scholarly journals Exomer: a coat complex for transport of select membrane proteins from the trans-Golgi network to the plasma membrane in yeast

2006 ◽  
Vol 174 (7) ◽  
pp. 973-983 ◽  
Author(s):  
Chao-Wen Wang ◽  
Susan Hamamoto ◽  
Lelio Orci ◽  
Randy Schekman
Keyword(s):  
Plasma Membrane ◽  
Membrane Proteins ◽  
Cell Surface ◽  
Protein Complex ◽  
Adaptor Protein ◽  
Nucleotide Exchange ◽  
Trans Golgi Network ◽  
Peripheral Proteins ◽  
Golgi Network

Ayeast plasma membrane protein, Chs3p, transits to the mother–bud neck from a reservoir comprising the trans-Golgi network (TGN) and endosomal system. Two TGN/endosomal peripheral proteins, Chs5p and Chs6p, and three Chs6p paralogues form a complex that is required for the TGN to cell surface transport of Chs3p. The role of these peripheral proteins has not been clear, and we now provide evidence that they create a coat complex required for the capture of membrane proteins en route to the cell surface. Sec7p, a Golgi protein required for general membrane traffic and functioning as a nucleotide exchange factor for the guanosine triphosphate (GTP)–binding protein Arf1p, is required to recruit Chs5p to the TGN surface in vivo. Recombinant forms of Chs5p, Chs6p, and the Chs6p paralogues expressed in baculovirus form a complex of approximately 1 MD that binds synthetic liposomes in a reaction requiring acidic phospholipids, Arf1p, and the nonhydrolyzable GTPγS. The complex remains bound to liposomes centrifuged on a sucrose density gradient. Thin section electron microscopy reveals a spiky coat structure on liposomes incubated with the full complex, Arf1p, and GTPγS. We termed the novel coat exomer for its role in exocytosis from the TGN to the cell surface. Unlike other coats (e.g., coat protein complex I, II, and clathrin/adaptor protein complex), the exomer does not form buds or vesicles on liposomes.

Interaction of amphiphysins with AP-1 clathrin adaptors at the membrane

2013 ◽  
Vol 450 (1) ◽  
pp. 73-83 ◽  
Author(s):  
Sonja Huser ◽  
Gregor Suri ◽  
Pascal Crottet ◽  
Martin Spiess
Keyword(s):  
Plasma Membrane ◽  
Brefeldin A ◽  
Adaptor Protein ◽  
Trans Golgi Network ◽  
Src Homology 3 ◽  
Amphiphysin 1 ◽  
Src Homology ◽  
Golgi Network

The assembly of clathrin/AP (adaptor protein)-1-coated vesicles on the trans-Golgi network and endosomes is much less studied than that of clathrin/AP-2 vesicles at the plasma membrane for endocytosis. In vitro, the association of AP-1 with protein-free liposomes had been shown to require phosphoinositides, Arf1 (ADP-ribosylation factor 1)–GTP and additional cytosolic factor(s). We have purified an active fraction from brain cytosol and found it to contain amphiphysin 1 and 2 and endophilin A1, three proteins known to be involved in the formation of AP-2/clathrin coats at the plasma membrane. Assays with bacterially expressed and purified proteins showed that AP-1 stabilization on liposomes depends on amphiphysin 2 or the amphiphysin 1/2 heterodimer. Activity is independent of the SH3 (Src homology 3) domain, but requires interaction of the WDLW motif with γ-adaptin. Endogenous amphiphysin in neurons and transfected protein in cell lines co-localize perinuclearly with AP-1 at the trans-Golgi network. This localization depends on interaction of clathrin and the adaptor sequence in the amphiphysins and is sensitive to brefeldin A, which inhibits Arf1-dependent AP-1 recruitment. Interaction between AP-1 and amphiphysin 1/2 in vivo was demonstrated by co-immunoprecipitation after cross-linking. These results suggest an involvement of amphiphysins not only with AP-2 at the plasma membrane, but also in AP-1/clathrin coat formation at the trans-Golgi network.

scholarly journals Role of the Endocytic Machinery in the Sorting of Lysosome-associated Membrane Proteins

2005 ◽  
Vol 16 (9) ◽  
pp. 4231-4242 ◽  
Author(s):  
Katy Janvier ◽  
Juan S. Bonifacino
Keyword(s):  
Plasma Membrane ◽  
Coat Protein ◽  
Membrane Proteins ◽  
Adaptor Protein ◽  
The Other ◽  
Sorting Signals ◽  
Efficient Delivery ◽  
Endocytic Machinery

The limiting membrane of the lysosome contains a group of transmembrane glycoproteins named lysosome-associated membrane proteins (Lamps). These proteins are targeted to lysosomes by virtue of tyrosine-based sorting signals in their cytosolic tails. Four adaptor protein (AP) complexes, AP-1, AP-2, AP-3, and AP-4, interact with such signals and are therefore candidates for mediating sorting of the Lamps to lysosomes. However, the role of these complexes and of the coat protein, clathrin, in sorting of the Lamps in vivo has either not been addressed or remains controversial. We have used RNA interference to show that AP-2 and clathrin—and to a lesser extent the other AP complexes—are required for efficient delivery of the Lamps to lysosomes. Because AP-2 is exclusively associated with plasma membrane clathrin coats, our observations imply that a significant population of Lamps traffic via the plasma membrane en route to lysosomes.

scholarly journals Phosphorylation of the cation-independent mannose 6-phosphate receptor is closely associated with its exit from the trans-Golgi network.

1993 ◽  
Vol 120 (1) ◽  
pp. 67-75 ◽  
Author(s):  
S Méresse ◽  
B Hoflack
Keyword(s):  
Cell Surface ◽  
Specific Inhibitor ◽  
Cytoplasmic Domain ◽  
Single Step ◽  
Coated Vesicles ◽  
Coated Pits ◽  
Trans Golgi Network ◽  
Mannose 6 Phosphate ◽  
Golgi Network

We have previously shown that two serine residues present in two conserved regions of the bovine cation-independent mannose 6-phosphate receptor (CI-MPR) cytoplasmic domain are phosphorylated in vivo (residues 2421 and 2492 of the full length bovine CI-MPR precursor). In this study, we have used CHO cells to investigate the phosphorylation state of these two serines along the different steps of the CI-MPR exocytic and endocytic recycling pathways. Transport and phosphorylation of the CI-MPR in the biosynthetic pathway were examined using deoxymannojirimycin (dMM), a specific inhibitor of the cis-Golgi processing enzyme alpha-mannosidase I which leads to the accumulation of N-linked high mannose oligosaccharides on glycoproteins. Upon removal of dMM, normal processing to complex-type oligosaccharides (galactosylation and then sialylation) occurs on the newly synthesized glycoproteins, including the CI-MPR which could then be purified and analyzed on lectin affinity columns. Phosphorylation of the newly synthesized CI-MPR was concomitant with the sialylation of its oligosaccharides and appeared as a major albeit transient modification. Phosphorylation of the cell surface CI-MPR was examined during its endocytosis as well as its return to the Golgi using antibody tagging and exogalactosylation. The cell surface CI-MPR was not phosphorylated when it entered clathrin-coated pits or when it moved to the early and late endosomes. In contrast, the surface CI-MPR was phosphorylated when it had been resialylated upon its return to the trans-Golgi network. Subcellular fractionation experiments showed that the phosphorylated CI-MPR and the corresponding kinase were found in clathrin-coated vesicles. Collectively, these results indicate that phosphorylation of the two serines in the CI-MPR cytoplasmic domain is associated with a single step of transport of its recycling pathways and occurs when this receptor is in the trans-Golgi network and/or has left this compartment via clathrin-coated vesicles.

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 Loss of P4 ATPases Drs2p and Dnf3p Disrupts Aminophospholipid Transport and Asymmetry in Yeast Post-Golgi Secretory Vesicles

2006 ◽  
Vol 17 (4) ◽  
pp. 1632-1642 ◽  
Author(s):  
Nele Alder-Baerens ◽  
Quirine Lisman ◽  
Lambert Luong ◽  
Thomas Pomorski ◽  
Joost C.M. Holthuis
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Plasma Membranes ◽  
Atp Hydrolysis ◽  
Budding Yeast ◽  
Secretory Vesicles ◽  
Trans Golgi Network ◽  
P Type ◽  
Golgi Network

Eukaryotic plasma membranes generally display asymmetric lipid distributions with the aminophospholipids concentrated in the cytosolic leaflet. This arrangement is maintained by aminophospholipid translocases (APLTs) that use ATP hydrolysis to flip phosphatidylserine (PS) and phosphatidylethanolamine (PE) from the external to the cytosolic leaflet. The identity of APLTs has not been established, but prime candidates are members of the P4 subfamily of P-type ATPases. Removal of P4 ATPases Dnf1p and Dnf2p from budding yeast abolishes inward translocation of 6-[(7-nitrobenz-2-oxa-1,3-diazol-4-yl)aminocaproyl] (NBD)-labeled PS, PE, and phosphatidylcholine (PC) across the plasma membrane and causes cell surface exposure of endogenous PE. Here, we show that yeast post-Golgi secretory vesicles (SVs) contain a translocase activity that flips NBD-PS, NBD-PE, and NBD-PC to the cytosolic leaflet. This activity is independent of Dnf1p and Dnf2p but requires two other P4 ATPases, Drs2p and Dnf3p, that reside primarily in the trans-Golgi network. Moreover, SVs have an asymmetric PE arrangement that is lost upon removal of Drs2p and Dnf3p. Our results indicate that aminophospholipid asymmetry is created when membrane flows through the Golgi and that P4-ATPases are essential for this process.

scholarly journals Chimeric Forms of Furin and Tgn38 Are Transported from the Plasma Membrane to the Trans-Golgi Network via Distinct Endosomal Pathways

1999 ◽  
Vol 146 (2) ◽  
pp. 345-360 ◽  
Author(s):  
William G. Mallet ◽  
Frederick R. Maxfield
Keyword(s):  
Plasma Membrane ◽  
Membrane Proteins ◽  
Chimeric Proteins ◽  
Endocytic Recycling ◽  
Single Pass ◽  
Trans Golgi Network ◽  
Late Endosomes ◽  
Recycling Pathway ◽  
Endosomal Transport ◽  
Golgi Network

Furin and TGN38 are membrane proteins that cycle between the plasma membrane and the trans-Golgi network (TGN), each maintaining a predominant distribution in the TGN. We have used chimeric proteins with an extracellular Tac domain and the cytoplasmic domain of TGN38 or furin to study the trafficking of these proteins in endosomes. Previously, we demonstrated that the postendocytic trafficking of Tac-TGN38 to the TGN is via the endocytic recycling pathway (Ghosh, R.N., W.G. Mallet, T.T. Soe, T.E. McGraw, and F.R. Maxfield. 1998. J. Cell Biol. 142:923–936). Here we show that internalized Tac-furin is delivered to the TGN through late endosomes, bypassing the endocytic recycling compartment. The transport of Tac-furin from late endosomes to the TGN appears to proceed via an efficient, single-pass mechanism. Delivery of Tac-furin but not Tac-TGN38 to the TGN is blocked by nocodazole, and the two pathways are also differentially affected by wortmannin. These studies demonstrate the existence of two independent pathways for endosomal transport of proteins to the TGN from the plasma membrane.

scholarly journals Visualization of the Dynamics of Synaptic Vesicle and Plasma Membrane Proteins in Living Axons

1998 ◽  
Vol 140 (3) ◽  
pp. 659-674 ◽  
Author(s):  
Takao Nakata ◽  
Sumio Terada ◽  
Nobutaka Hirokawa
Keyword(s):  
Plasma Membrane ◽  
Membrane Proteins ◽  
Synaptic Vesicle ◽  
Synaptic Vesicles ◽  
Fluorescent Protein ◽  
Plasma Membrane Proteins ◽  
Trans Golgi Network ◽  
Direction Of Movement ◽  
Green Fluorescent ◽  
Golgi Network

Newly synthesized membrane proteins are transported by fast axonal flow to their targets such as the plasma membrane and synaptic vesicles. However, their transporting vesicles have not yet been identified. We have successfully visualized the transporting vesicles of plasma membrane proteins, synaptic vesicle proteins, and the trans-Golgi network residual proteins in living axons at high resolution using laser scan microscopy of green fluorescent protein-tagged proteins after photobleaching. We found that all of these proteins are transported by tubulovesicular organelles of various sizes and shapes that circulate within axons from branch to branch and switch the direction of movement. These organelles are distinct from the endosomal compartments and constitute a new entity of membrane organelles that mediate the transport of newly synthesized proteins from the trans-Golgi network to the plasma membrane.

scholarly journals Adaptor protein complex 4 deficiency: a paradigm of childhood-onset hereditary spastic paraplegia caused by defective protein trafficking

2020 ◽  
Vol 29 (2) ◽  
pp. 320-334 ◽  
Author(s):  
Robert Behne ◽  
Julian Teinert ◽  
Miriam Wimmer ◽  
Angelica D’Amore ◽  
Alexandra K Davies ◽  
...  
Keyword(s):  
Protein Complex ◽  
Protein Trafficking ◽  
Hereditary Spastic Paraplegia ◽  
Adaptor Protein ◽  
Spastic Paraplegia ◽  
Childhood Onset ◽  
Trans Golgi Network ◽  
Wide Range ◽  
Golgi Network ◽  
The Impact

Abstract Deficiency of the adaptor protein complex 4 (AP-4) leads to childhood-onset hereditary spastic paraplegia (AP-4-HSP): SPG47 (AP4B1), SPG50 (AP4M1), SPG51 (AP4E1) and SPG52 (AP4S1). This study aims to evaluate the impact of loss-of-function variants in AP-4 subunits on intracellular protein trafficking using patient-derived cells. We investigated 15 patient-derived fibroblast lines and generated six lines of induced pluripotent stem cell (iPSC)-derived neurons covering a wide range of AP-4 variants. All patient-derived fibroblasts showed reduced levels of the AP4E1 subunit, a surrogate for levels of the AP-4 complex. The autophagy protein ATG9A accumulated in the trans-Golgi network and was depleted from peripheral compartments. Western blot analysis demonstrated a 3–5-fold increase in ATG9A expression in patient lines. ATG9A was redistributed upon re-expression of AP4B1 arguing that mistrafficking of ATG9A is AP-4-dependent. Examining the downstream effects of ATG9A mislocalization, we found that autophagic flux was intact in patient-derived fibroblasts both under nutrient-rich conditions and when autophagy is stimulated. Mitochondrial metabolism and intracellular iron content remained unchanged. In iPSC-derived cortical neurons from patients with AP4B1-associated SPG47, AP-4 subunit levels were reduced while ATG9A accumulated in the trans-Golgi network. Levels of the autophagy marker LC3-II were reduced, suggesting a neuron-specific alteration in autophagosome turnover. Neurite outgrowth and branching were reduced in AP-4-HSP neurons pointing to a role of AP-4-mediated protein trafficking in neuronal development. Collectively, our results establish ATG9A mislocalization as a key marker of AP-4 deficiency in patient-derived cells, including the first human neuron model of AP-4-HSP, which will aid diagnostic and therapeutic studies.

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