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.

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.

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 Intracellular Trafficking of a Palmitoylated Membrane-Associated Protein Component of Enveloped Vaccinia Virus

2003 ◽  
Vol 77 (16) ◽  
pp. 9008-9019 ◽  
Author(s):  
Matloob Husain ◽  
Bernard Moss
Keyword(s):  
Plasma Membrane ◽  
Vaccinia Virus ◽  
Intracellular Trafficking ◽  
Plasma Membranes ◽  
Secretory Pathway ◽  
Fluorescent Protein ◽  
Dominant Negative ◽  
Coated Pits ◽  
Early Endosomes ◽  
Green Fluorescent

ABSTRACT The F13L protein of vaccinia virus, an essential and abundant palmitoylated peripheral membrane component of intra- and extracellular enveloped virions, associates with Golgi, endosomal, and plasma membranes in the presence or absence of other viral proteins. In the present study, the trafficking of a fully functional F13L-green fluorescent protein (GFP) chimera in transfected and productively infected cells was analyzed using specific markers and inhibitors. We found that Sar1H79G, a trans-dominant-negative protein inhibitor of cargo transport from the endoplasmic reticulum, had no apparent effect on the intracellular distribution of F13L-GFP, suggesting that the initial membrane localization occurs at a downstream compartment of the secretory pathway. Recycling of F13L-GFP from the plasma membrane was demonstrated by partial colocalization with FM4-64, a fluorescent membrane marker of endocytosis. Punctate F13L-GFP fluorescence overlapped with clathrin and Texas red-conjugated transferrin, suggesting that endocytosis occurred via clathrin-coated pits. The inhibitory effects of chlorpromazine and trans-dominant-negative forms of dynamin and Eps15 protein on the recycling of F13L-GFP provided further evidence for clathrin-mediated endocytosis. In addition, the F13L protein was specifically coimmunoprecipitated with α-adaptin, a component of the AP-2 complex that interacts with Eps15. Nocodazole and wortmannin perturbed the intracellular trafficking of F13L-GFP, consistent with its entry into late and early endosomes through the secretory and endocytic pathways, respectively. The recycling pathway described here provides a mechanism for the reutilization of the F13L protein following its deposition in the plasma membrane during the exocytosis of enveloped virions.

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 Septins suppress the release of vaccinia virus from infected cells

2018 ◽  
Vol 217 (8) ◽  
pp. 2911-2929 ◽  
Author(s):  
Julia Pfanzelter ◽  
Serge Mostowy ◽  
Michael Way
Keyword(s):  
Plasma Membrane ◽  
Vaccinia Virus ◽  
Membrane Trafficking ◽  
Live Cell Imaging ◽  
Actin Polymerization ◽  
Antiviral Effect ◽  
Cellular Processes ◽  
Viral Egress ◽  
Infected Cells ◽  
Cell Spread

Septins are conserved components of the cytoskeleton that play important roles in many fundamental cellular processes including division, migration, and membrane trafficking. Septins can also inhibit bacterial infection by forming cage-like structures around pathogens such as Shigella. We found that septins are recruited to vaccinia virus immediately after its fusion with the plasma membrane during viral egress. RNA interference–mediated depletion of septins increases virus release and cell-to-cell spread, as well as actin tail formation. Live cell imaging reveals that septins are displaced from the virus when it induces actin polymerization. Septin loss, however, depends on the recruitment of the SH2/SH3 adaptor Nck, but not the activity of the Arp2/3 complex. Moreover, it is the recruitment of dynamin by the third Nck SH3 domain that displaces septins from the virus in a formin-dependent fashion. Our study demonstrates that septins suppress vaccinia release by “entrapping” the virus at the plasma membrane. This antiviral effect is overcome by dynamin together with formin-mediated actin polymerization.

Intracellular Trafficking of Factor V Subsequent to Its Endocytosis by Megakaryocytes.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1031-1031
Author(s):  
Lorna W. Seifert ◽  
Alexa Wahle-Ritchie ◽  
Beth A. Bouchard ◽  
Paula B. Tracy
Keyword(s):  
Intracellular Trafficking ◽  
Retrograde Transport ◽  
Factor V ◽  
Structural Element ◽  
Trans Golgi Network ◽  
Granule Protein ◽  
Fluorescent Markers ◽  
Early Endosomes ◽  
Golgi Network ◽  
Acidic Organelles

Abstract Recent studies by our laboratory have identified physical and functional differences between plasma- and platelet-derived factor V. Additional studies indicate that the platelet-derived molecule originates from megakaryocyte endocytosis of the plasma-derived cofactor via a receptor-mediated, clathrin-dependent mechanism, and is subsequently packaged and stored in platelet α-granules. We hypothesize that plasma-derived factor V is modified intracellularly following its endocytosis by megakarycytes to generate the unique platelet-derived cofactor molecule. Thus, the time-dependent, intracellular trafficking of fluorescently-labeled factor V by the megakaryocyte-like cell line, CMK, was determined by confocal microscopy using various organelle-specific, fluorescent markers. Previously, we had demonstrated that subsequent to its endocytosis factor V partially co-localizes with two other proteins known to be endocytosed by megakaryocytes, fibrinogen, an α -granule protein, and transferrin, an iron transport protein. In the current study, we demonstrated that subsequent to their endocytosis, factor V and transferrin partially co-localized to early endosomes as determined using an antibody directed against Rab5. Complete co-localization of anti-Rab5 with an antibody against early endosomal antigen-1 (EEA-1) confirmed the specificity of the anti-Rab5 antibody for early endosomes. Endocytosed factor V was also shown to partially co-localize with von Willebrand factor, an α -granule protein that is synthesized by megakaryocytes. Its synthesis by megakaryocytes was confirmed by partial co-localization of this antibody with anti-Golgi antibodies against GM130, a structural element of the Golgi apparatus, and p230 trans, a protein involved in vesicular transport from the trans-Golgi network. Factor V also partially co-localized with these Golgi markers, consistent with the hypothesis that factor V is modified intracellularly subsequent to its endocytosis. Co-localization studies were also performed using LysoSensor Blue, which partitions into acidic organelles with a pH ~5.1 exhibiting an increase in fluorescence intensity upon acidification. Neither factor V nor transferrin co-localized with LysoSensor Blue confirming that they are not trafficked to lysosomes subsequent to their endocytosis. In conclusion, these combined observations suggest that subsequent to its endocytosis by megakaryocytes factor V is trafficked through early endosomes and ultimately stored in the α -granule with vWF and fibrinogen. Further, these data suggest that prior to its packaging into α -granules factor V may undergo retrograde transport through and O-linked glycosylation in the trans-Golgi network, which is consistent with our previous observations that purified, platelet-derived factor V contains an N-acetyl glucosamine or galactosamine at Thr402 that is not observed in its plasma counterpart.

scholarly journals TSSC1 is novel component of the endosomal retrieval machinery

2016 ◽  
Vol 27 (18) ◽  
pp. 2867-2878 ◽  
Author(s):  
David C. Gershlick ◽  
Christina Schindler ◽  
Yu Chen ◽  
Juan S. Bonifacino
Keyword(s):  
Plasma Membrane ◽  
Affinity Purification ◽  
Critical Role ◽  
Gel Filtration ◽  
Retrograde Transport ◽  
Recycling Endosomes ◽  
B Subunit ◽  
Multiple Protein ◽  
Early Endosomes ◽  
Golgi Network

Endosomes function as a hub for multiple protein-sorting events, including retrograde transport to the trans-Golgi network (TGN) and recycling to the plasma membrane. These processes are mediated by tubular-vesicular carriers that bud from early endosomes and fuse with a corresponding acceptor compartment. Two tethering complexes named GARP (composed of ANG2, VPS52, VPS53, and VPS54 subunits) and EARP (composed of ANG2, VPS52, VPS53, and Syndetin subunits) were previously shown to participate in SNARE-dependent fusion of endosome-derived carriers with the TGN and recycling endosomes, respectively. Little is known, however, about other proteins that function with GARP and EARP in these processes. Here we identify a protein named TSSC1 as a specific interactor of both GARP and EARP and as a novel component of the endosomal retrieval machinery. TSSC1 is a predicted WD40/β-propeller protein that coisolates with both GARP and EARP in affinity purification, immunoprecipitation, and gel filtration analyses. Confocal fluorescence microscopy shows colocalization of TSSC1 with both GARP and EARP. Silencing of TSSC1 impairs transport of internalized Shiga toxin B subunit to the TGN, as well as recycling of internalized transferrin to the plasma membrane. Fluorescence recovery after photobleaching shows that TSSC1 is required for efficient recruitment of GARP to the TGN. These studies thus demonstrate that TSSC1 plays a critical role in endosomal retrieval pathways as a regulator of both GARP and EARP function.

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 Domains within the GARP Subunit Vps54 Confer Separate Functions in Complex Assembly and Early Endosome Recognition

2006 ◽  
Vol 17 (4) ◽  
pp. 1859-1870 ◽  
Author(s):  
Nicole R. Quenneville ◽  
Tzu-Yuan Chao ◽  
J. Michael McCaffery ◽  
Elizabeth Conibear
Keyword(s):  
Retrograde Transport ◽  
Complex Assembly ◽  
Terminal Domain ◽  
Late Endosomes ◽  
Early Endosomes ◽  
Donor And Acceptor ◽  
Single Complex ◽  
A Subunit ◽  
Golgi Network ◽  
Garp Complex

Tethering complexes contribute to the specificity of membrane fusion by recognizing organelle features on both donor and acceptor membranes. The Golgi-associated retrograde protein (GARP) complex is required for retrograde traffic from both early and late endosomes to the trans-Golgi network (TGN), presenting a paradox as to how a single complex can interact specifically with vesicles from multiple upstream compartments. We have found that a subunit of the GARP complex, Vps54, can be separated into N- and C-terminal regions that have different functions. Whereas the N-terminus of Vps54 is important for GARP complex assembly and stability, a conserved C-terminal domain mediates localization to an early endocytic compartment. Mutation of this C-terminal domain has no effect on retrograde transport from late endosomes. However, a specific defect in retrieval of Snc1 from early endosomes is observed when recycling from late endosomes to the Golgi is blocked. These data suggest that separate domains recruit tethering complexes to different upstream compartments to regulate individual trafficking pathways.

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