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 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.

Surfactant Protein A Enhances Constitutive Immune Functions of Clathrin Heavy Chain and Clathrin Adaptor Protein 2

2016 ◽  
Vol 55 (1) ◽  
pp. 92-104 ◽  
Author(s):  
Christina Moulakakis ◽  
Christine Steinhäuser ◽  
Dominika Biedziak ◽  
Katja Freundt ◽  
Norbert Reiling ◽  
...  
Keyword(s):  
Heavy Chain ◽  
Protein A ◽  
Adaptor Protein ◽  
Surfactant Protein ◽  
Surfactant Protein A ◽  
Immune Functions ◽  
Clathrin Heavy Chain ◽  
Clathrin Adaptor

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 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 Localization of integral membrane peptidylglycine alpha-amidating monooxygenase in neuroendocrine cells

1997 ◽  
Vol 110 (6) ◽  
pp. 695-706 ◽  
Author(s):  
S.L. Milgram ◽  
S.T. Kho ◽  
G.V. Martin ◽  
R.E. Mains ◽  
B.A. Eipper
Keyword(s):  
Steady State ◽  
Secretory Granules ◽  
Microscopic Level ◽  
Neuroendocrine Cells ◽  
Light Microscopic Level ◽  
Steady State Distribution ◽  
State Distribution ◽  
Golgi Membranes ◽  
Early Endosomes ◽  
Golgi Network

Peptidylglycine alpha-amidating monooxygenase (PAM) catalyzes the amidation of glycine-extended peptides in neuroendocrine cells. At steady state, membrane PAM is accumulated in a perinuclear compartment. We examined the distribution of membrane PAM in stably transfected AtT-20 cells and compared its localization to markers for the trans-Golgi network (TGN), endosomes, and lysosomes. At the light microscopic level, the distribution of membrane PAM does not overlap extensively with lysosomal markers but does overlap with TGN38 and with SCAMP, a component of post-Golgi membranes involved in recycling pathways. By immunoelectron microscopy, membrane PAM is present in tubulovesicular structures which constitute the TGN; some of these PAM-containing tubulovesicular structures are more distal to the Golgi stacks and do not contain TGN38. While some POMC-derived peptides are present in tubulovesicular structures like those that contain membrane PAM, the majority of the POMC-derived peptides are present in secretory granules. There is little overlap between the steady state distribution of membrane PAM and internalized FITC-transferrin in the early endosomes. Few of the perinuclear PAM-containing structures are labeled with HRP or WGA-HRP even following long incubations. Therefore, membrane PAM is localized to perinuclear tubulovesicular structures which are partially devoid of TGN38 and are not all endosomal in origin.

scholarly journals Retrograde transport of mannose-6-phosphate receptor depends on several sorting machineries as analyzed by sulfatable nanobodies

2019 ◽  
Author(s):  
Dominik P. Buser ◽  
Martin Spiess
Keyword(s):  
Plasma Membrane ◽  
Steady State ◽  
Protein Transport ◽  
Retrograde Transport ◽  
Lysosomal Hydrolases ◽  
Late Endosomes ◽  
Mannose 6 Phosphate ◽  
Clathrin Adaptor ◽  
Golgi Network

AbstractRetrograde protein transport from the cell surface and endosomes to the trans-Golgi network (TGN) is essential for membrane homeostasis in general and for the recycling of mannose-6-phosphate receptors (MPRs) for sorting of lysosomal hydrolases in particular. Several different sorting machineries have been implicated in retrieval from early or late endosomes to the TGN, mostly for the cation-independent MPR (CIMPR), mainly by analysis of steady-state localization and by interaction studies. We employed a nanobody-based sulfation tool to more directly determine transport kinetics from the plasma membrane to the TGN – the site of sulfation – for the cation-dependent MPR (CDMPR) with and without silencing of candidate machinery proteins. The clathrin adaptor AP-1 that operates bidirectionally at the TGN-to-endosome interface, which had been shown to cause reduced sulfation when rapidly depleted, produced hypersulfation of nanobodies internalized by CDMPR upon long-term silencing, reflecting accumulation in the TGN. In contrast, knockdown of retromer (Vps26), epsinR, or Rab9 reduced CDMPR arrival to the TGN. No effect was observed upon silencing of TIP47. Most surprisingly, depletion of the GGA (Golgi-localized, γ-adaptin ear-containing, Arf-binding) proteins inhibited retrograde transport rather than TGN exit. This study illustrates the usefulness of derivatized, sulfation-competent nanobodies to analyze retrograde protein transport to identify the contributions of different machineries.

Clathrin, adaptors and eps15 in endosomes containing activated epidermal growth factor receptors

1999 ◽  
Vol 112 (3) ◽  
pp. 317-327 ◽  
Author(s):  
T. Sorkina ◽  
A. Bild ◽  
F. Tebar ◽  
A. Sorkin
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Adaptor Protein ◽  
Receptor Internalization ◽  
Image Deconvolution ◽  
Protein Markers ◽  
Coated Pits ◽  
Early Endosomes ◽  
Epidermal Growth ◽  
Clathrin Adaptor

Activation of the epidermal growth factor receptor (EGFR) by EGF results in binding of clathrin adaptor protein complex AP-2 to the receptor cytoplasmic tail. The transient interaction with AP-2 is thought to be responsible for the selective recruitment of the EGFR into coated pits during endocytosis. In this study we found that EGF-induced EGFR/AP-2 association, measured by co-immunoprecipitation, persists after receptor internalization. Double-label immunofluorescence of EGF-treated A-431 and COS-1 cells revealed the presence of AP-2, clathrin and eps15, another component of the plasma membrane coated pits, in the large perinuclear endosomes loaded with EGFRs. By optical sectioning and image deconvolution, the immunoreactivities were seen to be distributed within vesicular and tubular elements of these endosomes. In addition, these compartments contained the transferrin receptors and a EEA.1 protein, markers of early endosomes. Furthermore, Golgi clathrin adaptor complex AP-1 was found in EGFR-containing endosomes and EGFR immunoprecipitates in A-431 cells. The direct interaction of the EGFR with micro1 as well as micro2 subunits of AP-1 and AP-2, correspondingly, was shown using the yeast two-hybrid assay. Brefeldin A, a drug that releases AP-1 from the trans-Golgi membranes, had no effect on AP-1 association with endosomes and its co-precipitation with EGFR. Taken together, the data suggest that endosomal EGFR-AP complexes make up a significant portion of the total amount of these complexes detectable by co-immunoprecipitation. It can be proposed that APs are capable of binding to the endosomal membrane via a mechanism that requires AP interaction with the intracellular tails of multimeric receptors like activated EGFR, which in turn allows recruitment of clathrin and eps15. The hypothesis that the competition between adaptor complexes for binding to the receptor tails in endosomes may regulate of the sorting of receptors is discussed.

scholarly journals Cluster of Differentiation Antigen 4 (CD4) Endocytosis and Adaptor Complex Binding Require Activation of the CD4 Endocytosis Signal by Serine Phosphorylation

1999 ◽  
Vol 10 (3) ◽  
pp. 677-691 ◽  
Author(s):  
Carol Pitcher ◽  
Stefan Höning ◽  
Anja Fingerhut ◽  
Katherine Bowers ◽  
Mark Marsh
Keyword(s):  
Phorbol Esters ◽  
Protein Complexes ◽  
Adaptor Protein ◽  
Cytoplasmic Domain ◽  
Serine Phosphorylation ◽  
Differentiation Antigen ◽  
Coated Pits ◽  
Early Endosomes ◽  
Clathrin Adaptor ◽  
Cluster Of Differentiation

Cluster of differentiation antigen 4 (CD4), the T lymphocyte antigen receptor component and human immunodeficiency virus coreceptor, is down-modulated when cells are activated by antigen or phorbol esters. During down-modulation CD4 dissociates from p56 lck , undergoes endocytosis through clathrin-coated pits, and is then sorted in early endosomes to late endocytic organelles where it is degraded. Previous studies have suggested that phosphorylation and a dileucine sequence are required for down-modulation. Using transfected HeLa cells, in which CD4 endocytosis can be studied in the absence of p56 lck , we show that the dileucine sequence in the cytoplasmic domain is essential for clathrin-mediated CD4 endocytosis. However, this sequence is only functional as an endocytosis signal when neighboring serine residues are phosphorylated. Phosphoserine is required for rapid endocytosis because CD4 molecules in which the cytoplasmic domain serine residues are substituted with glutamic acid residues are not internalized efficiently. Using surface plasmon resonance, we show that CD4 peptides containing the dileucine sequence bind weakly to clathrin adaptor protein complexes 2 and 1. The affinity of this interaction is increased 350- to 700-fold when the peptides also contain phosphoserine residues.

scholarly journals P4-ATPase Requirement for AP-1/Clathrin Function in Protein Transport from the trans-Golgi Network and Early Endosomes

2008 ◽  
Vol 19 (8) ◽  
pp. 3526-3535 ◽  
Author(s):  
Ke Liu ◽  
Kavitha Surendhran ◽  
Steven F. Nothwehr ◽  
Todd R. Graham
Keyword(s):  
Protein Transport ◽  
Early Endosome ◽  
Coated Vesicle ◽  
Transport Pathways ◽  
Vesicle Budding ◽  
Trans Golgi Network ◽  
Conditional Allele ◽  
Early Endosomes ◽  
Clathrin Adaptor ◽  
Golgi Network

Drs2p is a resident type 4 P-type ATPase (P4-ATPase) and potential phospholipid translocase of the trans-Golgi network (TGN) where it has been implicated in clathrin function. However, precise protein transport pathways requiring Drs2p and how it contributes to clathrin-coated vesicle budding remain unclear. Here we show a functional codependence between Drs2p and the AP-1 clathrin adaptor in protein sorting at the TGN and early endosomes of Saccharomyces cerevisiae. Genetic criteria indicate that Drs2p and AP-1 operate in the same pathway and that AP-1 requires Drs2p for function. In addition, we show that loss of AP-1 markedly increases Drs2p trafficking to the plasma membrane, but does not perturb retrieval of Drs2p from the early endosome back to the TGN. Thus AP-1 is required at the TGN to sort Drs2p out of the exocytic pathway, presumably for delivery to the early endosome. Moreover, a conditional allele that inactivates Drs2p phospholipid translocase (flippase) activity disrupts its own transport in this AP-1 pathway. Drs2p physically interacts with AP-1; however, AP-1 and clathrin are both recruited normally to the TGN in drs2Δ cells. These results imply that Drs2p acts independently of coat recruitment to facilitate AP-1/clathrin-coated vesicle budding from the TGN.

scholarly journals EHDS are serine phosphoproteins: EHD1 phosphorylation is enhanced by serum stimulation

2008 ◽  
Vol 13 (4) ◽  
Author(s):  
Boris Fichtman ◽  
Liat Ravid ◽  
Debora Rapaport ◽  
Mia Horowitz
Keyword(s):  
Adaptor Protein ◽  
Serine Phosphorylation ◽  
The Other ◽  
Serum Stimulation ◽  
Kinase C ◽  
Multiple Protein ◽  
Early Endosomes ◽  
Pkc Protein Kinase C ◽  
Clathrin Adaptor ◽  
Endocytic Pathways

AbstractEndocytic processes are mediated by multiple protein-protein interacting modules and regulated by phosphorylation and dephosphorylation. The Eps15 homology domain containing protein 1 (EHD1) has been implicated in regulating recycling of proteins, internalized both in clathrin-dependent and clathrin-independent endocytic pathways, from the recycling compartment to the plasma membrane. EHD1 was found in a complex with clathrin, adaptor protein complex-2 (AP-2) and insulin-like growth factor-1 receptor (IGF-1R), and was shown to interact with Rabenosyn-5, SNAP29, EHBP1 (EH domain binding protein 1) and syndapin I and II. In this study, we show that EHD1, like the other human EHDs, undergoes serine-phosphorylation. Our results also indicate that EHD1 is a serum-inducible serine-phosphoprotein and that PKC (protein kinase C) is one of its kinases. In addition, we show that inhibitors of clathrin-mediated endocytosis decrease EHD1 phosphorylation, while inhibitors of caveolinmediated endocytosis do not affect EHD1 phosphorylation. The results of experiments in which inhibitors of endocytosis were employed strongly suggest that EHD1 phosphorylation occurs between early endosomes and the endocytic recycling compartment.

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