scholarly journals Adaptor and Clathrin Exchange at the Plasma Membrane andtrans-Golgi Network

2003 ◽  
Vol 14 (2) ◽  
pp. 516-528 ◽  
Author(s):  
Xufeng Wu ◽  
Xiaohong Zhao ◽  
Rosa Puertollano ◽  
Juan S. Bonifacino ◽  
Evan Eisenberg ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Fluorescence Recovery After Photobleaching ◽  
Fluorescence Recovery ◽  
Coated Vesicle ◽  
Coated Pits ◽  
Trans Golgi Network ◽  
Rapid Exchange ◽  
Dynamic Structures ◽  
Golgi Network ◽  
Network Exchange

We previously demonstrated, using fluorescence recovery after photobleaching, that clathrin in clathrin-coated pits at the plasma membrane exchanges with free clathrin in the cytosol, suggesting that clathrin-coated pits are dynamic structures. We now investigated whether clathrin at the trans-Golgi network as well as the clathrin adaptors AP2 and AP1 in clathrin-coated pits at the plasma membrane and trans-Golgi network, respectively, also exchange with free proteins in the cytosol. We found that when the budding of clathrin-coated vesicle is blocked without significantly affecting the structure of clathrin-coated pits, both clathrin and AP2 at the plasma membrane and clathrin and AP1 at thetrans-Golgi network exchange rapidly with free proteins in the cytosol. In contrast, when budding of clathrin-coated vesicles was blocked at the plasma membrane or trans-Golgi network by hypertonic sucrose or K+ depletion, conditions that markedly affect the structure of clathrin-coated pits, clathrin exchange was blocked but AP2 at the plasma membrane and both AP1 and the GGA1 adaptor at the trans-Golgi network continue to rapidly exchange. We conclude that clathrin-coated pits are dynamic structures with rapid exchange of both clathrin and adaptors and that adaptors are able to exchange independently of clathrin when clathrin exchange is blocked.

Expression of auxilin or AP180 inhibits endocytosis by mislocalizing clathrin: evidence for formation of nascent pits containing AP1 or AP2 but not clathrin

2001 ◽  
Vol 114 (2) ◽  
pp. 353-365 ◽  
Author(s):  
X. Zhao ◽  
T. Greener ◽  
H. Al-Hasani ◽  
S.W. Cushman ◽  
E. Eisenberg ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Cultured Cells ◽  
Coated Vesicles ◽  
Coated Pits ◽  
Trans Golgi Network ◽  
J Domain ◽  
Almost All ◽  
Golgi Network ◽  
Assembly Proteins

Although uncoating of clathrin-coated vesicles is a key event in clathrin-mediated endocytosis it is unclear what prevents uncoating of clathrin-coated pits before they pinch off to become clathrin-coated vesicles. We have shown that the J-domain proteins auxilin and GAK are required for uncoating by Hsc70 in vitro. In the present study, we expressed auxilin in cultured cells to determine if this would block endocytosis by causing premature uncoating of clathrin-coated pits. We found that expression of auxilin indeed inhibited endocytosis. However, expression of auxilin with its J-domain mutated so that it no longer interacted with Hsc70 also inhibited endocytosis as did expression of the clathrin-assembly protein, AP180, or its clathrin-binding domain. Accompanying this inhibition, we observed a marked decrease in clathrin associated with the plasma membrane and the trans-Golgi network, which provided us with an opportunity to determine whether the absence of clathrin from clathrin-coated pits affected the distribution of the clathrin assembly proteins AP1 and AP2. Surprisingly we found almost no change in the association of AP2 and AP1 with the plasma membrane and the trans-Golgi network, respectively. This was particularly obvious when auxilin or GAK was expressed with functional J-domains since, in these cases, almost all of the clathrin was sequestered in granules that also contained Hsc70 and auxilin or GAK. We conclude that expression of clathrin-binding proteins inhibits clathrin-mediated endocytosis by sequestering clathrin so that it is no longer available to bind to nascent pits but that assembly proteins bind to these pits independently of clathrin.

μ1A deficiency induces a profound increase in MPR300/IGF-II receptor internalization rate

2001 ◽  
Vol 114 (24) ◽  
pp. 4469-4476 ◽  
Author(s):  
Christoph Meyer ◽  
Eeva-Liisa Eskelinen ◽  
Medigeshi Ramarao Guruprasad ◽  
Kurt von Figura ◽  
Peter Schu
Keyword(s):  
Plasma Membrane ◽  
Fold Increase ◽  
Retrograde Transport ◽  
Receptor Internalization ◽  
Recycling Rate ◽  
Coated Pits ◽  
Trans Golgi Network ◽  
Internalization Rate ◽  
Clathrin Adaptor ◽  
Golgi Network

The mannose-6-phosphate/IGF-II receptor MPR300 mediates sorting of lysosomal enzymes from the trans-Golgi network to endosomes and endocytosis of hormones, for example, of IGF-II. We analyzed transport of MPR300 in μ1A-adaptin-deficient fibroblasts, which lack a functional AP-1 clathrin adaptor complex. In μ1A-adaptin-deficient fibroblasts, the homologous MPR46 accumulates in endosomes due to a block in retrograde transport to the trans-Golgi network. The MPR300-mediated endocytosis is markedly enhanced. We demonstrate that the seven-fold increase in endocytosis is not associated with an increased steady-state concentration of receptors at the plasma membrane, but with an increased internalization rate of MPR300. Internalization of other receptors that are also endocytosed by AP-2 is not affected. More MPR300 receptors are found in clathrin-coated pits of the plasma membrane, whereas outside coated-areas, more MPR300 are concentrated in clusters and all intracellular receptors reside in endosomes, which are in equilibrium with the plasma membrane. Thus AP-1-mediated transport of MPR300 from endosomes to the TGN controls indirectly the recycling rate of the receptor between the plasma membrane and endosomes.

scholarly journals Clathrin Assembly Lymphoid Myeloid Leukemia (CALM) Protein: Localization in Endocytic-coated Pits, Interactions with Clathrin, and the Impact of Overexpression on Clathrin-mediated Traffic

1999 ◽  
Vol 10 (8) ◽  
pp. 2687-2702 ◽  
Author(s):  
Francesc Tebar ◽  
Stefan K. Bohlander ◽  
Alexander Sorkin
Keyword(s):  
Plasma Membrane ◽  
Heavy Chain ◽  
Myeloid Leukemia ◽  
Protein Localization ◽  
Adaptor Protein ◽  
Coated Pits ◽  
Trans Golgi Network ◽  
Clathrin Heavy Chain ◽  
Golgi Network ◽  
The Impact

The clathrin assembly lymphoid myeloid leukemia (CALM) gene encodes a putative homologue of the clathrin assembly synaptic protein AP180. Hence the biochemical properties, the subcellular localization, and the role in endocytosis of a CALM protein were studied. In vitro binding and coimmunoprecipitation demonstrated that the clathrin heavy chain is the major binding partner of CALM. The bulk of cellular CALM was associated with the membrane fractions of the cell and localized to clathrin-coated areas of the plasma membrane. In the membrane fraction, CALM was present at near stoichiometric amounts relative to clathrin. To perform structure–function analysis of CALM, we engineered chimeric fusion proteins of CALM and its fragments with the green fluorescent protein (GFP). GFP–CALM was targeted to the plasma membrane–coated pits and also found colocalized with clathrin in the Golgi area. High levels of expression of GFP–CALM or its fragments with clathrin-binding activity inhibited the endocytosis of transferrin and epidermal growth factor receptors and altered the steady-state distribution of the mannose-6-phosphate receptor in the cell. In addition, GFP–CALM overexpression caused the loss of clathrin accumulation in the trans-Golgi network area, whereas the localization of the clathrin adaptor protein complex 1 in the trans-Golgi network remained unaffected. The ability of the GFP-tagged fragments of CALM to affect clathrin-mediated processes correlated with the targeting of the fragments to clathrin-coated areas and their clathrin-binding capacities. Clathrin–CALM interaction seems to be regulated by multiple contact interfaces. The C-terminal part of CALM binds clathrin heavy chain, although the full-length protein exhibited maximal ability for interaction. Altogether, the data suggest that CALM is an important component of coated pit internalization machinery, possibly involved in the regulation of clathrin recruitment to the membrane and/or the formation of the coated pit.

scholarly journals Actin dynamics coupled to clathrin-coated vesicle formation at the trans-Golgi network

2004 ◽  
Vol 165 (6) ◽  
pp. 781-788 ◽  
Author(s):  
Sebastien Carreno ◽  
Åsa E. Engqvist-Goldstein ◽  
Claire X. Zhang ◽  
Kent L. McDonald ◽  
David G. Drubin
Keyword(s):  
Time Lapse ◽  
Actin Dynamics ◽  
Coated Vesicle ◽  
Cell Cortex ◽  
Actin Assembly ◽  
Trans Golgi Network ◽  
Lysosome Biogenesis ◽  
Diverse Species ◽  
Golgi Network ◽  
Golgi Organization

In diverse species, actin assembly facilitates clathrin-coated vesicle (CCV) formation during endocytosis. This role might be an adaptation specific to the unique environment at the cell cortex, or it might be fundamental, facilitating CCV formation on different membranes. Proteins of the Sla2p/Hip1R family bind to actin and clathrin at endocytic sites in yeast and mammals. We hypothesized that Hip1R might also coordinate actin assembly with clathrin budding at the trans-Golgi network (TGN). Using deconvolution and time-lapse microscopy, we showed that Hip1R is present on CCVs emerging from the TGN. These vesicles contain the mannose 6-phosphate receptor involved in targeting proteins to the lysosome, and the actin nucleating Arp2/3 complex. Silencing of Hip1R expression by RNAi resulted in disruption of Golgi organization and accumulation of F-actin structures associated with CCVs on the TGN. Hip1R silencing and actin poisons slowed cathepsin D exit from the TGN. These studies establish roles for Hip1R and actin in CCV budding from the TGN for lysosome biogenesis.

scholarly journals Lysosomal Hydrolase Mannose 6-Phosphate Uncovering Enzyme Resides in the trans-Golgi Network

2001 ◽  
Vol 12 (6) ◽  
pp. 1623-1631 ◽  
Author(s):  
Jack Rohrer ◽  
Rosalind Kornfeld
Keyword(s):  
Plasma Membrane ◽  
Fluorescent Protein ◽  
Lysosomal Hydrolases ◽  
Intracellular Location ◽  
Trans Golgi Network ◽  
Cytosolic Domain ◽  
Cytosolic Domains ◽  
Mannose 6 Phosphate ◽  
Green Fluorescent ◽  
Golgi Network

A crucial step in lysosomal biogenesis is catalyzed by “uncovering” enzyme (UCE), which removes a coveringN-acetylglucosamine from the mannose 6-phosphate (Man-6-P) recognition marker on lysosomal hydrolases. This study shows that UCE resides in the trans-Golgi network (TGN) and cycles between the TGN and plasma membrane. The cytosolic domain of UCE contains two potential endocytosis motifs: 488YHPL and C-terminal 511NPFKD. YHPL is shown to be the more potent of the two in retrieval of UCE from the plasma membrane. A green-fluorescent protein-UCE transmembrane-cytosolic domain fusion protein colocalizes with TGN 46, as does endogenous UCE in HeLa cells, showing that the transmembrane and cytosolic domains determine intracellular location. These data imply that the Man-6-P recognition marker is formed in the TGN, the compartment where Man-6-P receptors bind cargo and are packaged into clathrin-coated vesicles.

Identification of a di-leucine motif within the C terminus domain of the Menkes disease protein that mediates endocytosis from the plasma membrane

1999 ◽  
Vol 112 (11) ◽  
pp. 1721-1732 ◽  
Author(s):  
M.J. Francis ◽  
E.E. Jones ◽  
E.R. Levy ◽  
R.L. Martin ◽  
S. Ponnambalam ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Golgi Apparatus ◽  
Transmembrane Domain ◽  
Menkes Disease ◽  
Cytoplasmic Domain ◽  
Endocytic Pathway ◽  
Site Directed Mutagenesis ◽  
Trans Golgi Network ◽  
C Terminus ◽  
Golgi Network

The protein encoded by the Menkes disease gene (MNK) is localised to the Golgi apparatus and cycles between the trans-Golgi network and the plasma membrane in cultured cells on addition and removal of copper to the growth medium. This suggests that MNK protein contains active signals that are involved in the retention of the protein to the trans-Golgi network and retrieval of the protein from the plasma membrane. Previous studies have identified a signal involved in Golgi retention within transmembrane domain 3 of MNK. To identify a motif sufficient for retrieval of MNK from the plasma membrane, we analysed the cytoplasmic domain, downstream of transmembrane domain 7 and 8. Chimeric constructs containing this cytoplasmic domain fused to the reporter molecule CD8 localised the retrieval signal(s) to 62 amino acids at the C terminus. Further studies were performed on putative internalisation motifs, using site-directed mutagenesis, protein expression, chemical treatment and immunofluorescence. We observed that a di-leucine motif (L1487L1488) was essential for rapid internalisation of chimeric CD8 proteins and the full-length Menkes cDNA from the plasma membrane. We suggest that this motif mediates the retrieval of MNK from the plasma membrane into the endocytic pathway, via the recycling endosomes, but is not sufficient on its own to return the protein to the Golgi apparatus. These studies provide a basis with which to identify other motifs important in the sorting and delivery of MNK from the plasma membrane to the Golgi apparatus.

Vesicle Formation at the Plasma Membrane and Trans-Golgi Network: The Same but Different

Science ◽  
2006 ◽  
Vol 313 (5793) ◽  
pp. 1591-1594 ◽  
Author(s):  
M. A. McNiven ◽  
H. M. Thompson
Keyword(s):  
Plasma Membrane ◽  
Vesicle Formation ◽  
Trans Golgi Network ◽  
Golgi Network

scholarly journals AP-2/Eps15 Interaction Is Required for Receptor-mediated Endocytosis

1998 ◽  
Vol 140 (5) ◽  
pp. 1055-1062 ◽  
Author(s):  
Alexandre Benmerah ◽  
Christophe Lamaze ◽  
Bernadette Bègue ◽  
Sandra L. Schmid ◽  
Alice Dautry-Varsat ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Fusion Protein ◽  
Binding Sites ◽  
Fluorescent Protein ◽  
Coated Vesicle ◽  
Mutant Form ◽  
A431 Cells ◽  
Coated Pits ◽  
Receptor Mediated Endocytosis ◽  
Terminal Domain

We have previously shown that the protein Eps15 is constitutively associated with the plasma membrane adaptor complex, AP-2, suggesting its possible role in endocytosis. To explore the role of Eps15 and the function of AP-2/Eps15 association in endocytosis, the Eps15 binding domain for AP-2 was precisely delineated. The entire COOH-terminal domain of Eps15 or a mutant form lacking all the AP-2–binding sites was fused to the green fluorescent protein (GFP), and these constructs were transiently transfected in HeLa cells. Overexpression of the fusion protein containing the entire COOH-terminal domain of Eps15 strongly inhibited endocytosis of transferrin, whereas the fusion protein in which the AP-2–binding sites had been deleted had no effect. These results were confirmed in a cell-free assay that uses perforated A431 cells to follow the first steps of coated vesicle formation at the plasma membrane. Addition of Eps15-derived glutathione-S-transferase fusion proteins containing the AP-2–binding site in this assay inhibited not only constitutive endocytosis of transferrin but also ligand-induced endocytosis of epidermal growth factor. This inhibition could be ascribed to a competition between the fusion protein and endogenous Eps15 for AP-2 binding. Altogether, these results show that interaction of Eps15 with AP-2 is required for efficient receptor-mediated endocytosis and thus provide the first evidence that Eps15 is involved in the function of plasma membrane–coated pits.

Dual-color visualization of trans-Golgi network to plasma membrane traffic along microtubules in living cells

1999 ◽  
Vol 112 (1) ◽  
pp. 21-33 ◽  
Author(s):  
D. Toomre ◽  
P. Keller ◽  
J. White ◽  
J.C. Olivo ◽  
K. Simons
Keyword(s):  
Plasma Membrane ◽  
Protein Trafficking ◽  
Membrane Traffic ◽  
Living Cells ◽  
Tubular Structures ◽  
Trans Golgi Network ◽  
Vesicular Stomatitis ◽  
Golgi Network ◽  
Color Visualization

The mechanisms and carriers responsible for exocytic protein trafficking between the trans-Golgi network (TGN) and the plasma membrane remain unclear. To investigate the dynamics of TGN-to-plasma membrane traffic and role of the cytoskeleton in these processes we transfected cells with a GFP-fusion protein, vesicular stomatitis virus G protein tagged with GFP (VSVG3-GFP). After using temperature shifts to block VSVG3-GFP in the endoplasmic reticulum and subsequently accumulate it in the TGN, dynamics of TGN-to-plasma membrane transport were visualized in real time by confocal and video microscopy. Both small vesicles (<250 nm) and larger vesicular-tubular structures (>1.5 microm long) are used as transport containers (TCs). These TCs rapidly moved out of the Golgi along curvilinear paths with average speeds of approximately 0.7 micrometer/second. Automatic computer tracking objectively determined the dynamics of different carriers. Fission and fusion of TCs were observed, suggesting that these late exocytic processes are highly interactive. To directly determine the role of microtubules in post-Golgi traffic, rhodamine-tubulin was microinjected and both labeled cargo and microtubules were simultaneously visualized in living cells. These studies demonstrated that exocytic cargo moves along microtubule tracks and reveals that carriers are capable of switching between tracks.

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