scholarly journals Cargo-sorting signals promote polymerization of adaptor protein-1 in an Arf-1·GTP-independent manner

2008 ◽  
Vol 479 (1) ◽  
pp. 63-68 ◽  
Author(s):  
Intaek Lee ◽  
Matthew T. Drake ◽  
Linton M. Traub ◽  
Stuart Kornfeld
Keyword(s):  
Adaptor Protein ◽  
Independent Manner ◽  
Sorting Signals ◽  
Cargo Sorting

scholarly journals Binding of cargo sorting signals to AP-1 enhances its association with ADP ribosylation factor 1–GTP

2008 ◽  
Vol 180 (3) ◽  
pp. 467-472 ◽  
Author(s):  
Intaek Lee ◽  
Balraj Doray ◽  
Jennifer Govero ◽  
Stuart Kornfeld
Keyword(s):  
Adaptor Protein ◽  
Adenosine Diphosphate ◽  
Core Domain ◽  
Transport Vesicles ◽  
Sorting Signals ◽  
Stable Association ◽  
Golgi Network ◽  
Adp Ribosylation Factor ◽  
Prevailing View ◽  
Cargo Sorting

The adaptor protein AP-1 is the major coat protein involved in the formation of clathrin-coated vesicles at the trans-Golgi network. The prevailing view is that AP-1 recruitment involves coincident binding to multiple low-affinity sites comprising adenosine diphosphate ribosylation factor 1 (Arf-1)–guanosine triphosphate (GTP), cargo sorting signals, and phosphoinositides. We now show that binding of cargo signal peptides to AP-1 induces a conformational change in its core domain that greatly enhances its interaction with Arf-1–GTP. In addition, we provide evidence for cross talk between the dileucine and tyrosine binding sites within the AP-1 core domain such that binding of a cargo signal to one site facilitates binding to the other site. The stable association of AP-1 with Arf-1–GTP, which is induced by cargo signals, would serve to provide sufficient time for adaptor polymerization and clathrin recruitment while ensuring the packaging of cargo molecules into the forming transport vesicles.

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 Role of the Endocytic Machinery in the Sorting of Lysosome-associated Membrane Proteins

2005 ◽  
Vol 16 (9) ◽  
pp. 4231-4242 ◽  
Author(s):  
Katy Janvier ◽  
Juan S. Bonifacino
Keyword(s):  
Plasma Membrane ◽  
Coat Protein ◽  
Membrane Proteins ◽  
Adaptor Protein ◽  
The Other ◽  
Sorting Signals ◽  
Efficient Delivery ◽  
Endocytic Machinery

The limiting membrane of the lysosome contains a group of transmembrane glycoproteins named lysosome-associated membrane proteins (Lamps). These proteins are targeted to lysosomes by virtue of tyrosine-based sorting signals in their cytosolic tails. Four adaptor protein (AP) complexes, AP-1, AP-2, AP-3, and AP-4, interact with such signals and are therefore candidates for mediating sorting of the Lamps to lysosomes. However, the role of these complexes and of the coat protein, clathrin, in sorting of the Lamps in vivo has either not been addressed or remains controversial. We have used RNA interference to show that AP-2 and clathrin—and to a lesser extent the other AP complexes—are required for efficient delivery of the Lamps to lysosomes. Because AP-2 is exclusively associated with plasma membrane clathrin coats, our observations imply that a significant population of Lamps traffic via the plasma membrane en route to lysosomes.

scholarly journals Oligomerization and Dissociation of AP-1 Adaptors Are Regulated by Cargo Signals and by ArfGAP1-induced GTP Hydrolysis

2005 ◽  
Vol 16 (10) ◽  
pp. 4745-4754 ◽  
Author(s):  
Daniel M. Meyer ◽  
Pascal Crottet ◽  
Bohumil Maco ◽  
Elena Degtyar ◽  
Dan Cassel ◽  
...  
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Adaptor Proteins ◽  
Gtpase Activity ◽  
Gtpase Activating Protein ◽  
Gtp Hydrolysis ◽  
Sorting Signals ◽  
Initial Assembly ◽  
Cargo Sorting

The mechanism of AP-1/clathrin coat formation was analyzed using purified adaptor proteins and synthetic liposomes presenting tyrosine sorting signals. AP-1 adaptors recruited in the presence of Arf1·GTP and sorting signals were found to oligomerize to high-molecular-weight complexes even in the absence of clathrin. The appendage domains of the AP-1 adaptins were not required for oligomerization. On GTP hydrolysis induced by the GTPase-activating protein ArfGAP1, the complexes were disassembled and AP-1 dissociated from the membrane. AP-1 stimulated ArfGAP1 activity, suggesting a role of AP-1 in the regulation of the Arf1 “GTPase timer.” In the presence of cytosol, AP-1 could be recruited to liposomes without sorting signals, consistent with the existence of docking factors in the cytosol. Under these conditions, however, AP-1 remained monomeric, and recruitment in the presence of GTP was short-lived. Sorting signals allowed stable recruitment and oligomerization also in the presence of cytosol. These results suggest a mechanism whereby initial assembly of AP-1 with Arf1·GTP and ArfGAP1 on the membrane stimulates Arf1 GTPase activity, whereas interaction with cargo induces oligomerization and reduces the rate of GTP hydrolysis, thus contributing to efficient cargo sorting.

scholarly journals Cbl-dependent Ubiquitination Is Required for Progression of EGF Receptors into Clathrin-coated Pits

2004 ◽  
Vol 15 (8) ◽  
pp. 3591-3604 ◽  
Author(s):  
Espen Stang ◽  
Frøydis D. Blystad ◽  
Maja Kazazic ◽  
Vibeke Bertelsen ◽  
Tonje Brodahl ◽  
...  
Keyword(s):  
Egf Receptor ◽  
Point Mutations ◽  
Adaptor Protein ◽  
Protein S ◽  
Egf Receptors ◽  
Ubiquitinated Protein ◽  
Coated Pits ◽  
Independent Manner ◽  
Sh3 Domains

Ligand binding causes the EGF receptor (EGFR) to become ubiquitinated by Cbl upon association with the adaptor protein Grb2. We have investigated the role of ubiquitin and Grb2 in ligand-induced endocytosis of the EGFR. Incubation of cells with EGF on ice caused translocation of Grb2 and Cbl from the cytosol to the rim of coated pits. Grb2 with point mutations in both SH3 domains inhibited recruitment of the EGFR to clathrin-coated pits, in a Ras-independent manner. On overexpression of the Cbl-binding protein Sprouty, ubiquitination of the EGFR was inhibited, the EGFR was recruited only to the rim of coated pits, and endocytosis of the EGFR was inhibited. Conjugation-defective ubiquitin similarly inhibited recruitment of EGF-EGFR to clathrin-coated pits. Even though this does not prove that cargo must be ubiquitinated, this indicates the importance of interaction of ubiquitinated protein(s) with proteins harboring ubiquitin-interacting domains. We propose that Grb2 mediates transient anchoring of the EGFR to an Eps15-containing molecular complex at the rim of coated pits and that Cbl-induced ubiquitination of the EGFR allows relocation of EGFR from the rim to the center of clathrin-coated pits.

scholarly journals The alternate AP-1 adaptor subunit Apm2 interacts with the Mil1 regulatory protein and confers differential cargo sorting

2016 ◽  
Vol 27 (3) ◽  
pp. 588-598 ◽  
Author(s):  
Shawn T. Whitfield ◽  
Helen E. Burston ◽  
Björn D. M. Bean ◽  
Nandini Raghuram ◽  
Lymarie Maldonado-Báez ◽  
...  
Keyword(s):  
Protein Complexes ◽  
Regulatory Protein ◽  
Adaptor Protein ◽  
General Mechanism ◽  
Specific Expression ◽  
Membrane Recruitment ◽  
Early Endosomes ◽  
Cell Type Specific Expression ◽  
Clathrin Adaptor ◽  
Cargo Sorting

Heterotetrameric adaptor protein complexes are important mediators of cargo protein sorting in clathrin-coated vesicles. The cell type–specific expression of alternate μ chains creates distinct forms of AP-1 with altered cargo sorting, but how these subunits confer differential function is unclear. Whereas some studies suggest the μ subunits specify localization to different cellular compartments, others find that the two forms of AP-1 are present in the same vesicle but recognize different cargo. Yeast have two forms of AP-1, which differ only in the μ chain. Here we show that the variant μ chain Apm2 confers distinct cargo-sorting functions. Loss of Apm2, but not of Apm1, increases cell surface levels of the v-SNARE Snc1. However, Apm2 is unable to replace Apm1 in sorting Chs3, which requires a dileucine motif recognized by the γ/σ subunits common to both complexes. Apm2 and Apm1 colocalize at Golgi/early endosomes, suggesting that they do not associate with distinct compartments. We identified a novel, conserved regulatory protein that is required for Apm2-dependent sorting events. Mil1 is a predicted lipase that binds Apm2 but not Apm1 and contributes to its membrane recruitment. Interactions with specific regulatory factors may provide a general mechanism to diversify the functional repertoire of clathrin adaptor complexes.

scholarly journals Subunit-dependent and independent rules of AMPA receptor trafficking during long-term depression in hippocampal neurons

2020 ◽  
Author(s):  
Shinji Matsuda ◽  
Michisuke Yuzaki
Keyword(s):  
Hippocampal Neurons ◽  
Protein Complexes ◽  
Dominant Role ◽  
Adaptor Protein ◽  
Long Term Potentiation ◽  
Proximal Region ◽  
Phosphorylation Sites ◽  
Independent Manner ◽  
Ampar Trafficking

ABSTRACTLong-term potentiation (LTP) and depression (LTD) of excitatory neurotransmission are believed to be the neuronal basis of learning and memory. Both processes are primarily mediated by neuronal activity-induced transport of postsynaptic AMPA-type glutamate receptors (AMPARs). While AMPAR subunits and their specific phosphorylation sites mediate differential AMPAR trafficking, LTP and LTD could also occur in a subunit-independent manner. Thus, it remains unclear whether and how, certain AMPAR subunits with phosphorylation sites are preferentially recruited to or removed from synapses during LTP and LTD. Here, we show that phosphorylation of the membrane-proximal region (MPR), which only occurs in GluA1 AMPAR subunits, mediates the subunit-dependent endosomal transport of AMPARs during LTD. AP-2 and AP-3, adaptor protein complexes necessary for clathrin-mediated endocytosis and late endosomal/lysosomal trafficking, respectively, are reported to be recruited to AMPARs by binding to the AMPAR auxiliary subunit, stargazin (STG), in an AMPAR subunit-independent manner. However, the association of AP-3, but not AP-2, with STG was indirectly inhibited by the phosphomimetic mutation in the MPR of GluA1. Thus, although AMPARs containing the phosphomimetic mutation at the MPR of GluA1 were endocytosed by a LTD-inducing stimulus, they were quickly recycled back to the cell surface in hippocampal neurons. These results could explain how the phosphorylation status of GluA1-MPR plays a dominant role in subunit-independent STG-mediated AMPAR trafficking during LTD.

scholarly journals RIAM Interacts with the Hematopoietic-Specific Adaptor Protein Gads and Forms a LAT-Independent Node of Signal Integration That Regulates Activation of PLC-γ1

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4138-4138
Author(s):  
Kankana Bardhan ◽  
Nikolaos Patsoukis ◽  
Donna M Berry ◽  
Jane McGlade ◽  
Vassiliki A. Boussiotis
Keyword(s):  
T Cells ◽  
Plasma Membrane ◽  
T Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Adaptor Protein ◽  
Ph Domain ◽  
Activated T Cells ◽  
Independent Manner ◽  
C Terminus

Abstract TCR stimulation triggers the activation of protein tyrosine kinases resulting in phosphorylation of the adaptor protein LAT. SLP-76, interacts constitutively with PLC-γ1 and with the SH3 domain of Gads, which via its SH2 domain mediates inducible recruitment of SLP-76 and PLC-γ1 to LAT, upon T cell activation. PLC-γ1 hydrolyzes phosphatidylinositol-4, 5 bisphosphate [PI(4,5)P2], generating inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG), second messengers responsible for mediating intracellular calcium release and activation of downstream signals. The adaptor protein RIAM constitutively interacts with PLC-γ1 and is required for PLC-γ1 activation. RIAM is a multidomain protein with a small N-terminus proline-rich region, two coiled-coiled regions, sequential Ras association (RA) and pleckstrin homology (PH) domains, and a large C-terminus proline-rich region, which interacts with PLC-γ1. The RA domain of RIAM has specificity for Rap1-GTP whereas the PH domain binds to the PLC-γ1 substrate PI(4,5)P2. The RA-PH domain region of RIAM functions as a single structural unit and mediates translocation of RIAM to the plasma membrane upon T cell activation. Previously, we determined that RIAM deficiency results in impaired activation of PLC-γ1 in spite of the formation of the PLC-γ1-SLP-76-LAT complex, suggesting perhaps somewhat paradoxically, that PLC-γ1-SLP-76-LAT signalosome is not sufficient to mediate distal signaling in the absence of RIAM. This observation indicated that RIAM mediates its effects at a level distal to SLP-76-LAT or through a signaling pathway parallel but distinct from SLP-76-Gads-LAT. Here we investigated whether RIAM forms a signalosome parallel to PLC-γ1-SLP-76-Gads and whether such pathway might be involved in the activation of PLC-γ1. Using primary T lymphocytes and Jurkat T cells stimulated via TCR/CD3 and CD28 we determined that RIAM constitutively interacted with Gads as determined by immunoprecipitation with RIAM-specific antibody followed by Gads immunoblot. To determine whether the interaction between RIAM and Gads was direct, we employed an in vitro protein association assay. Glutathione S-transferase (GST) and GST-fusion protein of Gads were coupled to glutathione-sepharose and incubated with [35S]methionine-labeled RIAM or luciferase, as negative control. Gads bound to [35S]methionine-labeled RIAM indicating that RIAM interacts directly with Gads. We further examined domain-specific interaction of RIAM with endogenous Gads using GST fusion proteins of RIAM. We determined a constitutive interaction between Gads and GST fusion proteins of full-length RIAM or C-terminus region of RIAM. Although a number of tyrosine phosphorylated proteins were associated with the RIAM-Gads complex upon T cell activation, LAT was not detected among the components of this complex as determined by immunoblot with anti-phosphotyrosine-specific or LAT-specific antibodies. Using a GST fusion protein of the RA-PH domain of RIAM we determined that, surprisingly, Gads displayed activation-dependent interaction with the RA-PH domain, which mediates the recruitment of RIAM to the plasma membrane upon T cell activation. Furthermore, in addition to Gads, SLP-76 and PLC-γ1 were recruited to the RA-PH domain of RIAM in activated T cells. To determine whether RIAM and Gads had a synergistic effect on IL-2 transcription, we performed luciferase-based reporter assays using a reporter construct driven by the entire IL-2 promoter or by NFAT binding sequences. We found that RIAM and Gads had a synergistic effect on IL-2 and on NFAT-mediated transcriptional activation, which depends on PLC-γ1. Thus, via its C-terminus region, RIAM directly and constitutively interacts with Gads. In addition, via its RA-PH domain, RIAM mediates an activation-dependent interaction with Gads and serves as a docking site recruiting the PLC-γ1-SLP-76-Gads complex to the plasma membrane in a LAT-independent manner. These findings indicate a crosstalk between RIAM and SLP-76 in the activation of PLC-γ1 and reveal a previously unidentified, alternative signaling pathway leading to Gads-SLP-76 recruitment to the plasma membrane of activated T cells in a LAT-independent manner. Disclosures No relevant conflicts of interest to declare.

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