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 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 Phosphoserine acidic cluster motifs bind distinct basic regions on the μ subunits of clathrin adaptor protein complexes

2018 ◽  
Vol 293 (40) ◽  
pp. 15678-15690 ◽  
Author(s):  
Rajendra Singh ◽  
Charlotte Stoneham ◽  
Christopher Lim ◽  
Xiaofei Jia ◽  
Javier Guenaga ◽  
...  
Keyword(s):  
Protein Complexes ◽  
Adaptor Protein ◽  
Clathrin Adaptor

Structural studies of phosphoinositide 3-kinase-dependent traffic to multivesicular bodies

2007 ◽  
Vol 74 ◽  
pp. 47-57 ◽  
Author(s):  
David J. Gill ◽  
Hsiangling Teo ◽  
Ji Sun ◽  
Olga Perisic ◽  
Dmitry B. Veprintsev ◽  
...  
Keyword(s):  
Protein Complexes ◽  
Multivesicular Body ◽  
Binding Pocket ◽  
Lipid Binding ◽  
Pleckstrin Homology Domain ◽  
Class Iii ◽  
Membrane Recruitment ◽  
Early Endosomes ◽  
Phosphoinositide 3 Kinase ◽  
Vacuolar Protein

Three large protein complexes known as ESCRT I, ESCRT II and ESCRT III drive the progression of ubiquitinated membrane cargo from early endosomes to lysosomes. Several steps in this process critically depend on PtdIns3P, the product of the class III phosphoinositide 3-kinase. Our work has provided insights into the architecture, membrane recruitment and functional interactions of the ESCRT machinery. The fan-shaped ESCRT I core and the trilobal ESCRT II core are essential to forming stable, rigid scaffolds that support additional, flexibly-linked domains, which serve as gripping tools for recognizing elements of the MVB (multivesicular body) pathway: cargo protein, membranes and other MVB proteins. With these additional (non-core) domains, ESCRT I grasps monoubiquitinated membrane proteins and the Vps36 subunit of the downstream ESCRT II complex. The GLUE (GRAM-like, ubiquitin-binding on Eap45) domain extending beyond the core of the ESCRT II complex recognizes PtdIns3P-containing membranes, monoubiquitinated cargo and ESCRT I. The structure of this GLUE domain demonstrates that it has a split PH (pleckstrin homology) domain fold, with a non-typical phosphoinositide-binding pocket. Mutations in the lipid-binding pocket of the ESCRT II GLUE domain cause a strong defect in vacuolar protein sorting in yeast.

scholarly journals Nanobody-dependent delocalization of endocytic machinery in Arabidopsis root cells dampens their internalization capacity

2020 ◽  
Author(s):  
Joanna Winkler ◽  
Andreas De Meyer ◽  
Evelien Mylle ◽  
Peter Grones ◽  
Veronique Storme ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Protein Complexes ◽  
Function Analysis ◽  
Plant Cells ◽  
Adaptor Protein ◽  
Genetic Mutation ◽  
Loss Of Function ◽  
Expression Domain ◽  
Specific Expression ◽  
Root Cells

AbstractPlant cells perceive and adapt to an ever-changing environment by modifying their plasma membrane (PM) proteome. Whereas secretion deposits new integral membrane proteins, internalization by endocytosis removes membrane proteins and associated ligands, largely with the aid of adaptor protein complexes and the scaffolding molecule clathrin. Two adaptor protein complexes function in clathrin-mediated endocytosis at the PM in plant cells, the heterotetrameric Adaptor Protein 2 (AP-2) complex and the octameric TPLATE complex (TPC). Whereas single subunit mutants in AP-2 develop into viable plants, genetic mutation of a single TPC subunit causes fully penetrant male sterility and silencing single subunits leads to seedling lethality. To address TPC function in somatic root cells, while minimizing indirect effects on plant growth, we employed nanobody-dependent delocalization of a functional, GFP-tagged TPC subunit, TML, in its respective homozygous genetic mutant background. In order to decrease the amount of functional TPC at the PM, we targeted our nanobody construct to the mitochondria and fused it to TagBFP2 to visualize it independently of its bait. We furthermore limited the effect of our delocalization to those tissues that are easily accessible for live-cell imaging by expressing it from the PIN2 promotor, which is active in root epidermal and cortex cells. With this approach, we successfully delocalized TML from the PM. Moreover, we also show co-recruitment of TML-GFP and AP2A1-TagRFP to the mitochondria, suggesting that our approach delocalized complexes, rather than individual adaptor complex subunits. In line with the specific expression domain, we only observed minor effects on root growth, yet realized a clear reduction of endocytic flux in epidermal root cells. Nanobody-dependent delocalization in plants, here exemplified using a TPC subunit, has the potential to be widely applicable to achieve specific loss-of-function analysis of otherwise lethal mutants.

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 Two Clathrin Adaptor Protein Complexes Instruct Axon-Dendrite Polarity

Neuron ◽  
2016 ◽  
Vol 90 (3) ◽  
pp. 564-580 ◽  
Author(s):  
Pengpeng Li ◽  
Sean A. Merrill ◽  
Erik M. Jorgensen ◽  
Kang Shen
Keyword(s):  
Protein Complexes ◽  
Adaptor Protein ◽  
Clathrin Adaptor

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.

scholarly journals Functions of Adaptor Protein (AP)-3 and AP-1 in Tyrosinase Sorting from Endosomes to Melanosomes

2005 ◽  
Vol 16 (11) ◽  
pp. 5356-5372 ◽  
Author(s):  
Alexander C. Theos ◽  
Danièle Tenza ◽  
José A. Martina ◽  
Ilse Hurbain ◽  
Andrew A. Peden ◽  
...  
Keyword(s):  
Protein Complexes ◽  
Adaptor Protein ◽  
Specific Protein ◽  
Model System ◽  
Multivesicular Bodies ◽  
Substantial Fraction ◽  
Sorting Signal ◽  
Early Endosomes ◽  
Unique Model ◽  
Multivesicular Endosomes

Specialized cells exploit adaptor protein complexes for unique post-Golgi sorting events, providing a unique model system to specify adaptor function. Here, we show that AP-3 and AP-1 function independently in sorting of the melanocyte-specific protein tyrosinase from endosomes to the melanosome, a specialized lysosome-related organelle distinguishable from lysosomes. AP-3 and AP-1 localize in melanocytes primarily to clathrin-coated buds on tubular early endosomes near melanosomes. Both adaptors recognize the tyrosinase dileucine-based melanosome sorting signal, and tyrosinase largely colocalizes with each adaptor on endosomes. In AP-3-deficient melanocytes, tyrosinase accumulates inappropriately in vacuolar and multivesicular endosomes. Nevertheless, a substantial fraction still accumulates on melanosomes, concomitant with increased association with endosomal AP-1. Our data indicate that AP-3 and AP-1 function in partially redundant pathways to transfer tyrosinase from distinct endosomal subdomains to melanosomes and that the AP-3 pathway ensures that tyrosinase averts entrapment on internal membranes of forming multivesicular bodies.

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