Immobilization of the early secretory pathway by a virus glycoprotein that binds to microtubules

Membrane trafficking from the endoplasmic reticulum (ER) to the Golgi complex is mediated by pleiomorphic carrier vesicles that are driven along microtubule tracks by the action of motor proteins. Here we describe how NSP4, a rotavirus membrane glycoprotein, binds to microtubules and blocks ER-to-Golgi trafficking in vivo. NSP4 accumulates in a post-ER, microtubule-associated membrane compartment and prevents targeting of vesicular stomatitis virus glycoprotein (VSV-G) at a pre-Golgi step. NSP4 also redistributes beta-COP and ERGIC53, markers of a vesicular compartment that dynamically cycles between the ER and Golgi, to structures aligned along linear tracks radiating throughout the cytoplasm. This block in membrane trafficking is released when microtubules are depolymerized with nocodazole, indicating that vesicles containing NSP4 are tethered to the microtubule cytoskeleton. Disruption of microtubule-mediated membrane transport by a viral glycoprotein may represent a novel pathogenic mechanism and provides a new experimental tool for the dissection of early steps in exocytic transport.     

Caveolin cycles between plasma membrane caveolae and the Golgi complex by microtubule-dependent and microtubule-independent steps

Caveolin is a protein associated with the characteristic coats that decorate the cytoplasmic face of plasma membrane caveolae. Recently it was found that exposure of human fibroblasts to cholesterol oxidase (CO) rapidly induces caveolin to redistribute to the ER and then to the Golgi complex, and that subsequent removal of CO allows caveolin to return to the plasma membrane (Smart, E. J., Y.-S. Ying, P. A. Conrad, R. G. W. Anderson, J. Cell Biol. 1994, 127:1185-1197). We now present evidence that caveolin normally undergoes microtubule-dependent cycling between the plasma membrane and the Golgi. In cells that were treated briefly with nocodazole and then with a mixture of nocodazole plus CO, caveolin relocated from the plasma membrane to the ER and then to the ER/Golgi intermediate compartment (ERGIC), but subsequent movement to the Golgi was not observed. Even in the absence of CO, nocodazole caused caveolin to accumulate in the ERGIC. Nocodazole did not retard the movement of caveolin from the Golgi to the plasma membrane after removal of CO. Incubation of cells at 15 degrees followed by elevation of the temperature to 37 degrees caused caveolin to accumulate first in the ERGIC and then in the Golgi, before finally reestablishing its normal steady state distribution predominantly in plasma membrane caveolae. In cells released from a 15 degrees block, movement of caveolin from the Golgi to the plasma membrane was not inhibited by nocodazole. Taken together, these results imply that caveolin cycles constitutively between the plasma membrane and the Golgi by a multi- step process, one of which, ERGIC-to-Golgi transport, requires microtubules. This novel, bidirectional pathway may indicate roles for microtubules in the maintenance of caveolae, and for caveolin in shuttling fatty acids and cholesterol between the plasma membrane and the ER/Golgi system.     

Syntaxin 17 cycles between the ER and ERGIC and is required to maintain the architecture of ERGIC and Golgi

Background information. Syntaxin 17 is a SNARE (soluble N‐ethylmaleimide‐sensitive‐factor‐attachment protein receptor) protein that predominantly localizes to the ER (endoplasmic reticulum) and to some extent in the ERGIC (ER—Golgi intermediate compartment). Syntaxin 17 has been suggested to function as a receptor at the ER membrane that mediates trafficking between the ER and post‐ER compartments. It has a unique 33 amino acid luminal tail whose function is not known. Here we have investigated the structural requirements for localization of syntaxin 17 to the ERGIC and its role in trafficking. Results. Deletion analysis showed that syntaxin 17 required its cytoplasmic domain to exit the ER and localize to the ERGIC. Mutation of a conserved tyrosine residue in the cytoplasmic domain resulted in reduced localization of syntaxin 17 in the ERGIC and ER‐exit sites, suggesting the presence of a tyrosine‐based ER export motif. Syntaxin 17 also required its C‐terminal tail to localize to the ERES (ER exit sites) and ERGIC. Knockdown of syntaxin 17 destabilized the ERGIC organization and also caused fragmentation of the Golgi complex. Syntaxin 17 showed direct interaction with transmembrane proteins p23 and p25 (cargo receptors that cycle between the ER and Golgi) with the help of its C‐terminal tail. Overexpression of syntaxin 17 redistributed β‐COP (β‐coatomer protein) which required its C‐terminal tail. Overexpression of syntaxin 17 also blocked the anterograde transport of VSVG (vesicular stomatitis virus G‐protein) in the ERGIC. Conclusions. We show that syntaxin 17 has a tyrosine‐based motif which is required for its incorporation into COPII (coatomer protein II) vesicles, exit from the ER and localization to the ERGIC. Our results suggest that syntaxin 17 cycles between the ER and ERGIC through classical trafficking pathways involving COPII and COPI (coatomer protein I) vesicles, which requires its unique C‐terminal tail. We also show that syntaxin 17 is essential for maintaining the architecture of ERGIC and Golgi.     

Dynamics of transitional endoplasmic reticulum sites in vertebrate cells

A typical vertebrate cell contains several hundred sites of transitional ER (tER). Presumably, tER sites generate elements of the ER-Golgi intermediate compartment (ERGIC), and ERGIC elements then generate Golgi cisternae. Therefore, characterizing the mechanisms that influence tER distribution may shed light on the dynamic behavior of the Golgi. We explored the properties of tER sites using Sec13 as a marker protein. Fluorescence microscopy confirmed that tER sites are long-lived ER subdomains. tER sites proliferate during interphase but lose Sec13 during mitosis. Unlike ERGIC elements, tER sites move very little. Nevertheless, when microtubules are depolymerized with nocodazole, tER sites redistribute rapidly to form clusters next to Golgi structures. Hence, tER sites have the unusual property of being immobile, yet dynamic. These findings can be explained by a model in which new tER sites are created by retrograde membrane traffic from the Golgi. We propose that the tER-Golgi system is organized by mutual feedback between these two compartments.     

The cytoplasmic tail of the severe acute respiratory syndrome coronavirus spike protein contains a novel endoplasmic reticulum retrieval signal that binds COPI and promotes interaction with membrane protein

Like other coronaviruses, severe acute respiratory syndrome coronavirus (SARS CoV) assembles at and buds into the lumen of the endoplasmic reticulum (ER)-Golgi intermediate compartment (ERGIC). Accumulation of the viral envelope proteins at this compartment is a prerequisite for virus assembly. Previously, we reported the identification of a dibasic motif (KxHxx) in the cytoplasmic tail of the SARS CoV spike (S) protein that was similar to a canonical dilysine ER retrieval signal. Here we demonstrate that this motif is a novel and functional ER retrieval signal which reduced the rate of traffic of the full-length S protein through the Golgi complex. The KxHxx motif also partially retained two different reporter proteins in the ERGIC region and reduced their rates of trafficking, although the motif was less potent than the canonical dilysine signal. The dibasic motif bound the coatomer complex I (COPI) in an in vitro binding assay, suggesting that ER retrieval may contribute to the accumulation of SARS CoV S protein near the virus assembly site for interaction with other viral structural proteins. In support of this, we found that the dibasic motif on the SARS S protein was required for its localization to the ERGIC/Golgi region when coexpressed with SARS membrane (M) protein. Thus, the cycling of SARS S through the ER-Golgi system may be required for its incorporation into assembling virions in the ERGIC.     

Human cytomegalovirus pp28 (UL99) localizes to a cytoplasmic compartment which overlaps the endoplasmic reticulum-golgi-intermediate compartment

Although the assembly of herpesviruses has remained an active area of investigation, considerable controversy continues to surround the cellular location of tegument and envelope acquisition. This controversy is particularly evident when the proposed pathways for alpha- and beta-herpesvirus assembly are compared. We have approached this aspect of human cytomegalovirus (HCMV) assembly, specifically, envelopment, by investigating the intracellular trafficking of viral tegument proteins which localize in the cytoplasms of infected cells. In this study we have demonstrated that the virion tegument protein pp28 (UL99), a true late protein, was membrane associated as a result of myristoylation. A mutation in this protein which prevented incorporation of [(3)H]myristic acid also altered the detergent solubility and intracellular distribution of the protein when it was expressed in transfected cells. Using a panel of markers for intracellular compartments, we could localize the expression of wild-type pp28 to an intracellular compartment which colocalized with the endoplasmic reticulum-Golgi-intermediate compartment (ERGIC), a dynamic compartment of the secretory pathway which interfaces with both the ER and Golgi apparatus. The localization of this viral tegument protein within an early secretory compartment of the cell provided further evidence that the assembly of the HCMV tegument likely includes a cytoplasmic phase. Because pp28 has been shown to be localized to a cytoplasmic assembly compartment in HCMV-infected cells, our findings also suggested that viral tegument protein interactions within the secretory pathway may have an important role in the assembly of the virion.     

Sequence requirements for localization of human cytomegalovirus tegument protein pp28 to the virus assembly compartment and for assembly of infectious virus

The human cytomegalovirus UL99 open reading frame encodes a 190-amino-acid (aa) tegument protein, pp28, that is myristoylated and phosphorylated. pp28 is essential for assembly of infectious virus, and nonenveloped virions accumulate in the cytoplasm of cells infected with recombinant viruses with a UL99 deletion. pp28 is localized to the endoplasmic reticulum-Golgi intermediate compartment (ERGIC) in transfected cells, while in infected cells, it is localized together with other virion proteins in a juxtanuclear compartment termed the assembly compartment (AC). We investigated the sequence requirements for pp28 trafficking to the AC and assembly of infectious virus. Our studies indicated that the first 30 to 35 aa were required for localization of pp28 to the ERGIC in transfected cells. Mutant forms of pp28 containing only the first 35 aa localized with other virion structural proteins to cytoplasmic compartments early in infection, but localization to the AC at late times required a minimum of 50 aa. In agreement with previous reports, we demonstrated that the deletion of a cluster of acidic amino acids (aa 44 to 59) prevented wild-type trafficking of pp28 and recovery of infectious virus. A recombinant virus expressing only the first 50 aa was replication competent, and this mutant, pp28, localized to the AC in cells infected with this virus. These findings argued that localization of pp28 to the AC was essential for assembly of infectious virus and raised the possibility that amino acids in the amino terminus of pp28 have additional roles in the envelopment and assembly of the virion other than simply localizing pp28 to the AC.     

Microtubule-dependent retrograde transport of proteins into the ER in the presence of brefeldin A suggests an ER recycling pathway

Characteristics of brefeldin A (BFA)-induced redistribution of Golgi proteins into the endoplasmic reticulum (ER) and its relationship to an ER retrieval pathway were investigated. Retrograde movement of Golgi proteins into the ER occurred via long, tubulovesicular processes extending out of the Golgi along microtubules. Microtubule-disrupting agents (i.e., nocodazole), energy poisons, and reduced temperatures inhibited this pathway. In BFA-treated cells Golgi proteins appeared to cycle between the ER and an intermediate compartment marked by a 53 kd protein. Addition of nocodazole disrupted this dynamic cycle by preferentially inhibiting retrograde movement, causing Golgi proteins to accumulate in the intermediate compartment. In the absence of BFA, such an ER cycling pathway appeared to be followed normally by the 53 kd protein but not by Golgi proteins, as revealed by temperature shift experiments. We propose that BFA induces the interaction of the Golgi with an intermediate "recycling" compartment that utilizes a microtubule-dependent pathway into the ER.     

The Golgi apparatus maintains its organization independent of the endoplasmic reticulum

Under artificial conditions Golgi enzymes have the capacity to rapidly accumulate in the endoplasmic reticulum (ER). These observations prompted the idea that Golgi enzymes constitutively recycle through the ER. We have tested this hypothesis under physiological conditions through use of a procedure that captures Golgi enzymes in the ER. In the presence of rapamycin, which induces a tight association between FKBP (FK506-binding protein) and FRAP (FKBP-rapamycin-associated protein), an FKBP-tagged Golgi enzyme can be trapped when it visits the ER by an ER-retained protein fused to FRAP. We find that although FKBP-ERGIC-53 of the ER-Golgi intermediate compartment (ERGIC) rapidly cycles through the ER (30 min), FKBP-Golgi enzyme chimeras remain stably associated with Golgi membranes. We also demonstrate that Golgi dispersion upon nocodazole treatment mainly occurs through a mechanism that does not involve the recycling of Golgi membranes through the ER. Our findings suggest that the Golgi apparatus, as defined by its collection of resident enzymes, exists independent of the ER.     

Trafficking-deficient hERG K⁺ channels linked to long QT syndrome are regulated by a microtubule-dependent quality control compartment in the ER

The human ether-a-go-go related gene (hERG) encodes the voltage-gated K(+) channel that underlies the rapidly activating delayed-rectifier current in cardiac myocytes. hERG is synthesized in the endoplasmic reticulum (ER) as an "immature" N-linked glycoprotein and is terminally glycosylated in the Golgi apparatus. Most hERG missense mutations linked to long QT syndrome type 2 (LQT2) reduce the terminal glycosylation and functional expression. We tested the hypothesis that a distinct pre-Golgi compartment negatively regulates the trafficking of some LQT2 mutations to the Golgi apparatus. We found that treating cells in nocodazole, a microtubule depolymerizing agent, altered the subcellular localization, functional expression, and glycosylation of the LQT2 mutation G601S-hERG differently from wild-type hERG (WT-hERG). G601S-hERG quickly redistributed to peripheral compartments that partially colocalized with KDEL (Lys-Asp-Glu-Leu) chaperones but not calnexin, Sec31, or the ER golgi intermediate compartment (ERGIC). Treating cells in E-4031, a drug that increases the functional expression of G601S-hERG, prevented the accumulation of G601S-hERG to the peripheral compartments and increased G601S-hERG colocalization with the ERGIC. Coexpressing the temperature-sensitive mutant G protein from vesicular stomatitis virus, a mutant N-linked glycoprotein that is retained in the ER, showed it was not restricted to the same peripheral compartments as G601S-hERG at nonpermissive temperatures. We conclude that the trafficking of G601S-hERG is negatively regulated by a microtubule-dependent compartment within the ER. Identifying mechanisms that prevent the sorting or promote the release of LQT2 channels from this compartment may represent a novel therapeutic strategy for LQT2.     

Overexpression of the Dynamitin (p50) Subunit of the Dynactin Complex Disrupts Dynein-dependent Maintenance of Membrane Organelle Distribution

Dynactin is a multisubunit complex that plays an accessory role in cytoplasmic dynein function. Overexpression in mammalian cells of one dynactin subunit, dynamitin, disrupts the complex, resulting in dissociation of cytoplasmic dynein from prometaphase kinetochores, with consequent perturbation of mitosis (Echeverri, C.J., B.M. Paschal, K.T. Vaughan, and R.B. Vallee. 1996. J. Cell Biol. 132:617–634). Based on these results, dynactin was proposed to play a role in linking cytoplasmic dynein to kinetochores and, potentially, to membrane organelles. The current study reports on the dynamitin interphase phenotype. In dynamitin-overexpressing cells, early endosomes (labeled with antitransferrin receptor), as well as late endosomes and lysosomes (labeled with anti–lysosome-associated membrane protein-1 [LAMP-1]), were redistributed to the cell periphery. This redistribution was disrupted by nocodazole, implicating an underlying plus end–directed microtubule motor activity. The Golgi stack, monitored using sialyltransferase, galactosyltransferase, and N-acetylglucosaminyltransferase I, was dramatically disrupted into scattered structures that colocalized with components of the intermediate compartment (ERGIC-53 and ERD-2). The disrupted Golgi elements were revealed by EM to represent short stacks similar to those formed by microtubule-depolymerizing agents. Golgi-to-ER traffic of stack markers induced by brefeldin A was not inhibited by dynamitin overexpression. Time-lapse observations of dynamitin-overexpressing cells recovering from brefeldin A treatment revealed that the scattered Golgi elements do not undergo microtubule-based transport as seen in control cells, but rather, remain stationary at or near their ER exit sites. These results indicate that dynactin is specifically required for ongoing centripetal movement of endocytic organelles and components of the intermediate compartment. Results similar to those of dynamitin overexpression were obtained by microinjection with antidynein intermediate chain antibody, consistent with a role for dynactin in mediating interactions of cytoplasmic dynein with specific membrane organelles. These results suggest that dynamitin plays a pivotal role in regulating organelle movement at the level of motor–cargo binding.     

Mechanisms of procollagen and HSP47 sorting during ER-to-Golgi trafficking

Efficient quality control and export of procollagen from the cell is crucial for extracellular matrix homeostasis, yet it is still incompletely understood. One of the debated questions is the role of a collagen-specific ER chaperone HSP47 in these processes. Most ER chaperones preferentially bind to unfolded polypeptide chains, enabling selective export of natively folded proteins from the ER after chaperone release. In contrast, HSP47 preferentially binds to the natively folded procollagen and is believed to be released only in the ER-Golgi intermediate compartment (ERGIC) or cis-Golgi. HSP47 colocalization with procollagen in punctate structures observed by immunofluorescence imaging of fixed cells has thus been interpreted as evidence for HSP47 export from the ER together with procollagen in transport vesicles destined for ERGIC or Golgi. To understand the mechanism of this co-trafficking and its physiological significance, we imaged the dynamics of fluorescently tagged type I procollagen and HSP47 punctate structures in live MC3T3 murine osteoblasts with up to 120 nm spatial and 500 ms time resolution. Contrary to the prevailing model, we discovered that most bona fide carriers delivering procollagen from ER exit sites (ERESs) to Golgi contained no HSP47, unless the RDEL signal for ER retention in HSP47 was deleted or mutated. These transport intermediates exhibited characteristic rapid, directional motion along microtubules, while puncta with colocalized HSP47 and procollagen similar to the ones described before had only limited, stochastic motion. Live cell imaging and fluorescence recovery after photobleaching revealed that the latter puncta (including the ones induced by ARF1 inhibition) were dilated regions of ER lumen, ERESs, or autophagic structures surrounded by lysosomal membranes. Procollagen was colocalized with HSP47 and ERGIC53 at ERESs. It was colocalized with ERGIC53 but not HSP47 in Golgi-bound transport intermediates. Our results suggest that procollagen and HSP47 sorting occurs at ERES before procollagen is exported from the ER in Golgi-bound transport intermediates, providing new insights into mechanisms of procollagen trafficking.     

A study of the protein secretory pathway of Aspergillus niger using a glucoamylase-GFP fusion protein

The effect of various treatments that block protein secretion was visualized in Aspergillus niger using a strain expressing a glucoamylase-GFP fusion protein. Cold shock caused the retention of the fusion protein in a reticulate network (ER) with brighter nodes that may represent Golgi bodies. Treatment of germlings with brefeldin A (BFA) also initially caused accumulation within the ER but prolonged exposure led to the formation and targeting of the fusion protein to vacuoles from the ER. Disruption of actin with cytochalasin A initially led to a faint diffuse accumulation and ultimately to the formation of aggregated bodies which were not vacuoles, suggesting that the actin cytoskeleton is important in secretory vesicle transport. Disruption of microtubules with nocodazole led to hyperbranching but did not cause intracellular accumulation, suggesting that microtubules play a role in directing vesicle transport rather than vesicle movement per se. Treatment of regenerating protoplasts confirmed that BFA and cytochalasin but not nocodazole inhibited protein secretion. When germlings were subjected to carbon starvation, vacuolation was rapidly initiated throughout the hyphae and GFP fluorescence was visible in some of the vacuoles, indicating retargeting of the fusion protein from the secretory pathway to the vacuoles.     

Direct interaction of the Golgi membrane with the endoplasmic reticulum membrane caused by nordihydroguaiaretic acid

Nordihydroguaiaretic acid (NDGA), an inhibitor of lipoxygenase, blocks protein transport from the endoplasmic reticulum (ER) to the Golgi complex and induces the redistribution of Golgi proteins into the ER. We investigated characteristics of NDGA-induced retrograde movement of the Golgi proteins to the ER. At an early stage of incubation of cells with NDGA, the Golgi complex formed convoluted membrane aggregates. Electron microscopy revealed that these aggregates directly interact en bloc with the ER membrane. The direct interaction and subsequent incorporation of the Golgi proteins into the ER were found to be temperature-dependent. The protein of ER-Golgi intermediate compartment (ERGIC), ERGIC53, was rapidly accumulated in the Golgi upon treatment with NDGA. This accumulation was significantly inhibited by low temperature at 15 degrees C. Under the condition, the redistribution of the Golgi proteins into the ER as well as the direct interaction between the ER and the Golgi by NDGA were also inhibited, suggesting an important role of the ERGIC in the retrograde movement. In contrast, the low temperature did not inhibit formation of the Golgi aggregates by NDGA. Taken together, these results suggest that NDGA causes the redistribution of the Golgi proteins into the ER through the direct connections between the Golgi, the ERGIC, and the ER.     

COG-7-deficient Human Fibroblasts Exhibit Altered Recycling of Golgi Proteins

Recently, we reported that two siblings presenting with the clinical syndrome congenital disorders of glycosylation (CDG) have mutations in the gene encoding Cog7p, a member of the conserved oligomeric Golgi (COG) complex. In this study, we analyzed the localization and trafficking of multiple Golgi proteins in patient fibroblasts under a variety of conditions. Although the immunofluorescent staining pattern of several Golgi proteins was indistinguishable from normal, the staining of endoplasmic reticulum (ER)-Golgi intermediate compartment (ERGIC)-53 and the vesicular-soluble N-ethylmaleimide-sensitive factor attachment protein receptors GS15 and GS28 was abnormal, and the steady-state level of GS15 was greatly decreased. Retrograde transport of multiple Golgi proteins to the ER in patient fibroblasts via brefeldin A-induced tubules was significantly slower than occurs in normal fibroblasts, whereas anterograde protein trafficking was much less affected. After prolonged treatment with brefeldin A, several Golgi proteins were detected in clusters that colocalize with the microtubule-organizing center in patient cells. All of these abnormalities were normalized in COG7-corrected patient fibroblasts. These results serve to better define the role of the COG complex in facilitating protein trafficking between the Golgi and ER and provide a diagnostic framework for the identification of CDG defects involving trafficking proteins.     

Osmotically induced cell volume changes alter anterograde and retrograde transport, Golgi structure, and COPI dissociation

Physiological conditions that impinge on constitutive traffic and affect organelle structure are not known. We report that osmotically induced cell volume changes, which are known to occur under a variety of conditions, rapidly inhibited endoplasmic reticulum (ER)-to-Golgi transport in mammalian cells. Both ER export and ER Golgi intermediate compartment (ERGIC)-to-Golgi trafficking steps were blocked, but retrograde transport was active, and it mediated ERGIC and Golgi collapse into the ER. Extensive tubulation and relatively rapid Golgi resident redistribution were observed under hypo-osmotic conditions, whereas a slower redistribution of the same markers, without apparent tubulation, was observed under hyperosmotic conditions. The osmotic stress response correlated with the perturbation of COPI function, because both hypo- and hyperosmotic conditions slowed brefeldin A-induced dissociation of betaCOP from Golgi membranes. Remarkably, Golgi residents reemerged after several hours of sustained incubation in hypotonic or hypertonic medium. Reemergence was independent of new protein synthesis but required PKC, an activity known to mediate cell volume recovery. Taken together these results indicate the existence of a coupling between cell volume and constitutive traffic that impacts organelle structure through independent effects on anterograde and retrograde flow and that involves, in part, modulation of COPI function.     

Regulation of ER–Golgi Intermediate Compartment Tubulation and Mobility by COPI Coats,Motor Proteins and Microtubules

Little is known about the formation and regulation of endoplasmic reticulum (ER)–Golgi transport intermediates, although previous studies suggest that cargo is the main regulator of their morphology. In this study, we analyze the role of coat protein I (COPI) and cytoskeleton in the formation of tubular ER–Golgi intermediate compartment (ERGIC) and also show that partial COPI detachment by means of low temperature (15°C) or brefeldin A induces the formation of transient tubular ERGIC elements. Most of them moved from the cell periphery to the perinuclear area and were 2.5× slower than vesicles. Time‐lapse analysis of living cells demonstrates that the ERGIC elements are able to shift very fast from tubular to vesicular forms and vice versa, suggesting that the amount of cargo is not the determining factor for ERGIC morphology. Both the partial microtubule depolymerization and the inhibition of uncoating of the membranes result in the formation of long tubules that grow from round ERGICs and form at complex network. Interestingly, both COPI detachment and microtubule depolymerization induce a redistribution of kinesin from peripheral ERGIC elements to the Golgi area, while dynein distribution is not affected. However, both kinesin and dynein downregulation by RNA interference induced ERGIC tubulation. The tubules induced by kinesin depletion were static, whereas those resulting from dynein depletion were highly mobile. Our results strongly suggest that the interaction of motor proteins with COPI‐coated membranes and microtubules is a key regulator of ERGIC morphology and mobility.     

Molecular determinants and dynamics of hepatitis C virus secretion

The current model of hepatitis C virus (HCV) production involves the assembly of virions on or near the surface of lipid droplets, envelopment at the ER in association with components of VLDL synthesis, and egress via the secretory pathway. However, the cellular requirements for and a mechanistic understanding of HCV secretion are incomplete at best. We combined an RNA interference (RNAi) analysis of host factors for infectious HCV secretion with the development of live cell imaging of HCV core trafficking to gain a detailed understanding of HCV egress. RNAi studies identified multiple components of the secretory pathway, including ER to Golgi trafficking, lipid and protein kinases that regulate budding from the trans-Golgi network (TGN), VAMP1 vesicles and adaptor proteins, and the recycling endosome. Our results support a model wherein HCV is infectious upon envelopment at the ER and exits the cell via the secretory pathway. We next constructed infectious HCV with a tetracysteine (TC) tag insertion in core (TC-core) to monitor the dynamics of HCV core trafficking in association with its cellular cofactors. In order to isolate core protein movements associated with infectious HCV secretion, only trafficking events that required the essential HCV assembly factor NS2 were quantified. TC-core traffics to the cell periphery along microtubules and this movement can be inhibited by nocodazole. Sub-populations of TC-core localize to the Golgi and co-traffic with components of the recycling endosome. Silencing of the recycling endosome component Rab11a results in the accumulation of HCV core at the Golgi. The majority of dynamic core traffics in association with apolipoprotein E (ApoE) and VAMP1 vesicles. This study identifies many new host cofactors of HCV egress, while presenting dynamic studies of HCV core trafficking in infected cells.     

Intracellular trafficking of Pseudomonas ExoS, a type III cytotoxin

Pseudomonas aeruginosa ExoS is a bifunctional type III cytotoxin that disrupts Ras- and Rho-signaling pathways in mammalian cells. A hydrophobic region (residues 51-77, termed the membrane localization domain) targets ExoS to the plasma membrane (PM) and late endosomes of host cells. In the current study, metabolic inhibitors and dominant-negative proteins that disrupt known vesicle-trafficking pathways were used to define the intracellular trafficking of ExoS. Release of ExoS from PM was independent of dynamin and ADP ribosylation factor 6 but inhibited by methyl-beta-cyclodextrin, a cholesterol-depleting reagent, and perinuclear localization of ExoS was disrupted by nocodazole. p50 dynamitin, a dynein inhibitor partially disrupted perinuclear localization of ExoS. Methyl-beta-cyclodextrin and nocodazole inhibited the ability of type-III-delivered ExoS to ADP-ribosylated Golgi/endoplasmic reticulum-resident Ras. Methyl-beta-cyclodextrin also relocated ExoS from the perinuclear region to the PM, indicating that ExoS can cycle through anterograde as well as through retrograde trafficking pathways. These findings show that ExoS endocytosis is cholesterol dependent, and it utilizes host microtubules, for intracellular trafficking. Understanding how type III cytotoxins enter and traffic within mammalian cells may identify new targets for therapeutic intervention of gram-negative bacterial pathogens.     

Mannose-dependent endoplasmic reticulum (ER)-Golgi intermediate compartment-53-mediated ER to Golgi trafficking of coagulation factors V and VIII.

The endoplasmic reticulum-Golgi intermediate compartment (ERGIC) is the site of segregation of secretory proteins for anterograde transport, via packaging into COPII-coated transport vesicles. ERGIC-53 is a homo-hexameric transmembrane lectin localized to the ERGIC that exhibits mannose-selective properties in vitro. Null mutations in ERGIC-53 were recently shown to be responsible for the autosomal recessive bleeding disorder, combined deficiency of coagulation factors V and VIII. We have studied the effect of defective ER to Golgi cycling by ERGIC-53 on the secretion of factors V and VIII. The secretion efficiency of factor V and factor VIII was studied in a tetracycline-inducible HeLa cell line overexpressing a wild-type ERGIC-53 or a cytosolic tail mutant of ERGIC-53 (KKAA) that is unable to exit the ER due to mutation of two COOH-terminal phenylalanine residues to alanines. The results show that efficient trafficking of factors V and VIII requires a functional ERGIC-53 cycling pathway and that this trafficking is dependent on post-translational modification of a specific cluster of asparagine (N)-linked oligosaccharides to a fully glucose-trimmed, mannose9 structure.