scholarly journals Diacylglycerol Is Required for the Formation of COPI Vesicles in the Golgi-to-ER Transport Pathway

2007 ◽  
Vol 18 (9) ◽  
pp. 3250-3263 ◽  
Author(s):  
Inés Fernández-Ulibarri ◽  
Montserrat Vilella ◽  
Francisco Lázaro-Diéguez ◽  
Elisabet Sarri ◽  
Susana E. Martínez ◽  
...  
Keyword(s):  
Secretory Pathway ◽  
Electron Tomography ◽  
Critical Role ◽  
Retrograde Transport ◽  
Transport Pathway ◽  
Surface Transport ◽  
Early Secretory Pathway ◽  
Membrane Fission ◽  
Functional Relevance ◽  
Golgi Network

Diacylglycerol is necessary for trans-Golgi network (TGN) to cell surface transport, but its functional relevance in the early secretory pathway is unclear. Although depletion of diacylglycerol did not affect ER-to-Golgi transport, it led to a redistribution of the KDEL receptor to the Golgi, indicating that Golgi-to-ER transport was perturbed. Electron microscopy revealed an accumulation of COPI-coated membrane profiles close to the Golgi cisternae. Electron tomography showed that the majority of these membrane profiles originate from coated buds, indicating a block in membrane fission. Under these conditions the Golgi-associated pool of ARFGAP1 was reduced, but there was no effect on the binding of coatomer or the membrane fission protein CtBP3/BARS to the Golgi. The addition of 1,2-dioctanoyl-sn-glycerol or the diacylglycerol analogue phorbol 12,13-dibutyrate reversed the effects of endogenous diacylglycerol depletion. Our findings implicate diacylglycerol in the retrograde transport of proteins from Golgi to the ER and suggest that it plays a critical role at a late stage of COPI vesicle formation.

The trials and tubule-ations of Rab6 involvement in Golgi-to-ER retrograde transport

2014 ◽  
Vol 42 (5) ◽  
pp. 1453-1459 ◽  
Author(s):  
Linda F. Heffernan ◽  
Jeremy C. Simpson
Keyword(s):  
Secretory Pathway ◽  
Size Composition ◽  
Retrograde Transport ◽  
Retrograde Flow ◽  
Transport Pathway ◽  
Membrane Flow ◽  
Transport Pathways ◽  
Complex Needs ◽  
Early Secretory Pathway ◽  
Regulatory Machinery

In the early secretory pathway, membrane flow in the anterograde direction from the endoplasmic reticulum (ER) to the Golgi complex needs to be tightly co-ordinated with retrograde flow to maintain the size, composition and functionality of these two organelles. At least two mechanisms of transport move material in the retrograde direction: one regulated by the cytoplasmic coatomer protein I complex (COPI), and a second COPI-independent pathway utilizing the small GTP-binding protein Rab6. Although the COPI-independent pathway was discovered 15 years ago, it remains relatively poorly characterized, with only a handful of machinery molecules associated with its operation. One feature that makes this pathway somewhat unusual, and potentially difficult to study, is that the transport carriers predominantly seem to be tubular rather than vesicular in nature. This suggests that the regulatory machinery is likely to be different from that associated with vesicular transport pathways controlled by conventional coat complexes. In the present mini-review, we have highlighted the key experiments that have characterized this transport pathway so far and also have discussed the challenges that lie ahead with respect to its further characterization.

scholarly journals The transmembrane protein p23 contributes to the organization of the Golgi apparatus

2000 ◽  
Vol 113 (6) ◽  
pp. 1043-1057 ◽  
Author(s):  
M. Rojo ◽  
G. Emery ◽  
V. Marjomaki ◽  
A.W. McDowall ◽  
R.G. Parton ◽  
...  
Keyword(s):  
Golgi Apparatus ◽  
Secretory Pathway ◽  
Transmembrane Protein ◽  
Ectopic Expression ◽  
Retrograde Transport ◽  
Early Secretory Pathway ◽  
Golgi Membranes ◽  
Constant Diameter ◽  
Golgi Network

In previous studies we have shown that p23, a member of the p24-family of small transmembrane proteins, is highly abundant in membranes of the cis-Golgi network (CGN), and is involved in sorting/trafficking in the early secretory pathway. In the present study, we have further investigated the role of p23 after ectopic expression. We found that ectopically expressed p23 folded and oligomerized properly, even after overexpression. However, in contrast to endogenous p23, exogenous p23 molecules did not localize to the CGN, but induced a significant expansion of characteristic smooth ER membranes, where they accumulated in high amounts. This ER-derived, p23-rich subdomain displayed a highly regular morphology, consisting of tubules and/or cisternae of constant diameter, which were reminiscent of the CGN membranes containing p23 in control cells. The expression of exogenous p23 also led to the specific relocalization of endogenous p23, but not of other proteins, to these specialized ER-derived membranes. Relocalization of p23 modified the ultrastructure of the CGN and Golgi membranes, but did not affect anterograde and retrograde transport reactions to any significant extent. We conclude (i) that p23 has a morphogenic activity that contributes to the morphology of CGN-membranes; and (ii) that the presence of p23 in the CGN is necessary for the proper organization of the Golgi apparatus.

scholarly journals Requirement for Neo1p in Retrograde Transport from the Golgi Complex to the Endoplasmic Reticulum

2003 ◽  
Vol 14 (12) ◽  
pp. 4971-4983 ◽  
Author(s):  
Zhaolin Hua ◽  
Todd R. Graham
Keyword(s):  
Endoplasmic Reticulum ◽  
Golgi Complex ◽  
Secretory Pathway ◽  
Protein Transport ◽  
Retrograde Transport ◽  
Transport Pathway ◽  
Transport Defect ◽  
Copii Vesicles ◽  
P Type ◽  
Adp Ribosylation Factor

Neo1p from Saccharomyces cerevisiae is an essential P-type ATPase and potential aminophospholipid translocase (flippase) in the Drs2p family. We have previously implicated Drs2p in protein transport steps in the late secretory pathway requiring ADP-ribosylation factor (ARF) and clathrin. Here, we present evidence that epitope-tagged Neo1p localizes to the endoplasmic reticulum (ER) and Golgi complex and is required for a retrograde transport pathway between these organelles. Using conditional alleles of NEO1, we find that loss of Neo1p function causes cargo-specific defects in anterograde protein transport early in the secretory pathway and perturbs glycosylation in the Golgi complex. Rer1-GFP, a protein that cycles between the ER and Golgi complex in COPI and COPII vesicles, is mislocalized to the vacuole in neo1-ts at the nonpermissive temperature. These phenotypes suggest that the anterograde protein transport defect is a secondary consequence of a defect in a COPI-dependent retrograde pathway. We propose that loss of lipid asymmetry in the cis Golgi perturbs retrograde protein transport to the ER.

scholarly journals A Novel Mechanism for Localizing Membrane Proteins to YeastTrans-Golgi Network Requires Function of Synaptojanin-like Protein

2001 ◽  
Vol 12 (10) ◽  
pp. 3175-3190 ◽  
Author(s):  
Seon-Ah Ha ◽  
Jeremy T. Bunch ◽  
Hiroko Hama ◽  
Daryll B. DeWald ◽  
Steven F. Nothwehr
Keyword(s):  
Membrane Proteins ◽  
Secretory Pathway ◽  
Protein A ◽  
Retrograde Transport ◽  
Cell Fractionation ◽  
Gene Encoding ◽  
Phosphoinositide Phosphatase ◽  
Endosomal Membranes ◽  
Delivery Mechanism ◽  
Golgi Network

Localization of resident membrane proteins to the yeasttrans-Golgi network (TGN) involves both their retrieval from a prevacuolar/endosomal compartment (PVC) and a “slow delivery” mechanism that inhibits their TGN-to-PVC transport. A screen for genes required for the slow delivery mechanism uncoveredINP53, a gene encoding a phosphoinositide phosphatase. A retrieval-defective model TGN protein, A(F→A)-ALP, was transported to the vacuole in inp53 mutants approximately threefold faster than in wild type. Inp53p appears to function in a process distinct from PVC retrieval because combining inp53 with mutations that block retrieval resulted in a much stronger phenotype than either mutation alone. In vps27 strains defective for both anterograde and retrograde transport out of the PVC, a loss of Inp53p function markedly accelerated the rate of transport of TGN residents A-ALP and Kex2p into the PVC. Inp53p function is cargo specific because a loss of Inp53p function had no effect on the rate of Vps10p transport to the PVC in vps27 cells. The rate of early secretory pathway transport appeared to be unaffected ininp53 mutants. Cell fractionation experiments suggested that Inp53p associates with Golgi or endosomal membranes. Taken together, these results suggest that a phosphoinositide signaling event regulates TGN-to-PVC transport of select cargo proteins.

scholarly journals Syntaxin 5-Dependent Retrograde Transport to thetrans-Golgi Network Is Required for Adeno-Associated Virus Transduction

2014 ◽  
Vol 89 (3) ◽  
pp. 1673-1687 ◽  
Author(s):  
Mathieu E. Nonnenmacher ◽  
Jean-Christophe Cintrat ◽  
Daniel Gillet ◽  
Thomas Weber
Keyword(s):  
Gene Delivery ◽  
Golgi Apparatus ◽  
Intracellular Transport ◽  
Brefeldin A ◽  
Retrograde Transport ◽  
Transport Pathway ◽  
Adeno Associated Virus ◽  
Viral Attachment ◽  
Golgi Network ◽  
Endosomal System

ABSTRACTIntracellular transport of recombinant adeno-associated virus (AAV) is still incompletely understood. In particular, the trafficking steps preceding the release of incoming AAV particles from the endosomal system into the cytoplasm, allowing subsequent nuclear import and the initiation of gene expression, remain to be elucidated fully. Others and we previously showed that a significant proportion of viral particles are transported to the Golgi apparatus and that Golgi apparatus disruption caused by the drug brefeldin A efficiently blocks AAV serotype 2 (AAV2) transduction. However, because brefeldin A is known to exert pleiotropic effects on the entire endosomal system, the functional relevance of transport to the Golgi apparatus for AAV transduction remains to be established definitively. Here, we show that AAV2 trafficking toward thetrans-Golgi network (TGN) and the Golgi apparatus correlates with transduction efficiency and relies on a nonclassical retrograde transport pathway that is independent of the retromer complex, late endosomes, and recycling endosomes. AAV2 transduction is unaffected by the knockdown of syntaxins 6 and 16, which are two major effectors in the retrograde transport of both exogenous and endogenous cargo. On the other hand, inhibition of syntaxin 5 function by small interfering RNA silencing or treatment with cyclized Retro-2 strongly decreases AAV2 transduction and transport to the Golgi apparatus. This inhibition of transduction is observed with several AAV serotypes and a number of primary and immortalized cells. Together, our data strongly suggest that syntaxin 5-mediated retrograde transport to the Golgi apparatus is a broadly conserved feature of AAV trafficking that appears to be independent of the identity of the receptors used for viral attachment.IMPORTANCEGene therapy constitutes a promising approach for the treatment of life-threatening conditions refractory to any other form of remedy. Adeno-associated virus (AAV) vectors are currently being evaluated for the treatment of diseases such as Duchenne muscular dystrophy, hemophilia, heart failure, Parkinson's disease, and others. Despite their promise as gene delivery vehicles, a better understanding of the biology of AAV-based vectors is necessary to improve further their efficacy. AAV vectors must reach the nucleus in order to deliver their genome, and their intracellular transport is not fully understood. Here, we dissect an important step of the intracellular journey of AAV by showing that retrograde transport of capsids to thetrans-Golgi network is necessary for gene delivery. We show that the AAV trafficking route differs from that of known Golgi apparatus-targeted cargos, and we raise the possibility that this nonclassical pathway is shared by most AAV variants, regardless of their attachment receptors.

scholarly journals Molecular basis for KDEL-mediated retrieval of escaped ER-resident proteins – SWEET talking the COPs

2020 ◽  
Vol 133 (19) ◽  
pp. jcs250100
Author(s):  
Simon Newstead ◽  
Francis Barr
Keyword(s):  
Secretory Pathway ◽  
Signal Sequence ◽  
Structural Similarity ◽  
Retrograde Transport ◽  
Coated Vesicles ◽  
Early Secretory Pathway ◽  
Cargo Proteins ◽  
Protein Localisation ◽  
And Function ◽  
Trafficking Receptor

ABSTRACTProtein localisation in the cell is controlled through the function of trafficking receptors, which recognise specific signal sequences and direct cargo proteins to different locations. The KDEL receptor (KDELR) was one of the first intracellular trafficking receptors identified and plays an essential role in maintaining the integrity of the early secretory pathway. The receptor recognises variants of a canonical C-terminal Lys-Asp-Glu-Leu (KDEL) signal sequence on ER-resident proteins when these escape to the Golgi, and targets these proteins to COPI- coated vesicles for retrograde transport back to the ER. The empty receptor is then recycled from the ER back to the Golgi by COPII-coated vesicles. Crystal structures of the KDELR show that it is structurally related to the PQ-loop family of transporters that are found in both pro- and eukaryotes, and shuttle sugars, amino acids and vitamins across cellular membranes. Furthermore, analogous to PQ-loop transporters, the KDELR undergoes a pH-dependent and ligand-regulated conformational cycle. Here, we propose that the striking structural similarity between the KDELR and PQ-loop transporters reveals a connection between transport and trafficking in the cell, with important implications for understanding trafficking receptor evolution and function.

scholarly journals The Secretory Apparatus of an Ancient Eukaryote: Protein Sorting to Separate Export Pathways Occurs Before Formation of Transient Golgi-like Compartments

2003 ◽  
Vol 14 (4) ◽  
pp. 1433-1447 ◽  
Author(s):  
Matthias Marti ◽  
Yajie Li ◽  
Elisabeth M. Schraner ◽  
Peter Wild ◽  
Peter Köhler ◽  
...  
Keyword(s):  
Secretory Pathway ◽  
Protein Sorting ◽  
Protozoan Parasite ◽  
Retrograde Transport ◽  
Surface Proteins ◽  
Cyst Form ◽  
Early Secretory Pathway ◽  
Vertebrate Hosts ◽  
Er Export ◽  
Secretory Apparatus

Transmission of the protozoan parasite Giardia intestinalis to vertebrate hosts presupposes the encapsulation of trophozoites into an environmentally resistant and infectious cyst form. We have previously shown that cyst wall proteins were faithfully sorted to large encystation-specific vesicles (ESVs), despite the absence of a recognizable Golgi apparatus. Here, we demonstrate that sorting to a second constitutively active pathway transporting variant-specific surface proteins (VSPs) to the surface depended on the cytoplasmic VSP tail. Moreover, pulsed endoplasmic reticulum (ER) export of chimeric reporters containing functional signals for both pathways showed that protein sorting was done at or very soon after export from the ER. Correspondingly, we found that a limited number of novel transitional ER-like structures together with small transport intermediates were generated during encystation. Colocalization of transitional ER regions and early ESVs with coat protein (COP) II and of maturing ESVs with COPI and clathrin strongly suggested that ESVs form by fusion of ER-derived vesicles and subsequently undergo maturation by retrograde transport. Together, the data supported the hypothesis that in Giardia, a primordial secretory apparatus is in operation by which proteins are sorted in the early secretory pathway, and the developmentally induced ESVs carry out at least some Golgi functions.

scholarly journals Retrograde Transport from Early Endosomes to thetrans-Golgi Network Enables Membrane Wrapping and Egress of Vaccinia Virus Virions

2016 ◽  
Vol 90 (19) ◽  
pp. 8891-8905 ◽  
Author(s):  
Gilad Sivan ◽  
Andrea S. Weisberg ◽  
Jeffrey L. Americo ◽  
Bernard Moss
Keyword(s):  
Vaccinia Virus ◽  
Actin Polymerization ◽  
Secretory Pathway ◽  
Retrograde Transport ◽  
Double Membrane ◽  
Retrograde Trafficking ◽  
Early Endosomes ◽  
Trafficking Inhibitor ◽  
Golgi Network ◽  
Cell Spread

ABSTRACTThe anterograde pathway, from the endoplasmic reticulum through thetrans-Golgi network to the cell surface, is utilized bytrans-membrane and secretory proteins. The retrograde pathway, which directs traffic in the opposite direction, is used following endocytosis of exogenous molecules and recycling of membrane proteins. Microbes exploit both routes: viruses typically use the anterograde pathway for envelope formation prior to exiting the cell, whereas ricin and Shiga-like toxins and some nonenveloped viruses use the retrograde pathway for cell entry. Mining a human genome-wide RNA interference (RNAi) screen revealed a need for multiple retrograde pathway components for cell-to-cell spread of vaccinia virus. We confirmed and extended these results while discovering that retrograde trafficking was required for virus egress rather than entry. Retro-2, a specific retrograde trafficking inhibitor of protein toxins, potently prevented spread of vaccinia virus as well as monkeypox virus, a human pathogen. Electron and confocal microscopy studies revealed that Retro-2 prevented wrapping of virions with an additional double-membrane envelope that enables microtubular transport, exocytosis, and actin polymerization. The viral B5 and F13 protein components of this membrane, which are required for wrapping, normally colocalize in thetrans-Golgi network. However, only B5 traffics through the secretory pathway, suggesting that F13 uses another route to thetrans-Golgi network. The retrograde route was demonstrated by finding that F13 was largely confined to early endosomes and failed to colocalize with B5 in the presence of Retro-2. Thus, vaccinia virus makes novel use of the retrograde transport system for formation of the viral wrapping membrane.IMPORTANCEEfficient cell-to-cell spread of vaccinia virus and other orthopoxviruses depends on the wrapping of infectious particles with a double membrane that enables microtubular transport, exocytosis, and actin polymerization. Interference with wrapping or subsequent steps results in severe attenuation of the virus. Some previous studies had suggested that the wrapping membrane arises from thetrans-Golgi network, whereas others suggested an origin from early endosomes. Some nonenveloped viruses use retrograde trafficking for entry into the cell. In contrast, we provided evidence that retrograde transport from early endosomes to thetrans-Golgi network is required for the membrane-wrapping step in morphogenesis of vaccinia virus and egress from the cell. The potentin vitroinhibition of this step by the drug Retro-2 suggests that derivatives with enhanced pharmacological properties might serve as useful antipoxviral agents.

scholarly journals Retrograde transport defects in Munc18-1 null neurons explain abnormal Golgi morphology

2020 ◽  
Author(s):  
Annemiek A. van Berkel ◽  
Tatiana C. Santos ◽  
Hesho Shaweis ◽  
Jan R.T. van Weering ◽  
Ruud F. Toonen ◽  
...  
Keyword(s):  
Cell Death ◽  
Secretory Pathway ◽  
Retrograde Transport ◽  
Cellular Mechanisms ◽  
Anterograde Transport ◽  
Synaptic Vesicle Exocytosis ◽  
Vesicle Exocytosis ◽  
Regulated Secretory Pathway ◽  
Cellular Phenotypes ◽  
Golgi Network

AbstractLoss of the exocytic Sec1/MUNC18 protein MUNC18-1 or its t-SNARE partners SNAP25 and syntaxin-1 results in rapid, cell-autonomous and unexplained neurodegeneration, which is independent of their known role in synaptic vesicle exocytosis. cis-Golgi abnormalities are the earliest cellular phenotypes before degeneration occurs. Here, we investigated whether these Golgi abnormalities cause defects in the constitutive and regulated secretory pathway that may explain neurodegeneration. Electron microscopy confirmed that loss of MUNC18-1 expression results in a smaller cis-Golgi. In addition, we now show that medial-Golgi and the trans-Golgi Network are also affected. However, stacking and cisternae ultrastructure of the Golgi were normal. Overall ultrastructure of null mutant neurons was remarkably normal just hours before cell death occurred. Anterograde ER-to-Golgi and Golgi exit of endogenous and exogenous proteins were normal. In contrast, loss of MUNC18-1 caused reduced retrograde Cholera Toxin transport from the plasma membrane to the Golgi. In addition, MUNC18-1-deficiency resulted in abnormalities in retrograde TrkB trafficking. We conclude that MUNC18-1 deficient neurons have normal anterograde yet reduced retrograde transport to the Golgi. This imbalance in transport routes provides a plausible explanation for the observed Golgi abnormalities and cell death in MUNC18-1 deficient neurons.Significance statementLoss of MUNC18-1 or its t-SNAREs SNAP25 and syntaxin-1 leads to massive, yet unexplained, neurodegeneration. Previous research showed that Golgi abnormalities are the earliest, shared phenotype. Golgi abnormalities are also an early feature in neurodegenerative diseases, such as Alzheimer’s Disease or Amyotrophic Lateral Sclerosis. This study elucidates the mechanism underlying the Golgi phenotype upon loss of MUNC18-1. By systematically assessing transport routes to and from the Golgi, we show that retrograde endosome-to-Golgi, but not anterograde transport from the Golgi, is disturbed. This imbalance in transport routes provides a plausible explanation for the Golgi phenotype, and may explain the neurodegeneration. The findings in this study contributes to new insights in cellular mechanisms of neurodegeneration.

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