scholarly journals Regulation of Clathrin-Mediated Endocytosis

2018 ◽  
Vol 87 (1) ◽  
pp. 871-896 ◽  
Author(s):  
Marcel Mettlen ◽  
Ping-Hung Chen ◽  
Saipraveen Srinivasan ◽  
Gaudenz Danuser ◽  
Sandra L. Schmid
Keyword(s):  
Conformational Changes ◽  
Posttranslational Modifications ◽  
Mammalian Cells ◽  
Environmental Changes ◽  
Protein Complexes ◽  
Adaptor Protein ◽  
Endocytic Pathway ◽  
Coat Proteins ◽  
Membrane Composition ◽  
Coated Pits

Clathrin-mediated endocytosis (CME) is the major endocytic pathway in mammalian cells. It is responsible for the uptake of transmembrane receptors and transporters, for remodeling plasma membrane composition in response to environmental changes, and for regulating cell surface signaling. CME occurs via the assembly and maturation of clathrin-coated pits that concentrate cargo as they invaginate and pinch off to form clathrin-coated vesicles. In addition to the major coat proteins, clathrin triskelia and adaptor protein complexes, CME requires a myriad of endocytic accessory proteins and phosphatidylinositol lipids. CME is regulated at multiple steps—initiation, cargo selection, maturation, and fission—and is monitored by an endocytic checkpoint that induces disassembly of defective pits. Regulation occurs via posttranslational modifications, allosteric conformational changes, and isoform and splice-variant differences among components of the CME machinery, including the GTPase dynamin. This review summarizes recent findings on the regulation of CME and the evolution of this complex process.

scholarly journals De novo design of tunable, pH-driven conformational changes

Science ◽  
2019 ◽  
Vol 364 (6441) ◽  
pp. 658-664 ◽  
Author(s):  
Scott E. Boyken ◽  
Mark A. Benhaim ◽  
Florian Busch ◽  
Mengxuan Jia ◽  
Matthew J. Bick ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Mammalian Cells ◽  
Large Scale ◽  
Environmental Changes ◽  
De Novo ◽  
Hydrophobic Interactions ◽  
De Novo Design ◽  
General Strategy ◽  
Histidine Residues ◽  
Hydrogen Bond Networks

The ability of naturally occurring proteins to change conformation in response to environmental changes is critical to biological function. Although there have been advances in the de novo design of stable proteins with a single, deep free-energy minimum, the design of conformational switches remains challenging. We present a general strategy to design pH-responsive protein conformational changes by precisely preorganizing histidine residues in buried hydrogen-bond networks. We design homotrimers and heterodimers that are stable above pH 6.5 but undergo cooperative, large-scale conformational changes when the pH is lowered and electrostatic and steric repulsion builds up as the network histidine residues become protonated. The transition pH and cooperativity can be controlled through the number of histidine-containing networks and the strength of the surrounding hydrophobic interactions. Upon disassembly, the designed proteins disrupt lipid membranes both in vitro and after being endocytosed in mammalian cells. Our results demonstrate that environmentally triggered conformational changes can now be programmed by de novo protein design.

scholarly journals Asymmetric Recognition of Nonconsensus AP-1 Sites by Fos-Jun and Jun-Jun Influences Transcriptional Cooperativity with NFAT1

2003 ◽  
Vol 23 (5) ◽  
pp. 1737-1749 ◽  
Author(s):  
Vladimir Ramirez-Carrozzi ◽  
Tom Kerppola
Keyword(s):  
Transcription Factors ◽  
Conformational Changes ◽  
Mammalian Cells ◽  
Protein Complexes ◽  
Regulatory Protein ◽  
Transcription Activation ◽  
Regulatory Elements ◽  
Preferred Orientation ◽  
Structural And Functional Properties ◽  
Half Site

ABSTRACT Many regulatory elements in eukaryotic promoters do not correspond to optimal recognition sequences for the transcription factors that regulate promoter function by binding to the elements. The sequence of the binding site may influence the structural and functional properties of regulatory protein complexes. Fos-Jun heterodimers were found to bind nonconsensus AP-1 sites in a preferred orientation. Oriented Fos-Jun heterodimer binding was attributed to nonidentical recognition of the two half-sites by Fos and Jun. Jun bound preferentially to the consensus half-site, whereas Fos was able to bind nonconsensus half-sites. The orientation of heterodimer binding affected the transcriptional cooperativity of Fos-Jun-NFAT1 complexes at composite regulatory elements in mammalian cells. Jun dimerization with Fos versus ATF2 caused it to bind opposite half-sites at nonconsensus AP-1 elements. Similarly, ATF2 bound to opposite half-sites in Fos-ATF2-NFAT1 and ATF2-Jun-NFAT1 complexes. The orientations of nonconsensus AP-1 sites within composite regulatory elements affected the cooperativity of Fos-Jun as well as Jun-Jun binding with NFAT1. Since Jun homodimers cannot bind to AP-1 sites in a preferred orientation, the effects of the orientations of nonconsensus AP-1 sites on the stabilities of Jun-Jun-NFAT1 complexes are likely to be due to asymmetric conformational changes in the two subunits of the homodimer. Nonconsensus AP-1 site orientation also affected the synergy of transcription activation between Jun homodimers and NFAT1 at composite regulatory elements. The asymmetric recognition of nonconsensus AP-1 sites can therefore influence the transcriptional activities of Fos and Jun both through effects on the orientation of heterodimer binding and through differential conformational changes in the two subunits of the dimer.

scholarly journals Association and Colocalization of Eps15 with Adaptor Protein-2 and Clathrin

1997 ◽  
Vol 136 (4) ◽  
pp. 811-821 ◽  
Author(s):  
Sanne van Delft ◽  
Christopher Schumacher ◽  
Willem Hage ◽  
Arie J. Verkleij ◽  
Paul M.P. van Bergen en Henegouwen
Keyword(s):  
Egf Receptor ◽  
Critical Role ◽  
Adaptor Protein ◽  
Coated Vesicles ◽  
Endocytic Pathway ◽  
Egf Receptors ◽  
Coated Pits ◽  
Early Endosomes ◽  
Endosomal Membranes ◽  
Cell Fractions

Eps15 has been identified as a substrate of the EGF receptor tyrosine kinase. In this report, we show that activation of the EGF receptor by either EGF or TGF-α results in phosphorylation of Eps15. Stimulation of cells with PDGF or insulin did not lead to Eps15 phosphorylation, suggesting that phosphorylation of Eps15 is a receptor-specific process. We demonstrate that Eps15 is constitutively associated with both α-adaptin and clathrin. Upon EGF stimulation, Eps15 and α-adaptin are recruited to the EGF receptor. Using a truncated EGF receptor mutant, we demonstrate that the regulatory domain of the cytoplasmic tail of the EGF receptor is essential for the binding of Eps15. Fractionation studies reveal that Eps15 is present in cell fractions enriched for plasma membrane and endosomal membranes. Immunofluorescence studies show that Eps15 colocalizes with adaptor protein-2 (AP-2) and partially with clathrin. No colocalization of Eps15 was observed with the early endosomal markers rab4 and rab5. These observations indicate that Eps15 is present in coated pits and coated vesicles of the clathrin-mediated endocytic pathway, but not in early endosomes. Neither AP-2 nor clathrin are required for the binding of Eps15 to coated pits or coated vesicles, since in membranes lacking AP-2 and clathrin, Eps15 still shows the same staining pattern. These findings suggest that Eps15 may play a critical role in the recruitment of active EGF receptors into coated pit regions before endocytosis of ligand-occupied EGF receptors.

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 Characterization of a Fourth Adaptor-related Protein Complex

1999 ◽  
Vol 10 (8) ◽  
pp. 2787-2802 ◽  
Author(s):  
Jennifer Hirst ◽  
Nicholas A. Bright ◽  
Brian Rous ◽  
Margaret S. Robinson
Keyword(s):  
Expressed Sequence Tag ◽  
Protein Complexes ◽  
Brefeldin A ◽  
Adaptor Protein ◽  
Cell Types ◽  
Small Gtpase ◽  
Immunogold Electron Microscopy ◽  
Coat Proteins ◽  
Cargo Proteins ◽  
Golgi Network

Adaptor protein complexes (APs) function as vesicle coat components in different membrane traffic pathways; however, there are a number of pathways for which there is still no candidate coat. To find novel coat components related to AP complexes, we have searched the expressed sequence tag database and have identified, cloned, and sequenced a new member of each of the four AP subunit families. We have shown by a combination of coimmunoprecipitation and yeast two-hybrid analysis that these four proteins (ε, β4, μ4, and ς4) are components of a novel adaptor-like heterotetrameric complex, which we are calling AP-4. Immunofluorescence reveals that AP-4 is localized to ∼10–20 discrete dots in the perinuclear region of the cell. This pattern is disrupted by treating the cells with brefeldin A, indicating that, like other coat proteins, the association of AP-4 with membranes is regulated by the small GTPase ARF. Immunogold electron microscopy indicates that AP-4 is associated with nonclathrin-coated vesicles in the region of the trans-Golgi network. The μ4 subunit of the complex specifically interacts with a tyrosine-based sorting signal, indicating that, like the other three AP complexes, AP-4 is involved in the recognition and sorting of cargo proteins with tyrosine-based motifs. AP-4 is of relatively low abundance, but it is expressed ubiquitously, suggesting that it participates in a specialized trafficking pathway but one that is required in all cell types.

scholarly journals The dyslexia-associated protein KIAA0319 interacts with adaptor protein 2 and follows the classical clathrin-mediated endocytosis pathway

2009 ◽  
Vol 297 (1) ◽  
pp. C160-C168 ◽  
Author(s):  
Clotilde Levecque ◽  
Antonio Velayos-Baeza ◽  
Zoe G. Holloway ◽  
Anthony P. Monaco
Keyword(s):  
Neuronal Migration ◽  
Mammalian Cells ◽  
Adaptor Protein ◽  
Surface Expression ◽  
Learning Disorders ◽  
Endocytic Pathway ◽  
Fine Tuning ◽  
Genetic Studies ◽  
Binding Partner ◽  
Endocytosis Pathway

Recently, genetic studies have implicated KIAA0319 in developmental dyslexia, the most common of the childhood learning disorders. The first functional data indicated that the KIAA0319 protein is expressed on the plasma membrane and may be involved in neuronal migration. Further analysis of the subcellular distribution of the overexpressed protein in mammalian cells indicates that KIAA0319 can colocalize with the early endosomal marker early endosome antigen 1 (EEA1) in large intracellular vesicles, suggesting that it is endocytosed. Antibody internalization assays with full-length KIAA0319 and deletion constructs confirmed that KIAA0319 is internalized and showed the importance of the cytoplasmic juxtamembranal region in this process. The present study has identified the medium subunit (μ2) of adaptor protein 2 (AP-2) as a binding partner of KIAA0319 in a yeast two-hybrid screen. Using Rab5 mutants or depletion of the μ-subunit of AP-2 or clathrin heavy chain by RNA interference, we demonstrate that KIAA0319 follows a clathrin-mediated endocytic pathway. We also identify tyrosine-995 of KIAA0319 as a critical amino acid required for the interaction with AP-2 and subsequent internalization. These results suggest the surface expression of KIAA0319 is regulated by endocytosis, supporting the idea that the internalization and recycling of the protein may be involved in fine tuning its role in neuronal migration.

CXCR7 heterodimerizes with CXCR4 and regulates CXCL12-mediated G protein signaling

Blood ◽  
2009 ◽  
Vol 113 (24) ◽  
pp. 6085-6093 ◽  
Author(s):  
Angélique Levoye ◽  
Karl Balabanian ◽  
Françoise Baleux ◽  
Françoise Bachelerie ◽  
Bernard Lagane
Keyword(s):  
Conformational Changes ◽  
Mammalian Cells ◽  
Protein Complexes ◽  
G Protein Signaling ◽  
Protein Signaling ◽  
Protein Activation ◽  
Conformational Rearrangements ◽  
Stromal Cell Derived Factor ◽  
Pathologic Processes ◽  
Gαi Protein

AbstractThe stromal cell-derived factor-1/CXCL12 chemokine engages the CXCR4 and CXCR7 receptors and regulates homeostatic and pathologic processes, including organogenesis, leukocyte homeostasis, and tumorigenesis. Both receptors are widely expressed in mammalian cells, but how they cooperate to respond to CXCL12 is not well understood. Here, we show that CXCR7 per se does not trigger Gαi protein–dependent signaling, although energy transfer assays indicate that it constitutively interacts with Gαi proteins and undergoes CXCL12-mediated conformational changes. Moreover, when CXCR4 and CXCR7 are coexpressed, we show that receptor heterodimers form as efficiently as receptor homodimers, thus opening the possibility that CXCR4/CXCR7 heterodimer formation has consequences on CXCL12-mediated signals. Indeed, expression of CXCR7 induces conformational rearrangements within preassembled CXCR4/Gαi protein complexes and impairs CXCR4-promoted Gαi-protein activation and calcium responses. Varying CXCR7 expression levels and blocking CXCL12/CXCR7 interactions in primary T cells suggest that CXCR4/CXCR7 heterodimers form in primary lymphocytes and regulate CXCL12-promoted chemotaxis. Taken together, these results identify CXCR4/CXCR7 heterodimers as distinct functional units with novel properties, which can contribute to the functional plasticity of CXCL12.

scholarly journals Flat-to-curved transition during clathrin-mediated endocytosis correlates with a change in clathrin-adaptor ratio and is regulated by membrane tension

2017 ◽  
Author(s):  
Delia Bucher ◽  
Felix Frey ◽  
Kem A. Sochacki ◽  
Susann Kummer ◽  
Jan-Philip Bergeest ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Adaptor Protein ◽  
Membrane Tension ◽  
Coated Pits ◽  
Lattice Curvature ◽  
Cellular Processes ◽  
Molecular Events ◽  
Electron And Light Microscopy ◽  
Mechanical Factors ◽  
Clathrin Adaptor

AbstractAlthough essential for many cellular processes, the sequence of structural and molecular events during clathrin-mediated endocytosis remains elusive. While it was believed that clathrin-coated pits grow with a constant curvature, it was recently suggested that clathrin first assembles to form a flat structure and then bends while maintaining a constant surface area. Here, we combine correlative electron and light microscopy and mathematical modelling to quantify the sequence of ultrastructural rearrangements of the clathrin coat during endocytosis in mammalian cells. We confirm that clathrin-coated structures can initially grow flat and that lattice curvature does not show a direct correlation with clathrin coat assembly. We demonstrate that curvature begins when 70% of the final clathrin content is acquired. We find that this transition is marked by a change in the clathrin to clathrin-adaptor protein AP2 ratio and that membrane tension suppresses this transition. Our results support the model that mammalian cells dynamically regulate the flat-to-curved transition in clathrin-mediated endocytosis by both biochemical and mechanical factors.

scholarly journals Role of AP1 and Gadkin in the traffic of secretory endo-lysosomes

2011 ◽  
Vol 22 (12) ◽  
pp. 2068-2082 ◽  
Author(s):  
Karine Laulagnier ◽  
Nicole L. Schieber ◽  
Tanja Maritzen ◽  
Volker Haucke ◽  
Robert G. Parton ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Secretory Pathway ◽  
Protein Complexes ◽  
Adaptor Protein ◽  
Cytosolic Calcium ◽  
Endocytic Pathway ◽  
Secretory Process ◽  
Microtubule Depolymerization ◽  
Lysosome Related Organelles ◽  
Endocytic Compartments

Whereas lysosome-related organelles (LRO) of specialized cells display both exocytic and endocytic features, lysosomes in nonspecialized cells can also acquire the property to fuse with the plasma membrane upon an acute rise in cytosolic calcium. Here, we characterize this unconventional secretory pathway in fibroblast-like cells, by monitoring the appearance of Lamp1 on the plasma membrane and the release of lysosomal enzymes into the medium. After sequential ablation of endocytic compartments in living cells, we find that donor membranes primarily derive from a late compartment, but that an early compartment is also involved. Strikingly, this endo-secretory process is not affected by treatments that inhibit endosome dynamics (microtubule depolymerization, cholesterol accumulation, overexpression of Rab7 or its effector Rab-interacting lysosomal protein [RILP], overexpression of Rab5 mutants), but depends on Rab27a, a GTPase involved in LRO secretion, and is controlled by F-actin. Moreover, we find that this unconventional endo-secretory pathway requires the adaptor protein complexes AP1, Gadkin (which recruits AP1 by binding to the γ1 subunit), and AP2, but not AP3. We conclude that a specific fraction of the AP2-derived endocytic pathway is dedicated to secretory purposes under the control of AP1 and Gadkin.

scholarly journals Phosphoinositide–Ap-2 Interactions Required for Targeting to Plasma Membrane Clathrin-Coated Pits

1999 ◽  
Vol 146 (4) ◽  
pp. 755-764 ◽  
Author(s):  
Ibragim Gaidarov ◽  
James H. Keen
Keyword(s):  
Plasma Membrane ◽  
Mammalian Cells ◽  
Limited Proteolysis ◽  
Adaptor Protein ◽  
Dominant Negative ◽  
Coated Pits ◽  
Intact Cells ◽  
Α Subunit ◽  
General Role ◽  
Receptor Complexes

The clathrin-associated AP-2 adaptor protein is a major polyphosphoinositide-binding protein in mammalian cells. A high affinity binding site has previously been localized to the NH2-terminal region of the AP-2 α subunit (Gaidarov et al. 1996. J. Biol. Chem. 271:20922–20929). Here we used deletion and site- directed mutagenesis to determine that α residues 21–80 comprise a discrete folding and inositide-binding domain. Further, positively charged residues located within this region are involved in binding, with a lysine triad at positions 55–57 particularly critical. Mutant peptides and protein in which these residues were changed to glutamine retained wild-type structural and functional characteristics by several criteria including circular dichroism spectra, resistance to limited proteolysis, and clathrin binding activity. When expressed in intact cells, mutated α subunit showed defective localization to clathrin-coated pits; at high expression levels, the appearance of endogenous AP-2 in coated pits was also blocked consistent with a dominant-negative phenotype. These results, together with recent work indicating that phosphoinositides are also critical to ligand-dependent recruitment of arrestin-receptor complexes to coated pits (Gaidarov et al. 1999. EMBO (Eur. Mol. Biol. Organ.) J. 18:871–881), suggest that phosphoinositides play a critical and general role in adaptor incorporation into plasma membrane clathrin-coated pits.

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