19 (CML19), is a member of the EF-hand superfamily of calcium (Ca2+)-binding proteins. In addition to the notion that CML19 interacts with the nucleotide excision repair protein RAD4, CML19 was suggested to be a component of the transcription export complex 2 (TREX-2) by interacting with SAC3B. However, the molecular determinants of this interaction have remained largely unknown. Herein, we identified a CML19-binding site within the C-terminus of SAC3B and characterized the binding properties of the corresponding 26-residue peptide (SAC3Bp), which exhibits the hydrophobic triad centrin-binding motif in a reversed orientation (I8W4W1). Using a combination of spectroscopic and calorimetric experiments, we shed light on the SAC3Bp–CML19 complex structure in solution. We demonstrated that the peptide interacts not only with Ca2+-saturated CML19, but also with apo-CML19 to form a protein–peptide complex with a 1 : 1 stoichiometry. Both interactions involve hydrophobic and electrostatic contributions and include the burial of Trp residues of SAC3Bp. However, the peptide likely assumes different conformations upon binding to apo-CML19 or Ca2+-CML19. Importantly, the peptide dramatically increases the affinity for Ca2+ of CML19, especially of the C-lobe, suggesting that in vivo the protein would be Ca2+-saturated and bound to SAC3B even at resting Ca2+-levels. Our results, providing direct evidence that Arabidopsis SAC3B is a CML19 target and proposing that CML19 can bind to SAC3B through its C-lobe independent of a Ca2+ stimulus, support a functional role for these proteins in TREX-2 complex and mRNA export.

Is the Rigidity of SARS-CoV-2 Spike Receptor-Binding Motif the Hallmark for Its Enhanced Infectivity and Pathogenesis?

2020 ◽  
Author(s):  
Angelo Spinello ◽  
Andrea Saltalamacchia ◽  
Alessandra Magistrato
Keyword(s):  
Health Economic ◽  
Global Health ◽  
Receptor Binding ◽  
Binding Motif ◽  
Spike Glycoprotein ◽  
High Affinity ◽  
Global Pandemic ◽  
Medical Countermeasures ◽  
High Infectivity ◽  
Human Receptor

<p>The latest outbreak of a new pathogenic coronavirus (SARS-CoV-2) is provoking a global health, economic and societal crisis. All-atom simulations enabled us to uncover the key molecular traits underlying the high affinity of SARS-CoV-2 spike glycoprotein towards its human receptor, providing a rationale to its high infectivity. Harnessing this knowledge can boost developing effective medical countermeasures to fight the current global pandemic.</p>

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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