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 The trans Golgi Network Is Lost from Cells Infected with African Swine Fever Virus

2001 ◽  
Vol 75 (23) ◽  
pp. 11755-11765 ◽  
Author(s):  
Mari McCrossan ◽  
Miriam Windsor ◽  
Sreenivasan Ponnambalam ◽  
John Armstrong ◽  
Thomas Wileman
Keyword(s):  
Membrane Proteins ◽  
Secretory Pathway ◽  
African Swine Fever Virus ◽  
African Swine Fever ◽  
Fever Virus ◽  
Host Proteins ◽  
Viral Membrane ◽  
Membrane Compartments ◽  
Hydrophobic Amino Acids ◽  
Golgi Network

ABSTRACT The cellular secretory pathway is important during the assembly and envelopment of viruses and also controls the transport of host proteins, such as cytokines and major histocompatibility proteins, that function during the elimination of viruses by the immune system. African swine fever virus (ASFV) encodes at least 26 proteins with stretches of hydrophobic amino acids suggesting entry into the secretory pathway (R. J. Yanez, J. M. Rodriguez, M. L. Nogal, L. Yuste, C. Enriquez, J. F. Rodriguez, and E. Vinuela, Virology 208:249–278, 1995). To predict how and where these potential membrane proteins function, we have studied the integrity of the secretory pathway in cells infected with ASFV. Remarkably, ASFV caused complete loss of immunofluorescence signal for the trans Golgi network (TGN) marker protein TGN46 and dispersed the AP1 TGN adapter complex. Loss of TGN46 signal was not due to degradation of TGN46, suggesting redistribution of TGN46 to other membrane compartments. ASFV markedly slowed transport of cathepsin D to lysosomes, demonstrating that loss of TGN structure correlated with loss of TGN function. ASFV shows a tropism for macrophages, and it is possible that ASFV compromises TGN function to augment the activity of viral membrane proteins or to suppress the function of host immunoregulatory proteins.

scholarly journals ER-Associated Degradation of Membrane Proteins in Yeast

2006 ◽  
Vol 6 ◽  
pp. 967-983 ◽  
Author(s):  
Cédric Pety de Thozée ◽  
Michel Ghislain
Keyword(s):  
Quality Control ◽  
Membrane Proteins ◽  
Control Systems ◽  
Protein Unfolding ◽  
Secretory Pathway ◽  
Retrograde Transport ◽  
26S Proteasome ◽  
Post Translational Modifications ◽  
Final Destination ◽  
Polypeptide Folding

Proteins destined for the secretory pathway are translocated into the endoplasmic reticulum (ER), where they are subjected to a variety of post-translational modifications before they reach their final destination. Newly synthesized proteins that have defect in polypeptide folding or subunit assembly are recognized by quality control systems and eliminated by the 26S proteasome, a cytosolic ATP-dependent proteolytic machinery. Delivery of non-native ER proteins to the proteasome requires retrograde transport across the ER membrane and depends on a protein-unfolding machine consisting of Cdc48p, Ufd1p, and Npl4p. Recent studies in yeast have highlighted the possible function of the Sar1p/COPII machinery in ER-associated degradation of some lumenal and membrane proteins.

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.

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.

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 Regulation of retromer recruitment to endosomes by sequential action of Rab5 and Rab7

2008 ◽  
Vol 183 (3) ◽  
pp. 513-526 ◽  
Author(s):  
Raul Rojas ◽  
Thijs van Vlijmen ◽  
Gonzalo A. Mardones ◽  
Yogikala Prabhu ◽  
Adriana L. Rojas ◽  
...  
Keyword(s):  
Cathepsin D ◽  
Retrograde Transport ◽  
Phosphatidylinositol 3 Kinase ◽  
Sorting Nexin ◽  
Retromer Complex ◽  
Endosomal Membranes ◽  
Guanosine Triphosphatase ◽  
Vacuolar Protein ◽  
Golgi Network ◽  
Phosphatidylinositol 3

The retromer complex mediates retrograde transport of transmembrane cargo from endosomes to the trans-Golgi network (TGN). Mammalian retromer is composed of a sorting nexin (SNX) dimer that binds to phosphatidylinositol 3-phosphate–enriched endosomal membranes and a vacuolar protein sorting (Vps) 26/29/35 trimer that participates in cargo recognition. The mammalian SNX dimer is necessary but not sufficient for recruitment of the Vps26/29/35 trimer to membranes. In this study, we demonstrate that the guanosine triphosphatase Rab7 contributes to this recruitment. The Vps26/29/35 trimer specifically binds to Rab7–guanosine triphosphate (GTP) and localizes to Rab7-containing endosomal domains. Interference with Rab7 function causes dissociation of the Vps26/29/35 trimer but not the SNX dimer from membranes. This blocks retrieval of mannose 6-phosphate receptors to the TGN and impairs cathepsin D sorting. Rab5-GTP does not bind to the Vps26/29/35 trimer, but perturbation of Rab5 function causes dissociation of both the SNX and Vps26/29/35 components from membranes through inhibition of a pathway involving phosphatidylinositol 3-kinase. These findings demonstrate that Rab5 and Rab7 act in concert to regulate retromer recruitment to endosomes.

scholarly journals Recycling endosomes can serve as intermediates during transport from the Golgi to the plasma membrane of MDCK cells

2004 ◽  
Vol 167 (3) ◽  
pp. 531-543 ◽  
Author(s):  
Agnes Lee Ang ◽  
Tomohiko Taguchi ◽  
Stephen Francis ◽  
Heike Fölsch ◽  
Lindsay J. Murrells ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Secretory Pathway ◽  
Mdck Cells ◽  
Basolateral Membrane ◽  
Cell Fractionation ◽  
Recycling Endosomes ◽  
Glycoprotein G ◽  
Clathrin Adaptor ◽  
Dependent Transport ◽  
Golgi Network

The AP-1B clathrin adaptor complex is responsible for the polarized transport of many basolateral membrane proteins in epithelial cells. Localization of AP-1B to recycling endosomes (REs) along with other components (exocyst subunits and Rab8) involved in AP-1B–dependent transport suggested that RE might be an intermediate between the Golgi and the plasma membrane. Although the involvement of endosomes in the secretory pathway has long been suspected, we now present direct evidence using four independent methods that REs play a role in basolateral transport in MDCK cells. Newly synthesized AP-1B–dependent cargo, vesicular stomatitis virus glycoprotein G (VSV-G), was found by video microscopy, immunoelectron microscopy, and cell fractionation to enter transferrin-positive REs within a few minutes after exit from the trans-Golgi network. Although transient, RE entry appears essential because enzymatic inactivation of REs blocked VSV-G delivery to the cell surface. Because an apically targeted VSV-G mutant behaved similarly, these results suggest that REs not only serve as an intermediate but also as a common site for polarized sorting on the endocytic and secretory pathways.

scholarly journals Vps52p, Vps53p, and Vps54p Form a Novel Multisubunit Complex Required for Protein Sorting at the Yeast Late Golgi

2000 ◽  
Vol 11 (1) ◽  
pp. 305-323 ◽  
Author(s):  
Elizabeth Conibear ◽  
Tom H. Stevens
Keyword(s):  
Membrane Proteins ◽  
Membrane Trafficking ◽  
Secretory Pathway ◽  
Protein Sorting ◽  
Retrograde Transport ◽  
Carboxypeptidase Y ◽  
Golgi Membrane ◽  
Triple Mutant ◽  
Protein Traffic ◽  
Golgi Compartment

The late Golgi of the yeast Saccharomyces cerevisiaereceives membrane traffic from the secretory pathway as well as retrograde traffic from post-Golgi compartments, but the machinery that regulates these vesicle-docking and fusion events has not been characterized. We have identified three components of a novel protein complex that is required for protein sorting at the yeast late Golgi compartment. Mutation of VPS52, VPS53, orVPS54 results in the missorting of 70% of the vacuolar hydrolase carboxypeptidase Y as well as the mislocalization of late Golgi membrane proteins to the vacuole, whereas protein traffic through the early part of the Golgi complex is unaffected. Avps52/53/54 triple mutant strain is phenotypically indistinguishable from each of the single mutants, consistent with the model that all three are required for a common step in membrane transport. Native coimmunoprecipitation experiments indicate that Vps52p, Vps53p, and Vps54p are associated in a 1:1:1 complex that sediments as a single peak on sucrose velocity gradients. This complex, which exists both in a soluble pool and as a peripheral component of a membrane fraction, colocalizes with markers of the yeast late Golgi by immunofluorescence microscopy. Together, the phenotypic and biochemical data suggest that VPS52, VPS53, andVPS54 are required for the retrograde transport of Golgi membrane proteins from an endosomal/prevacuolar compartment. The Vps52/53/54 complex joins a growing list of distinct multisubunit complexes that regulate membrane-trafficking events.

Targeting of Shiga Toxin B-Subunit to Retrograde Transport Route in Association with Detergent-resistant Membranes

2001 ◽  
Vol 12 (8) ◽  
pp. 2453-2468 ◽  
Author(s):  
Thomas Falguières ◽  
Frédéric Mallard ◽  
Carole Baron ◽  
Daniel Hanau ◽  
Clifford Lingwood ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Hela Cells ◽  
Shiga Toxin ◽  
Secretory Pathway ◽  
Retrograde Transport ◽  
Toxin B ◽  
B Subunit ◽  
Early Endosomes ◽  
Golgi Network ◽  
Triton X

In HeLa cells, Shiga toxin B-subunit is transported from the plasma membrane to the endoplasmic reticulum, via early endosomes and the Golgi apparatus, circumventing the late endocytic pathway. We describe here that in cells derived from human monocytes, i.e., macrophages and dendritic cells, the B-subunit was internalized in a receptor-dependent manner, but retrograde transport to the biosynthetic/secretory pathway did not occur and part of the internalized protein was degraded in lysosomes. These differences correlated with the observation that the B-subunit associated with Triton X-100-resistant membranes in HeLa cells, but not in monocyte-derived cells, suggesting that retrograde targeting to the biosynthetic/secretory pathway required association with specialized microdomains of biological membranes. In agreement with this hypothesis we found that in HeLa cells, the B-subunit resisted extraction by Triton X-100 until its arrival in the target compartments of the retrograde pathway, i.e., the Golgi apparatus and the endoplasmic reticulum. Furthermore, destabilization of Triton X-100-resistant membranes by cholesterol extraction potently inhibited B-subunit transport from early endosomes to thetrans-Golgi network, whereas under the same conditions, recycling of transferrin was not affected. Our data thus provide first evidence for a role of lipid asymmetry in membrane sorting at the interface between early endosomes and the trans-Golgi network.

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