scholarly journals A versatile nanobody-based toolkit to analyze retrograde transport from the cell surface

2018 ◽  
Vol 115 (27) ◽  
pp. E6227-E6236 ◽  
Author(s):  
Dominik P. Buser ◽  
Kai D. Schleicher ◽  
Cristina Prescianotto-Baschong ◽  
Martin Spiess
Keyword(s):  
Electron Microscopy ◽  
Cell Surface ◽  
Adaptor Protein ◽  
Retrograde Transport ◽  
Transport Pathways ◽  
Consensus Sequences ◽  
Retrograde Traffic ◽  
Biochemical Detection ◽  
Golgi Network ◽  
Kinetics Of

Retrograde transport of membranes and proteins from the cell surface to the Golgi and beyond is essential to maintain homeostasis, compartment identity, and physiological functions. To study retrograde traffic biochemically, by live-cell imaging or by electron microscopy, we engineered functionalized anti-GFP nanobodies (camelid VHH antibody domains) to be bacterially expressed and purified. Tyrosine sulfation consensus sequences were fused to the nanobody for biochemical detection of trans-Golgi arrival, fluorophores for fluorescence microscopy and live imaging, and APEX2 (ascorbate peroxidase 2) for electron microscopy and compartment ablation. These functionalized nanobodies are specifically captured by GFP-modified reporter proteins at the cell surface and transported piggyback to the reporters’ homing compartments. As an application of this tool, we have used it to determine the contribution of adaptor protein-1/clathrin in retrograde transport kinetics of the mannose-6-phosphate receptors from endosomes back to the trans-Golgi network. Our experiments establish functionalized nanobodies as a powerful tool to demonstrate and quantify retrograde transport pathways.

Characterization of Endocytic Pathways by Quantitative Fluorescence Microscopy

1998 ◽  
Vol 4 (S2) ◽  
pp. 1024-1025
Author(s):  
Frederick R. Maxfield ◽  
Richik N. Ghosh ◽  
William G. Mallet ◽  
Thwe Thwe Soe ◽  
Philip L. Leopold ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Cell Surface ◽  
Light And Electron Microscopy ◽  
Endocytic Pathway ◽  
Early Endosomes ◽  
Pass Through ◽  
Golgi Network ◽  
Quantitative Fluorescence ◽  
Endocytic Pathways

We have used light and electron microscopy to analyze endocytic trafficking pathways. In one set of studies, we have used fluorescently labeled antibodies to trace an endocytic pathway from the cell surface to the trans- Golgi network (TGN). Cells were transfected with a construct consisting of the transmembrane and cytoplasmic domains of TGN38 and the extracellular domain of Tac. TGN38 is predominantly in the TGN, but a small fraction is found on the cell surface. We used FITC-labeled anti-Tac monoclonal IgG to analyze the pathway from the surface to the TGN. We compared the distribution of internalized Tac-TGN38 to internalized transferrin. We found that most Tac-TGN38 enters the same early endosomes as transferrin. Furthermore, most Tac-TGN38 returns to the cell surface from the endocytic recycling compartment (ERC) at the same rate as transferrin. However, on each pass through the cell approximately 18% of Tac-TGN is retained, and this Tac-TGN38 is delivered to the TGN.

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 The COG complex interacts directly with Syntaxin 6 and positively regulates endosome-to-TGN retrograde transport

2011 ◽  
Vol 194 (3) ◽  
pp. 459-472 ◽  
Author(s):  
Orly Laufman ◽  
WanJin Hong ◽  
Sima Lev
Keyword(s):  
Steady State ◽  
State Level ◽  
Retrograde Transport ◽  
Snare Complex ◽  
Steady State Level ◽  
Cog Complex ◽  
Target Membrane ◽  
Steady State Levels ◽  
Retrograde Traffic ◽  
Golgi Network

The conserved oligomeric Golgi (COG) complex has been implicated in the regulation of endosome to trans-Golgi network (TGN) retrograde trafficking in both yeast and mammals. However, the exact mechanisms by which it regulates this transport route remain largely unknown. In this paper, we show that COG interacts directly with the target membrane SNARE (t-SNARE) Syntaxin 6 via the Cog6 subunit. In Cog6-depleted cells, the steady-state level of Syntaxin 6 was markedly reduced, and concomitantly, endosome-to-TGN retrograde traffic was significantly attenuated. Cog6 knockdown also affected the steady-state levels and/or subcellular distributions of Syntaxin 16, Vti1a, and VAMP4 and impaired the assembly of the Syntaxin 6–Syntaxin16–Vti1a–VAMP4 SNARE complex. Remarkably, overexpression of VAMP4, but not of Syntaxin 6, bypassed the requirement for COG and restored endosome-to-TGN trafficking in Cog6-depleted cells. These results suggest that COG directly interacts with specific t-SNAREs and positively regulates SNARE complex assembly, thereby affecting their associated trafficking steps.

scholarly journals ARF1 and ARF4 regulate recycling endosomal morphology and retrograde transport from endosomes to the Golgi apparatus

2013 ◽  
Vol 24 (16) ◽  
pp. 2570-2581 ◽  
Author(s):  
Waka Nakai ◽  
Yumika Kondo ◽  
Akina Saitoh ◽  
Tomoki Naito ◽  
Kazuhisa Nakayama ◽  
...  
Keyword(s):  
Golgi Apparatus ◽  
Retrograde Transport ◽  
Class Ii ◽  
Small Gtpases ◽  
Class I ◽  
Transport Pathways ◽  
Recycling Endosomes ◽  
Late Endosomes ◽  
Recycling Pathway ◽  
Golgi Network

Small GTPases of the ADP-ribosylation factor (ARF) family, except for ARF6, mainly localize to the Golgi apparatus, where they trigger formation of coated carrier vesicles. We recently showed that class I ARFs (ARF1 and ARF3) localize to recycling endosomes, as well as to the Golgi, and are redundantly required for recycling of endocytosed transferrin. On the other hand, the roles of class II ARFs (ARF4 and ARF5) are not yet fully understood, and the complementary or overlapping functions of class I and class II ARFs have been poorly characterized. In this study, we find that simultaneous depletion of ARF1 and ARF4 induces extensive tubulation of recycling endosomes. Moreover, the depletion of ARF1 and ARF4 inhibits retrograde transport of TGN38 and mannose-6-phosphate receptor from early/recycling endosomes to the trans-Golgi network (TGN) but does not affect the endocytic/recycling pathway of transferrin receptor or inhibit retrograde transport of CD4-furin from late endosomes to the TGN. These observations indicate that the ARF1+ARF4 and ARF1+ARF3 pairs are both required for integrity of recycling endosomes but are involved in distinct transport pathways: the former pair regulates retrograde transport from endosomes to the TGN, whereas the latter is required for the transferrin recycling pathway from endosomes to the plasma membrane.

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.

scholarly journals Targeting of the Human Immunodeficiency Virus Type 1 Envelope to the trans-Golgi Network through Binding to TIP47 Is Required for Env Incorporation into Virions and Infectivity

2003 ◽  
Vol 77 (12) ◽  
pp. 6931-6945 ◽  
Author(s):  
Guillaume Blot ◽  
Katy Janvier ◽  
Sophie Le Panse ◽  
Richard Benarous ◽  
Clarisse Berlioz-Torrent
Keyword(s):  
Human Immunodeficiency Virus ◽  
Cell Surface ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Retrograde Transport ◽  
Cytoplasmic Domain ◽  
Immunodeficiency Virus ◽  
Golgi Network ◽  
Hiv 1

ABSTRACT Here, we report that human immunodeficiency virus type 1 (HIV-1) Env glycoprotein is located mainly in the trans-Golgi network (TGN) due to determinants present in the cytoplasmic domain of the transmembrane gp41 glycoprotein (TMgp41). Internalization assays demonstrated that Env present at the cell surface returns to the TGN. We found that the cytoplasmic domain of TMgp41 binds to TIP47, a protein required for the transport of mannose-6-phosphate receptors from endosomes to the TGN. Overexpression of a mutant of TIP47 affected the transport of Env from endosomes to the TGN. Retrograde transport of Env to the TGN requires a Y802W803 diaromatic motif present in the TMgp41 cytoplasmic domain. Mutation of this motif abolished both targeting to the TGN as well as interaction with TIP47. These data support the view that binding of TIP47 to HIV-1 Env facilitates its delivery to the TGN. Lastly, we show that virus mutated in the Y802W803 motif is poorly infectious and presents a defect in Env incorporation, supporting a model in which retrograde transport of Env is implicated in the optimization of fully infectious HIV-1 production.

scholarly journals FAPP2 is involved in the transport of apical cargo in polarized MDCK cells

2005 ◽  
Vol 170 (4) ◽  
pp. 521-526 ◽  
Author(s):  
Otilia V. Vieira ◽  
Paul Verkade ◽  
Aki Manninen ◽  
Kai Simons
Keyword(s):  
Fluorescent Protein ◽  
Mdck Cells ◽  
Basolateral Membrane ◽  
Adaptor Protein ◽  
Pleckstrin Homology Domain ◽  
Transport Pathways ◽  
Protein Encoding ◽  
Phosphatidylinositol 4 ◽  
Golgi Network ◽  
Darby Canine Kidney

Phosphatidylinositol-4-phosphate (PI(4)P) is the main phosphoinositide in the Golgi complex and has been reported to play a pleiotropic role in transport of cargo from the trans-Golgi network to the plasma membrane (PM) in polarized Madin–Darby canine kidney (MDCK) cells. Overexpression of the chimeric fluorescent protein encoding the pleckstrin homology domain, which is specific for PI(4)P, inhibited both apical and basolateral transport pathways. The transport of apical cargo from the Golgi was shown to be specifically decreased by adenovirus-mediated RNA interference directed against PI(4)P adaptor protein (FAPP) 2. FAPP1 depletion had no effect on transport. On the other hand, FAPP2 was not involved in the Golgi-to-PM transport of cargo that was targeted to the basolateral membrane domain. Thus, we conclude that FAPP2 plays a specific role in apical transport in MDCK cells.

scholarly journals FIP1/RCP Binding to Golgin-97 Regulates Retrograde Transport from Recycling Endosomes to the trans-Golgi Network

2010 ◽  
Vol 21 (17) ◽  
pp. 3041-3053 ◽  
Author(s):  
Jian Jing ◽  
Jagath R. Junutula ◽  
Christine Wu ◽  
Jemima Burden ◽  
Hugo Matern ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Binding Protein ◽  
Retrograde Transport ◽  
Recycling Endosome ◽  
Transport Pathways ◽  
Recycling Endosomes ◽  
Trans Golgi Network ◽  
C Terminus ◽  
Transport Vesicles ◽  
Golgi Network

Many proteins are retrieved to the trans-Golgi Network (TGN) from the endosomal system through several retrograde transport pathways to maintain the composition and function of the TGN. However, the molecular mechanisms involved in these distinct retrograde pathways remain to be fully understood. Here we have used fluorescence and electron microscopy as well as various functional transport assays to show that Rab11a/b and its binding protein FIP1/RCP are both required for the retrograde delivery of TGN38 and Shiga toxin from early/recycling endosomes to the TGN, but not for the retrieval of mannose-6-phosphate receptor from late endosomes. Furthermore, by proteomic analysis we identified Golgin-97 as a FIP1/RCP-binding protein. The FIP1/RCP-binding domain maps to the C-terminus of Golgin-97, adjacent to its GRIP domain. Binding of FIP1/RCP to Golgin-97 does not affect Golgin-97 recruitment to the TGN, but appears to regulate the targeting of retrograde transport vesicles to the TGN. Thus, we propose that FIP1/RCP binding to Golgin-97 is required for tethering and fusion of recycling endosome-derived retrograde transport vesicles to the TGN.

scholarly journals AP-4 regulates neuronal lysosome composition, function and transport via regulating export of critical lysosome receptor proteins at the trans-Golgi network

2021 ◽  
Author(s):  
Piyali Majumder ◽  
Daisy Edmison ◽  
Catherine Rodger ◽  
Evan Reid ◽  
Swetha Gowrishankar
Keyword(s):  
Adaptor Protein ◽  
Complex Form ◽  
Retrograde Transport ◽  
Protein Traffic ◽  
Trans Golgi Network ◽  
Lysosome Biogenesis ◽  
And Function ◽  
Golgi Network ◽  
Human Ipsc ◽  
Autophagosome Maturation

The adaptor protein complex-4 or AP-4 is known to mediate autophagosome maturation through regulating sorting of transmembrane cargo such as ATG9A at the Golgi. There is a need to understand AP-4 function in neurons, as mutations in any of its four subunits cause a complex form of hereditary spastic paraplegia (HSP) with intellectual disability. While AP-4 has been implicated in regulating trafficking and distribution of cargo such as ATG9A and APP, little is known about its effect on neuronal lysosomal protein traffic, lysosome biogenesis and function. In this study, we demonstrate that in human iPSC-derived neurons AP-4 regulates lysosome composition, function and transport via regulating export of critical lysosomal receptors, including Sortilin 1, from the trans-Golgi network to endo-lysosomes. Additionally, loss of AP-4 causes endo-lysosomes to stall and build up in axonal swellings potentially through reduced recruitment of retrograde transport machinery to the organelle. These findings of axonal lysosome build-up are highly reminiscent of those observed in Alzheimer disease as well as in neurons modelling the most common form of HSP, caused by spastin mutations. Our findings implicate AP-4 as a critical regulator of neuronal lysosome biogenesis and altered lysosome function and axonal endo-lysosome transport as an underlying defect in AP-4 deficient HSP.

Steady-state localization of a medial-Golgi glycosyltransferase involves transit through the trans-Golgi network

2001 ◽  
Vol 358 (1) ◽  
pp. 33-40 ◽  
Author(s):  
Andrew S. OPAT ◽  
Fiona HOUGHTON ◽  
Paul A. GLEESON
Keyword(s):  
Steady State ◽  
Cho Cells ◽  
Chinese Hamster ◽  
Retrograde Transport ◽  
Asymmetric Distribution ◽  
Transport Pathways ◽  
Golgi Stack ◽  
Trans Golgi Network ◽  
Golgi Network

The steady-state localization of medial-Golgi enzymes is likely to involve retrograde transport pathways; however, the trafficking of these resident enzymes through the Golgi stack is unclear. To investigate if the medial-Golgi enzyme β-1,2-N-acetylglucosaminyltransferase I (GlcNAc-TI) is transported to the late Golgi, a modified GlcNAc-TI bearing an N-glycan site on the C-terminus was constructed. The modified GlcNAc-TI was demonstrated to be functionally active in vivo, and was localized to the Golgi stack of transfected cells. In stable Chinese-hamster ovary (CHO) cell clones, the N-glycosylated GlcNAc-TI carried sialylated complex N-glycan chains. Pulse-chase studies showed that the majority of GlcNAc-TI was sialylated within 60min of synthesis. Treatment of transfected CHO cells with Brefeldin A resulted in the glycosylated GlcNAc-TI bearing endo-β-N-acetylglucosaminidase H resistant chains; however, the sialylation of glycosylated GlcNAc-TI was dramatically reduced. These data imply that, in CHO cells, newly synthesized GlcNAc-TI is transported rapidly through the Golgi stack to the trans-Golgi network, suggesting that GlcNAc-TI continuously recycles from the late Golgi. Furthermore, this data suggests that retrograde transport pathways play an important role in establishing the asymmetric distribution of GlcNAc-TI within the Golgi stack.

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