COPI mediates recycling of an exocytic SNARE by recognition of a ubiquitin sorting signal

The COPI coat forms transport vesicles from the Golgi complex and plays a poorly defined role in endocytic trafficking. Here we show that COPI binds K63-linked polyubiquitin and this interaction is crucial for trafficking of a ubiquitinated yeast SNARE (Snc1). Snc1 is a v-SNARE that drives fusion of exocytic vesicles with the plasma membrane, and then recycles through the endocytic pathway to the Golgi for reuse in exocytosis. Removal of ubiquitin from Snc1, or deletion of a β'-COP subunit propeller domain that binds K63-linked polyubiquitin, disrupts Snc1 recycling causing aberrant accumulation in internal compartments. Moreover, replacement of the β'-COP propeller domain with unrelated ubiquitin-binding domains restores Snc1 recycling. These results indicate that ubiquitination, a modification well known to target membrane proteins to the lysosome or vacuole for degradation, can also function as recycling signal to sort a SNARE into COPI vesicles in a non-degradative pathway.

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COPI mediates recycling of an exocytic SNARE from endosomes by recognition of a ubiquitin sorting signal

10.1101/133546 ◽

2017 ◽

Author(s):

Peng Xu ◽

Hannah M. Hankins ◽

Chris Macdonald ◽

Samuel J. Erlinger ◽

Meredith N. Frazier ◽

...

Keyword(s):

Plasma Membrane ◽

Membrane Proteins ◽

Golgi Complex ◽

Budding Yeast ◽

Sorting Signal ◽

Binding Domains ◽

Transport Vesicles ◽

Early Endosomes ◽

Ubiquitin Binding ◽

Target Membrane

ABSTRACTThe COPI coat forms transport vesicles from the Golgi complex and plays a poorly defined role in endocytic trafficking. Here we show that COPI mediates delivery of a budding yeast SNARE (Snc1) from early endosomes to the Golgi complex through recognition of a polyubiquitin sorting signal. Snc1 is a v-SNARE that drives fusion of exocytic vesicles with the plasma membrane, and then recycles through early endosomes back to the Golgi for reuse. Removal of ubiquitin from Snc1, or deletion of a β’-COP subunit propeller domain that binds K63-linked polyubiquitin, causes aberrant accumulation of Snc1 in early endosomes. Moreover, replacement of the β’-COP propeller domain with unrelated ubiquitin-binding domains restores Snc1 recycling. These results indicate that ubiquitination, a modification well known to target membrane proteins to the lysosome or vacuole for degradation, can also function as recycling signal to sort a SNARE into COPI vesicles at early endosomes for Golgi delivery.

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Cargo induces retromer-mediated membrane remodeling on membranes

Molecular Biology of the Cell ◽

10.1091/mbc.e18-06-0339 ◽

2018 ◽

Vol 29 (22) ◽

pp. 2709-2719 ◽

Cited By ~ 6

Author(s):

Latha Kallur Purushothaman ◽

Christian Ungermann

Keyword(s):

Plasma Membrane ◽

Simple Model ◽

Membrane Proteins ◽

Golgi Complex ◽

Exact Order ◽

Membrane Remodeling ◽

Sorting Nexin ◽

Low Concentrations ◽

Sorting Station ◽

Specific Trigger

Endosomes serve as a central sorting station of lipids and proteins that arrive via vesicular carrier from the plasma membrane and the Golgi complex. At the endosome, retromer complexes sort selected receptors and membrane proteins into tubules or vesicles that bud off the endosome. The mature endosome finally fuses with the lysosome. Retromer complexes consist of a cargo selection complex (CSC) and a membrane remodeling part (sorting nexin [SNX]-Bin/amphiphysin/Rvs [BAR], or Snx3 in yeast) and different assemblies of retromer mediate recycling of different cargoes. Due to this complexity, the exact order of events that results in carrier formation is not yet understood. Here, we reconstituted this process on giant unilamellar vesicles together with purified retromer complexes from yeast and selected cargoes. Our data reveal that the membrane remodeling activity of both Snx3 and the SNX-BAR complex is strongly reduced at low concentrations, which can be reactivated by CSC. At even lower concentrations, these complexes still associate with membranes, but only remodel membranes in the presence of their specific cargoes. Our data thus favor a simple model, where cargo functions as a specific trigger of retromer-mediated sorting on endosomes.

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Clathrin Hub Expression Affects Early Endosome Distribution with Minimal Impact on Receptor Sorting and Recycling

Molecular Biology of the Cell ◽

10.1091/mbc.12.9.2790 ◽

2001 ◽

Vol 12 (9) ◽

pp. 2790-2799 ◽

Cited By ~ 27

Author(s):

Elizabeth M. Bennett ◽

Sharron X. Lin ◽

Mhairi C. Towler ◽

Frederick R. Maxfield ◽

Frances M. Brodsky

Keyword(s):

Plasma Membrane ◽

Early Endosome ◽

Dominant Negative ◽

Endocytic Pathway ◽

Endocytic Trafficking ◽

Minimal Impact ◽

Recycling Endosomes ◽

Receptor Mediated Endocytosis ◽

Early Endosomes ◽

Endosomal Membranes

Clathrin-coated vesicles execute receptor-mediated endocytosis at the plasma membrane. However, a role for clathrin in later endocytic trafficking processes, such as receptor sorting and recycling or maintaining the organization of the endocytic pathway, has not been thoroughly characterized. The existence of clathrin-coated buds on endosomes suggests that clathrin might mediate later endocytic trafficking events. To investigate the function of clathrin-coated buds on endosomal membranes, endosome function and distribution were analyzed in a HeLa cell line that expresses the dominant-negative clathrin inhibitor Hub in an inducible manner. As expected, Hub expression reduced receptor-mediated endocytosis at the plasma membrane. Hub expression also induced a perinuclear aggregation of early endosome antigen 1-positive early endosomes, such that sorting and recycling endosomes were found tightly concentrated in the perinuclear region. Despite the dramatic redistribution of endosomes, Hub expression did not affect the overall kinetics of receptor sorting or recycling. These data show that clathrin function is necessary to maintain proper cellular distribution of early endosomes but does not play a prominent role in sorting and recycling events. Thus, clathrin's role on endosomal membranes is to influence organelle localization and is distinct from its role in trafficking pathways at the plasma membrane and trans-Golgi network.

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The VE-cadherin cytoplasmic domain undergoes proteolytic processing during endocytosis

Molecular Biology of the Cell ◽

10.1091/mbc.e16-09-0658 ◽

2017 ◽

Vol 28 (1) ◽

pp. 76-84 ◽

Cited By ~ 18

Author(s):

Wenji Su ◽

Andrew P. Kowalczyk

Keyword(s):

Plasma Membrane ◽

Adhesion Strength ◽

Turnover Rate ◽

Cytoplasmic Tail ◽

Cytoplasmic Domain ◽

Proteolytic Processing ◽

Endocytic Pathway ◽

Cytoplasmic Domains ◽

P120 Catenin ◽

Binding Domains

VE-cadherin trafficking to and from the plasma membrane has emerged as a critical mechanism for regulating cadherin surface levels and adhesion strength. In addition, proteolytic processing of cadherin extracellular and cytoplasmic domains has been reported to regulate cadherin adhesion and signaling. Here we provide evidence that VE-cadherin is cleaved by calpain upon entry into clathrin-enriched domains. This cleavage event occurs between the β-catenin and p120-binding domains within the cadherin cytoplasmic tail. Of interest, VE-cadherin mutants that are resistant to endocytosis are similarly resistant to cleavage. Furthermore, p120-catenin overexpression blocks cadherin internalization and cleavage, coupling entry into the endocytic pathway with proteolytic processing. Of importance, the cleavage of the VE-cadherin tail alters the postendocytic trafficking itinerary of the cadherin, resulting in a higher turnover rate due to decreased recycling and increased degradation. In conclusion, this study identifies a novel proteolytic event that regulates the trafficking of VE-cadherin after endocytosis.

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Estimation of the amount of internalized ricin that reaches the trans-Golgi network

The Journal of Cell Biology ◽

10.1083/jcb.106.2.253 ◽

1988 ◽

Vol 106 (2) ◽

pp. 253-267 ◽

Cited By ~ 139

Author(s):

B van Deurs ◽

K Sandvig ◽

OW Petersen ◽

S Olsnes ◽

K Simons ◽

...

Keyword(s):

Plasma Membrane ◽

G Protein ◽

Golgi Complex ◽

Endocytic Pathway ◽

Membrane Glycoproteins ◽

Double Labeling ◽

Trans Golgi Network ◽

A Chain ◽

Golgi Network ◽

Ricin A

We have used a protocol for internalization of ricin, a ligand binding to plasma membrane glycoproteins and glycolipids with terminal galactosyl residues, and infection with the vesicular stomatitis virus ts 045 mutant in BHK-21 cells to determine whether internalized plasma membrane molecules tagged by ricin reach distinct compartments of the biosynthetic-exocytic pathway. At 39.5 degrees C newly synthesized G protein of ts 045 was largely prevented from leaving the endoplasmic reticulum. At the same temperature ricin was endocytosed and reached, in addition to endosomes and lysosomes, elements of the Golgi complex. When the temperature was lowered to 19.5 degrees C, no more ricin was delivered to the Golgi complex, but now G protein accumulated in the Golgi stacks and the trans-Golgi network (TGN). Double-labeling immunogold cytochemistry on ultracryosections was used to detect G protein and ricin simultaneously. These data, combined with stereological and biochemical methods, showed that approximately 5% of the total amount of ricin within the cells, corresponding to 6-8 X 10(4) molecules per cell, colocalized with G protein in the Golgi complex after 60 min at 39.5 degrees C. Of this amount approximately 70-80% was present in the TGN. Since most of the ricin molecules remain bound to their binding sites at the low pH prevailing in compartments of the endocytic pathway, the results indicate that a fraction of the internalized plasma membrane molecules with terminal galactose are not recycled directly from endosomes or delivered to lysosomes, but are routed to the Golgi complex. Also, the results presented here, in combination with other recent studies on ricin internalization, suggest that translocation of the toxic ricin A-chain to the cytosol occurs in the TGN.

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Segregation in the Golgi complex precedes export of endolysosomal proteins in distinct transport carriers

The Journal of Cell Biology ◽

10.1083/jcb.201707172 ◽

2017 ◽

Vol 216 (12) ◽

pp. 4141-4151 ◽

Cited By ~ 38

Author(s):

Yu Chen ◽

David C. Gershlick ◽

Sang Yoon Park ◽

Juan S. Bonifacino

Keyword(s):

Plasma Membrane ◽

Membrane Proteins ◽

Golgi Complex ◽

Adaptor Proteins ◽

Transmembrane Domains ◽

The Other ◽

Present Evidence ◽

Sorting Signals ◽

Vesicular Carriers ◽

Classical Models

Biosynthetic sorting of newly synthesized transmembrane cargos to endosomes and lysosomes is thought to occur at the TGN through recognition of sorting signals in the cytosolic tails of the cargos by adaptor proteins, leading to cargo packaging into coated vesicles destined for the endolysosomal system. Here we present evidence for a different mechanism in which two sets of endolysosomal proteins undergo early segregation to distinct domains of the Golgi complex by virtue of the proteins’ luminal and transmembrane domains. Proteins in one Golgi domain exit into predominantly vesicular carriers by interaction of sorting signals with adaptor proteins, but proteins in the other domain exit into predominantly tubular carriers shared with plasma membrane proteins, independently of signal–adaptor interactions. These findings demonstrate that sorting of endolysosomal proteins begins at an earlier stage and involves mechanisms that partly differ from those described by classical models.

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The adaptor protein AP-4 as a component of the clathrin coat machinery: a morphological study

Biochemical Journal ◽

10.1042/bj20041010 ◽

2005 ◽

Vol 385 (2) ◽

pp. 503-510 ◽

Cited By ~ 28

Author(s):

Nicolas BAROIS ◽

Oddmund BAKKE

Keyword(s):

Golgi Complex ◽

Intracellular Trafficking ◽

Intracellular Localization ◽

Adaptor Protein ◽

Immunogold Labelling ◽

Endocytic Pathway ◽

Transport Vesicles ◽

Cargo Proteins ◽

The Difference ◽

First Time

The four members of the AP (adaptor protein) family are heterotetrameric cytosolic complexes that are involved in the intracellular trafficking of cargo proteins between different organelles. They interact with motifs present in the cytoplasmic tails of their specific cargo proteins at different intracellular locations. While AP-1, AP-2 and AP-3 have been investigated extensively, very few studies have focused on the fourth member, AP-4. In the present study, we report on the intracellular localization of AP-4 in the MDCK (Madin–Darby canine kidney) and MelJuSo cell lines after immunogold labelling of ultrathin cryosections. We find that AP-4 is localized mainly in the Golgi complex, as well as on endosomes and transport vesicles. Interestingly, we show for the first time that AP-4 is localized with the clathrin coat machinery in the Golgi complex and in the endocytic pathway. Furthermore, we find that AP-4 is localized with the CI-MPR (cation-independent mannose 6-phosphate receptor), but not with the transferrin receptor, LAMP-2 (lysosomal-associated membrane protein-2) or invariant chain. The difference in morphology between CI-MPR/AP-4-positive vesicles and CI-MPR/AP-1-positive vesicles raises the possibility that AP-4 acts at a location different from that of AP-1 in the intracellular trafficking pathway of CI-MPR.

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PLRP2 selectively localizes synaptic membrane proteins via acyl-chain remodeling of phospholipids

The Journal of Lipid Research ◽

10.1194/jlr.ra120001087 ◽

2020 ◽

Vol 61 (12) ◽

pp. 1747-1763

Author(s):

Hideaki Kuge ◽

Izumi Miyamoto ◽

Ken-ichi Yagyu ◽

Koichi Honke

Keyword(s):

Plasma Membrane ◽

Membrane Proteins ◽

Transmembrane Domain ◽

Protein Localization ◽

Acyl Chain ◽

Synaptic Membrane ◽

Membrane Organization ◽

Membrane Phospholipids ◽

Lipid Domain ◽

Transport Vesicles

The plasma membrane of neurons consists of distinct domains, each of which carries specialized functions and a characteristic set of membrane proteins. While this compartmentalized membrane organization is essential for neuronal functions, it remains controversial how neurons establish these domains on the laterally fluid membrane. Here, using immunostaining, lipid-MS analysis and gene ablation with the CRISPR/Cas9 system, we report that the pancreatic lipase-related protein 2 (PLRP2), a phospholipase A1 (PLA1), is a key organizer of membrane protein localization at the neurite tips of PC12 cells. PLRP2 produced local distribution of 1-oleoyl-2-palmitoyl-PC at these sites through acyl-chain remodeling of membrane phospholipids. The resulting lipid domain assembled the syntaxin 4 (Stx4) protein within itself by selectively interacting with the transmembrane domain of Stx4. The localized Stx4, in turn, facilitated the fusion of transport vesicles that contained the dopamine transporter with the domain of the plasma membrane, which led to the localized distribution of the transporter to that domain. These results revealed the pivotal roles of PLA1, specifically PLRP2, in the formation of functional domains in the plasma membrane of neurons. In addition, our results suggest a mode of membrane organization in which the local acyl-chain remodeling of membrane phospholipids controls the selective localization of membrane proteins by regulating both lipid-protein interactions and the fusion of transport vesicles to the lipid domain.

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Evidence for Apical Endocytosis in Polarized Hepatic Cells: Phosphoinositide 3-Kinase Inhibitors Lead to the Lysosomal Accumulation of Resident Apical Plasma Membrane Proteins

The Journal of Cell Biology ◽

10.1083/jcb.145.5.1089 ◽

1999 ◽

Vol 145 (5) ◽

pp. 1089-1102 ◽

Cited By ~ 57

Author(s):

Pamela L. Tuma ◽

Catherine M. Finnegan ◽

Ji-Hyun Yi ◽

Ann L. Hubbard

Keyword(s):

Plasma Membrane ◽

Membrane Proteins ◽

Epithelial Cells ◽

Kinase Inhibitors ◽

Apical Membrane ◽

Membrane Dynamics ◽

Endocytic Pathway ◽

Apical Plasma Membrane ◽

Plasma Membrane Proteins ◽

Phosphoinositide 3 Kinase

The architectural complexity of the hepatocyte canalicular surface has prevented examination of apical membrane dynamics with methods used for other epithelial cells. By adopting a pharmacological approach, we have documented for the first time the internalization of membrane proteins from the hepatic apical surface. Treatment of hepatocytes or WIF-B cells with phosphoinositide 3-kinase inhibitors, wortmannin or LY294002, led to accumulation of the apical plasma membrane proteins, 5′-nucleotidase and aminopeptidase N in lysosomal vacuoles. By monitoring the trafficking of antibody-labeled molecules, we determined that the apical proteins in vacuoles came from the apical plasma membrane. Neither newly synthesized nor transcytosing apical proteins accumulated in vacuoles. In wortmannin-treated cells, transcytosing apical proteins traversed the subapical compartment (SAC), suggesting that this intermediate in the basolateral-to-apical transcytotic pathway remained functional. Ultrastructural analysis confirmed these results. However, apically internalized proteins did not travel through SAC en route to lysosomal vacuoles, indicating that SAC is not an intermediate in the apical endocytic pathway. Basolateral membrane protein distributions did not change in treated cells, uncovering another difference in endocytosis from the two domains. Similar effects were observed in polarized MDCK cells, suggesting conserved patterns of phosphoinositide 3-kinase regulation among epithelial cells. These results confirm a long-held but unproven assumption that lysosomes are the final destination of apical membrane proteins in hepatocytes. Significantly, they also confirm our hypothesis that SAC is not an apical endosome.

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Isoforms of Ankyrin-3 That Lack the NH2-terminal Repeats Associate with Mouse Macrophage Lysosomes

The Journal of Cell Biology ◽

10.1083/jcb.136.5.1059 ◽

1997 ◽

Vol 136 (5) ◽

pp. 1059-1070 ◽

Cited By ~ 52

Author(s):

Thomas C. Hoock ◽

Luanne L. Peters ◽

Samuel E. Lux

Keyword(s):

Plasma Membrane ◽

Plasma Membranes ◽

Endocytic Pathway ◽

Cdna Clones ◽

Mouse Macrophage ◽

Regulatory Domains ◽

Binding Domains ◽

Mouse Macrophages ◽

Repeat Domain ◽

Terminal Repeats

We have recently cloned and characterized ankyrin-3 (also called ankyrinG), a new ankyrin that is widely distributed, especially in epithelial tissues, muscle, and neuronal axons (Peters, L.L., K.M. John, F.M. Lu, E.M. Eicher, A. Higgins, M. Yialamas, L.C. Turtzo, A.J. Otsuka, and S.E. Lux. 1995. J. Cell Biol. 130: 313–330). Here we show that in mouse macrophages, ankyrin-3 is expressed exclusively as two small isoforms (120 and 100 kD) that lack the NH2-terminal repeats. Sequence analysis of isolated Ank3 cDNA clones, obtained by reverse transcription and amplification of mouse macrophage RNA (GenBank Nos. U89274 and U89275), reveals spectrin-binding and regulatory domains identical to those in kidney ankyrin-3 (GenBank No. L40631) preceded by a 29–amino acid segment of the membrane (“repeat”) domain, beginning near the end of the last repeat. Antibodies specific for the regulatory and spectrin-binding domains of ankyrin-3 localize the protein to the surface of intracellular vesicles throughout the macrophage cytoplasm. It is not found on the plasma membrane. Also, epitope-tagged mouse macrophage ankyrin-3, transiently expressed in COS cells, associates with intracellular, not plasma, membranes. In contrast, ankyrin-1 (erythrocyte ankyrin, ankyrinR), which is also expressed in mouse macrophages, is located exclusively on the plasma membrane. The ankyrin-3–positive vesicles appear dark on phasecontrast microscopy. Two observations suggest that they are lysosomes. First, they are a late compartment in the endocytic pathway. They are only accessible to a fluorescent endocytic tracer (FITC-dextran) after a 24-h incubation, at which time all of the FITC-dextran– containing vesicles contain ankyrin-3 and vice versa. Second, the ankyrin-3–positive vesicles contain lysosomal-associated membrane glycoprotein (LAMP-1), a recognized lysosomal marker. This is the first evidence for the association of an ankyrin with lysosomes and is an example of two ankyrins present in the same cell that segregate to different locations.

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