Perinuclear Endoplasmic Reticulum Research Articles

Ice2p is important for the distribution and structure of the cortical ER network in Saccharomyces cerevisiae

In Saccharomyces cerevisiae, the endoplasmic reticulum (ER) is found along the cell periphery (cortical ER) and nucleus (perinuclear ER). In this study, we characterize a novel ER protein called Ice2p that localizes to the cortical and perinuclear ER. Ice2p is predicted to be a type-III transmembrane protein. Cells carrying a genomic disruption of ICE2 display defects in the distribution of cortical ER in mother and daughter cells. Furthermore, fluorescence imaging of ice2delta cells reveals an abnormal cortical ER tubular network morphology in both the mother cell and the developing bud. Subcellular fractionation analysis using sucrose gradients corroborate the data from the fluorescence studies. Our findings indicate that Ice2p plays a role in forming and/or maintaining the cortical ER network in budding yeast.

On the biogenesis of lipid bodies in ancient eukaryotes: synthesis of triacylglycerols by a Toxoplasma DGAT1-related enzyme

In mammalian cells, the main stored neutral lipids are triacylglycerol and cholesteryl esters, which are produced by two related enzymes, acyl-CoA:diacylglycerol acyltransferase (DGAT) and acyl-CoA:cholesterol acyltransferase (ACAT), respectively. Very little is known about the metabolism, intracellular storage and function of neutral lipids in many pathogenic lower eukaryotes. In this paper, we have characterized the activity of an important triacylglycerol synthetic enzyme in the protozoan Toxoplasma gondii. A full-length cDNA and gene encoding a T. gondii DGAT1-related enzyme were identified and designated TgDGAT1. The gene is composed of 15 exons and 14 introns, and encodes a protein with a predicted M(r) 63.5kDa, containing signature motifs characteristic of the DGAT1 family. The native protein migrates at 44kDa under reducing conditions. TgDGAT1 is an integral membrane protein localized to the parasite cortical and perinuclear endoplasmic reticulum, with the C-terminus oriented to the lumen of the organelle. When a Saccharomyces cerevisiae mutant strain lacking neutral lipid production is transformed with TgDGAT1 cDNA, a significant DGAT activity is reconstituted, resulting in triacylglycerol synthesis and biogenesis of cytosolic lipid inclusions, resembling lipid bodies in T. gondii. No production of steryl esters is observed upon TgDGAT1 expression in yeast. In contrast to human DGAT1 lacking fatty acid specificity, TgDGAT1 preferentially incorporates palmitate. Our results indicate that parasitic protozoa are also neutral lipid accumulators and illustrate the first example of the existence of a functional DGAT gene in an ancient eukaryote, demonstrating that diacylglycerol esterification is evolutionarily conserved.

Drosophila Glucosylceramide Synthase: A NEGATIVE REGULATOR OF CELL DEATH MEDIATED BY PROAPOPTOTIC FACTORS

Glucosylceramide synthase (GlcT-1) catalyzes the formation of glucosylceramide (GlcCer), the core structure of major glycosphingolipids (GSLs), from ceramide and UDP-glucose. Ceramide and its metabolites, such as sphingosine-1-phosphate, are now known to be important mediators of apoptosis and cell survival. Recently, we have shown that GlcT-1 functions to regulate intracellular ceramide levels via glycosylation of ceramide. In this study, we employ the fruit fly Drosophila melanogaster as a model system for understanding the in vivo roles of GlcT-1. We isolated and characterized a GlcT-1 homologue (DGlcT-1) from Drosophila. When DGlcT-1 was expressed in GM-95 cells deficient in GSLs (because of the absence of GlcT-1 activity), these cells regained the ability to synthesize GSLs. Northern blot and in situ hybridization analyses revealed that the expression of DGlcT-1 mRNA was ubiquitous throughout development, suggesting that DGlcT-1 is important for development and differentiation. Indeed, RNA interference experiments demonstrated that the loss of GlcT-1 function enhances apoptotic cell death. Conversely, targeted expression of GlcT-1 partially rescued cell death caused by the proapoptotic factors Reaper and Grim, suggesting that ceramide generation might be one signal pathway that executes the cell death program. We also found that GlcT-1 localized not only in the Golgi apparatus but also in the perinuclear endoplasmic reticulum, providing the first visual evidence of GlcT-1 in membranes. These results indicate that GlcT-1 might down-regulate ceramide generated in these membranes.

Deficiencies in the endoplasmic reticulum (ER)-membrane protein Gab1p perturb transfer of glycosylphosphatidylinositol to proteins and cause perinuclear ER-associated actin bar formation.

The essential GAB1 gene, which encodes an endoplasmic reticulum (ER)-membrane protein, was identified in a screen for mutants defective in cellular morphogenesis. A temperature-sensitive gab1 mutant accumulates complete glycosylphosphatidylinositol (GPI) precursors, and its temperature sensitivity is suppressed differentially by overexpression of different subunits of the GPI transamidase, from strong suppression by Gpi8p and Gpi17p, to weak suppression by Gaa1p, and to no suppression by Gpi16p. In addition, both Gab1p and Gpi17p localize to the ER and are in the same protein complex in vivo. These findings suggest that Gab1p is a subunit of the GPI transamidase with distinct relationships to other subunits in the same complex. We also show that depletion of Gab1p or Gpi8p, but not Gpi17p, Gpi16p, or Gaa1p causes accumulation of cofilin-decorated actin bars that are closely associated with the perinuclear ER, which highlights a functional interaction between the ER network and the actin cytoskeleton.

A Fenton reaction at the endoplasmic reticulum is involved in the redox control of hypoxia-inducible gene expression

It has been proposed that hydroxyl radicals (.OH) generated in a perinuclear iron-dependent Fenton reaction are involved in O(2)-dependent gene expression. Thus, it was the aim of this study to localize the cellular compartment in which the Fenton reaction takes place and to determine whether scavenging of.OH can modulate hypoxia-inducible factor 1 (HIF-1)-dependent gene expression. The Fenton reaction was localized by using the nonfluorescent dihydrorhodamine (DHR) 123 that is irreversibly oxidized to fluorescent rhodamine 123 while scavenging.OH together with gene constructs allowing fluorescent labeling of mitochondria, endoplasmic reticulum (ER), Golgi apparatus, peroxisomes, or lysosomes. A 3D two-photon confocal laser scanning microscopy showed.OH generation in distinct hot spots of perinuclear ER pockets. This ER-based Fenton reaction was strictly pO(2)-dependent. Further colocalization experiments showed that the O(2)-sensitive transcription factor HIF-1alpha was present at the ER under normoxia, whereas HIF-1alpha was present only in the nucleus under hypoxia. Inhibition of the Fenton reaction by the.OH scavenger DHR attenuated HIF-prolyl hydroxylase activity and interaction with von Hippel-Lindau protein, leading to enhanced HIF-1alpha levels, HIF-1alpha transactivation, and activated expression of the HIF-1 target genes plasminogen activator inhibitor 1 and heme oxygenase 1. Further,.OH scavenging appeared to enhance redox factor 1 (Ref-1) binding and, thus, recruitment of p300 to the transactivation domain C because mutation of the Ref-1 binding site cysteine 800 abolished DHR-induced transactivation. Thus, the localized Fenton reaction appears to impact the expression of hypoxia-regulated genes by means of HIF-1alpha stabilization and coactivator recruitment.

A Novel Transport Pathway for a Yeast Plasma Membrane Protein Encoded by a Localized mRNA

Generally, plasma membrane (PM) proteins are cotranslationally inserted into the endoplasmic reticulum (ER) and travel in vesicles via the Golgi apparatus to the PM. In the yeast Saccharomyces cerevisiae, the polytopic membrane protein Ist2p is encoded by an mRNA that is localized to the cortex of daughter cells [1–3]. It has been suggested that IST2 mRNA localization leads to the accumulation of the protein at the PM of daughter cells [2]. Since small- and medium-sized daughter cells only contain cortical, but not perinuclear ER [4], this implies the local translation of Ist2p specifically at the cortical ER. Here, we show that localization of constitutively expressed IST2 mRNA is required for delivery of Ist2p to the PM of daughter, but not mother cells and that it does not result in daughter-specific Ist2p accumulation. In contrast to a PM-located hexose transporter (Hxt1p) that follows the standard secretory pathway, the trafficking of Ist2p is independent of myosin-mediated vesicular transport. Furthermore, colocalization experiments in mutants of the secretory pathway demonstrate that trafficking of Ist2p does not require the classical secretory machinery. These data suggest the existence of a novel trafficking pathway connecting specialized domains of the ER with the PM.

Membrane Synthesis, Specific Lipid Requirements, and Localized Lipid Composition Changes Associated with a Positive-Strand RNA Virus RNA Replication Protein

Multifunctional RNA replication protein 1a of brome mosaic virus (BMV), a positive-strand RNA virus, localizes to the cytoplasmic face of endoplasmic reticulum (ER) membranes and induces ER lumenal spherules in which viral RNA synthesis occurs. We previously showed that BMV RNA replication in yeast is severely inhibited prior to negative-strand RNA synthesis by a single-amino-acid substitution in the ole1w allele of yeast Delta9 fatty acid (FA) desaturase, which converts saturated FAs (SFAs) to unsaturated FAs (UFAs). Here we further define the relationships between 1a, membrane lipid composition, and RNA synthesis. We show that 1a expression increases total membrane lipids in wild-type (wt) yeast by 25 to 33%, consistent with recent results indicating that the numerous 1a-induced spherules are enveloped by invaginations of the outer ER membrane. 1a did not alter total membrane lipid composition in wt or ole1w yeast, but the ole1w mutation selectively depleted 18-carbon, monounsaturated (18:1) FA chains and increased 16:0 SFA chains, reducing the UFA-to-SFA ratio from approximately 2.5 to approximately 1.5. Thus, ole1w inhibition of RNA replication was correlated with decreased levels of UFA, membrane fluidity, and plasticity. The ole1w mutation did not alter 1a-induced membrane synthesis, 1a localization to the perinuclear ER, or colocalization of BMV 2a polymerase, nor did it block spherule formation. Moreover, BMV RNA replication templates were still recovered from cell lysates in a 1a-induced, 1a- and membrane-associated, and nuclease-resistant but detergent-susceptible state consistent with spherules. However, unlike nearby ER membranes, the membranes surrounding spherules in ole1w cells were not distinctively stained with osmium tetroxide, which interacts specifically with UFA double bonds. Thus, in ole1w cells, spherule-associated membranes were locally depleted in UFAs. This localized UFA depletion helps to explain why BMV RNA replication is more sensitive than cell growth to reduced UFA levels. The results imply that 1a preferentially interacts with one or more types of membrane lipids.

Replication of hepatitis C virus RNA occurs in a membrane-bound replication complex containing nonstructural viral proteins and RNA.

Biochemical studies revealed that nonstructural proteins of hepatitis C virus (HCV) interacted with each other and were associated with intracellular membranes. The goals of this study were to determine whether nonstructural viral proteins are colocalized at specific intracellular sites where HCV RNA is replicated and to identify the virus components of the HCV replication complex (RC). Immunofluorescence and subcellular fractionation studies were performed to determine the intracellular colocalization of nonstructural HCV proteins and the replicating RNA in a human hepatoma cell line, Huh7, in which a subgenomic HCV RNA was replicated persistently. The replicating HCV RNA was labelled with 5-bromouridine 5'-triphosphate (BrUTP). Results show that each of the nonstructural HCV proteins was colocalized predominantly with the newly synthesized HCV RNA labelled with BrUTP and an endoplasmic reticulum (ER) protein, calnexin. Consistent with these findings, subcellular fractionation and Western blot analyses revealed that the nonstructural HCV proteins were colocalized with HCV RNA mainly in the membrane fractions. Conversely, the viral nonstructural proteins and RNA remained in the soluble fractions upon treatment with detergent, confirming the membrane association of the HCV RC. HCV RNA in the membrane-bound RC was resistant to RNase treatment, whereas it became sensitive to RNases once the membranes were disrupted by treatment with detergent, suggesting that the HCV RC is assembled within membrane structures. Collectively, these findings demonstrate that HCV RNA replication occurs in the perinuclear ER membrane-bound HCV RC, containing nonstructural viral proteins and RNA.

Absence of superoxide dismutase activity in a soluble cellular isoform of prion protein produced by baculovirus expression system

A method for expression and purification of a soluble form of histidine (HIS)-tagged murine prion protein (bacMuPrP), which lacks the entire C-terminal cleavage and glycosyl phosphatidyl inositol (GPI) addition site, has been developed using a recombinant baculovirus expression system and purification with Ni–NTA agarose affinity chromatography. In mammalian sources, PrP C is attached to the cell membrane by a GPI anchor. However, in our system, bacMuPrP was secreted into the media, enabling its easy purification in abundance. Indirect immunofluorescence studies and immunoblot analysis localized not in cell membrane but in the perinuclear endoplasmic reticulum region in cells and is secreted into the media. Tunicamycin treatment revealed non-glycosylated proteins were secreted into the media, suggesting that glycosylation is not necessary for bacMuPrP secretion. Density-gradient sedimentation analysis demonstrated a sedimentation coefficient of secretory bacMuPrP as 2.3 S, indicating a monomeric form. Although affinity-purified PrP from mouse brain or recombinant prion protein (PrP) produced by Escherichia coli and refolded in the presence of copper has been reported to display superoxide dismutase (SOD) activity, bacMuPrP did not show SOD activity. These results suggest that bacMuPrP has a different biochemical and biophysical characterization from mammalian and bacterial-derived PrP. Furthermore, this simple expression system may provide an adequate source for structural, functional, and biochemical analyses of PrP.

Arachidonate release and prostaglandin production by group IVC phospholipase A2 (cytosolic phospholipase A2gamma).

While the role of the group IVA Ca(2+)-dependent cytosolic phospholipase A(2)alpha (cPLA(2)alpha) in arachidonic acid (AA) metabolism has been well documented, that of its paralogue, Ca(2+)-independent group IVC PLA(2) (cPLA(2)gamma), has remained uncertain. Here we show, using a transfection strategy, that cPLA(2)gamma has the ability to increase the spontaneous and stimulus-induced release of cellular fatty acids. The AA released by cPLA(2)gamma was metabolized further to prostaglandin E(2) via cyclo-oxygenase-1 (COX-1) in the immediate response, and via COX-2 in the delayed response. Mutation of the putative catalytic-centre residue Ser(82) abrogated the AA-releasing function of cPLA(2)gamma both in vitro and in vivo. Confocal microscopy revealed that cPLA(2)gamma was distributed in the perinuclear endoplasmic reticulum membranes. Mutating the C-terminal prenylation site of cPLA(2)gamma abrogated its intracellular membrane localization and cellular AA-releasing function, without reducing its enzyme activity in vitro. Our results indicate that cPLA(2)gamma is the second cPLA(2) enzyme that contributes to cellular AA metabolism and phospholipid remodelling under appropriate conditions.

Calcium signaling to nucleus via store-operated system during cell cycle in retinal neuroepithelium

Intracellular Ca 2+ is a regulatory signal for cell proliferation. To reveal Ca 2+ signal dynamics during cell cycle, we applied Ca 2+ fluorescence imaging to the neural retina of chick embryo, where the soma changes its position during the cell cycle. Purinoceptors were stimulated to cause Ca 2+ release from Ca 2+ stores, since the purinoceptor activation promotes DNA synthesis. Ca 2+ rises occurred in the nucleoplasm of cells at around S-phase. The soma of S-phase cell is located in the inner layer of the retinal neuroepithelium and issues an outer process, which extends to the ventricular surface. Fluorescent probes for endoplasmic reticulum (ER) showed that the ERs in the outer process and the nuclear envelope (NE) or peri-nuclear ER formed the Ca 2+ store. Depletion of the Ca 2+ store induced capacitative Ca 2+ entry (CCE), which caused Ca 2+ rises in the terminal of outer process and soma. The store-operated Ca 2+ signaling declined in M-phase cells and postmitotic cells (retinal ganglion cells (RGCs)) with the loss of the outer process. These results suggest that the Ca 2+ signaling to nucleus via the store-operated system including the ERs in the outer process is crucial for the cell cycle progression in the retinal neuroepithelium.

Ultrastructure of retinal ganglion cell death after axotomy in chick embryos.

Axotomy often leads to neuronal death, which occurs after a particularly short delay in immature animals. Tectal lesions were made in embryonic day (E) 12 chick embryos, thereby axotomizing the retinal ganglion cells of the contralateral eye, which then died within 3 days. We here describe the ultrastructural changes in the axotomized ganglion cells. The main changes were nuclear invagination and type 3B (cytoplasmic type) cell death characterized by dilation of the perinuclear space, endoplasmic reticulum, and Golgi apparatus. However, nuclear invagination was never seen in type 3B dying cells. All the axotomy-induced retinal ganglion cell death appears to have been of type 3B; apoptosis was not induced by axotomy, as was confirmed by additional light microscopic experiments showing that it did not increase the frequency of apoptotic markers revealed by terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling (the TUNEL method) labeling and immunoreactivity for activated caspase-3. However, the latter methods did show small numbers of apoptotic cells dying naturally even in control retinas. After the death of the axotomized ganglion cells, they were phagocytosed mainly in Müller processes. The present findings open up the chick tectal lesion model as a system for analyzing type 3B neuronal death in vivo.

The localization of two voltage-gated calcium channels in the pyloric network of the lobster stomatogastric ganglion

Voltage-gated calcium channels are critical to all aspects of nervous system function, with differing roles within the neuronal somata, at synaptic terminals, and at the neuromuscular junction. We have developed antibodies against two voltage-gated Ca 2+ channel genes from the spiny lobster, Panulirus interruptus, which are homologous to the Drosophila Ca1A (a P/Q-type channel) and Ca1D (an L-type channel) genes. Using these antibodies, we have found that each channel shows unique patterns of localization within the stomatogastric nervous system. Both antibodies stain somata of most of the neurons in the pyloric network to varying degrees. The high degree of variability in staining intensity within individual pyloric cell classes supports the hypothesis of Golowasch et al. (1999a,b) that individual cells can vary in their composition of ionic currents and still have similar firing properties. Anti-Ca1A stains structures in the neuropil, some of which are terminals of axons descending from higher ganglia; however, the majority of these are neither neurites nor blood vessels, but may instead be glial cells or other support elements. Anti-Ca1A labeling was also prominent in the peripheral axons of pyloric motoneurons as they enter muscles, indicating that this channel may be involved in regulation of synaptic transmission onto the foregut muscles. Anti-Ca1D does not label neurites in the neuropil of the stomatogastric ganglion. It stains glial cells in the stomatogastric ganglion in the region of their nuclei, presumably from protein being produced in the perinuclear rough endoplasmic reticulum, en route to the glial cell periphery. While anti-Ca1D labeling is seen in a patchy distribution along peripheral pyloric axons, it was never seen near the muscle. We conclude that the localization of these two calcium channels is tightly controlled within the stomatogastric nervous system, but we cannot conclusively demonstrate that Ca1A and/or Ca1D channels play roles in synaptic integration within the stomatogastric ganglion.

Ultrastructure of tetrasporogenesis in the red alga Osmundea spectabilis var. spectabilis (Rhodomelaceae: Ceramiales: Rhodophyta)

Tetrasporogenesis in Osmundeaspectabilis (Postels and Ruprecht) K.W. Nam var. spectabilis proceeds through three developmental stages. A uninucleate tetraspore mother cell with synaptonemal complexes depicted during early prophase of meiosis I comprises the youngest stage. Subsequently, the nuclear envelope obtains a highly irregular profile and is surrounded by perinuclear endoplasmic reticulum (ER) and numerous mitochondria. Proplastids with peripheral thylakoid and numerous straight-profiled dictyosomes are present. Dictyosome vesicles discharge their contents, initiating tetraspore wall formation. A four-nucleate tetraspore mother cell is formed prior to tetrahedral cleavage. During the second stage of tetraspore formation the nuclei are very irregular in outline and move toward the central region where they fuse. The formation of fibrous vacuoles originates from the fibrous vacuole associated organelles (FVAOs). Thylakoid formation from concentric lamellar bodies (CLBs) seems to occur in plastids. Extremely hypertrophied dictyosomes with two tightly compressed and densely laminated mid-regions produce vesicles which contribute to enlargement of the fibrous vacuoles. During the second stage of tetraspore development striated vesicles are formed and starch granule formation increases. Mature tetraspores are characterized by numerous cored vesicles and abundant starch granules. Plastids have a fully developed system of internal thylakoids, and cleavage is usually complete at this stage. Mature tetraspores are surrounded by a bi-layered wall and the tetrasporangial wall. Unbranched, tubular structures connecting the plasmalemma with the peripheral sheets of ER are possibly involved in wall deformation. A distinct blebbing nuclear envelope appears to sequester vesicles into the cytoplasm.

Identification of sequences in Brome mosaic virus replicase protein 1a that mediate association with endoplasmic reticulum membranes.

RNA replication of all positive-strand RNA viruses is closely associated with intracellular membranes. Brome mosaic virus (BMV) RNA replication occurs on the perinuclear region of the endoplasmic reticulum (ER), both in its natural plant host and in the yeast Saccharomyces cerevisiae. The only viral component in the BMV RNA replication complex that localizes independently to the ER is 1a, a multifunctional protein with an N-terminal RNA capping domain and a C-terminal helicase-like domain. The other viral replication components, the RNA polymerase-like protein 2a and the RNA template, depend on 1a for recruitment to the ER. We show here that, in membrane extracts, 1a is fully susceptible to proteolytic digestion in the absence of detergent and thus, a finding consistent with its roles in RNA replication, is wholly or predominantly on the cytoplasmic face of the ER with no detectable lumenal protrusions. Nevertheless, 1a association with membranes is resistant to high-salt and high-pH treatments that release most peripheral membrane proteins. Membrane flotation gradient analysis of 1a deletion variants and 1a segments fused to green fluorescent protein (GFP) showed that sequences in the N-terminal RNA capping module of 1a mediate membrane association. In particular, a region C-terminal to the core methyltransferase homology was sufficient for high-affinity ER membrane association. Confocal immunofluorescence microscopy showed that even though these determinants mediate ER localization, they fail to localize GFP to the narrow region of the perinuclear ER, where full-length 1a normally resides. Instead, they mediate a more globular or convoluted distribution of ER markers. Thus, additional sequences in 1a that are distinct from the primary membrane association determinants contribute to 1a's normal subcellular distribution, possibly through effects on 1a conformation, orientation, or multimerization on the membrane.

Molecular Characterization of Two Arabidopsis Ire1 Homologs, Endoplasmic Reticulum-Located Transmembrane Protein Kinases

A major response of eukaryotic cells to the presence of unfolded proteins in the lumen of the endoplasmic reticulum (ER) is to activate genes that encode ER-located molecular chaperones, such as the binding protein. This response, called the unfolded protein response, requires the transduction of a signal from the ER to the nucleus. In yeast (Saccharomyces cerevisiae) and mammalian cells, an ER-located transmembrane receptor protein kinase/ribonuclease called Ire1, with a sensor domain in the lumen of the ER, is the first component of this pathway. Here, we report the cloning and derived amino acid sequences of AtIre1-1 and AtIre1-2, two Arabidopsis homologs of Ire1. The two proteins are located in the perinuclear ER (based on heterologous expression of fusions with green fluorescent protein). The expression patterns of the two genes (using β-glucuronidase fusions) are nearly nonoverlapping. We also demonstrate functional complementation of the sensor domains of the two proteins in yeast and show that the Ire1-2 protein is capable of autotransphosphorylation. These and other findings are discussed in relation to the involvement of these genes in unfolded protein response signaling in plants.

Molecular Characterization of Two Arabidopsis Ire1 Homologs, Endoplasmic Reticulum-Located Transmembrane Protein Kinases

A major response of eukaryotic cells to the presence of unfolded proteins in the lumen of the endoplasmic reticulum (ER) is to activate genes that encode ER-located molecular chaperones, such as the binding protein. This response, called the unfolded protein response, requires the transduction of a signal from the ER to the nucleus. In yeast (Saccharomyces cerevisiae) and mammalian cells, an ER-located transmembrane receptor protein kinase/ribonuclease called Ire1, with a sensor domain in the lumen of the ER, is the first component of this pathway. Here, we report the cloning and derived amino acid sequences of AtIre1-1 and AtIre1-2, two Arabidopsis homologs of Ire1. The two proteins are located in the perinuclear ER (based on heterologous expression of fusions with green fluorescent protein). The expression patterns of the two genes (using beta-glucuronidase fusions) are nearly nonoverlapping. We also demonstrate functional complementation of the sensor domains of the two proteins in yeast and show that the Ire1-2 protein is capable of autotransphosphorylation. These and other findings are discussed in relation to the involvement of these genes in unfolded protein response signaling in plants.

Mutation of host delta9 fatty acid desaturase inhibits brome mosaic virus RNA replication between template recognition and RNA synthesis.

All positive-strand RNA viruses assemble their RNA replication complexes on intracellular membranes. Brome mosaic virus (BMV) replicates its RNA in endoplasmic reticulum (ER)-associated complexes in plant cells and the yeast Saccharomyces cerevisiae. BMV encodes RNA replication factors 1a, with domains implicated in RNA capping and helicase functions, and 2a, with a central polymerase-like domain. Factor 1a interacts independently with the ER membrane, viral RNA templates, and factor 2a to form RNA replication complexes on the perinuclear ER. We show that BMV RNA replication is severely inhibited by a mutation in OLE1, an essential yeast chromosomal gene encoding delta9 fatty acid desaturase, an integral ER membrane protein and the first enzyme in unsaturated fatty acid synthesis. OLE1 deletion and medium supplementation show that BMV RNA replication requires unsaturated fatty acids, not the Ole1 protein, and that viral RNA replication is much more sensitive than yeast growth to reduced unsaturated fatty acid levels. In ole1 mutant yeast, 1a still becomes membrane associated, recruits 2a to the membrane, and recognizes and stabilizes viral RNA templates normally. However, RNA replication is blocked prior to initiation of negative-strand RNA synthesis. The results show that viral RNA synthesis is highly sensitive to lipid composition and suggest that proper membrane fluidity or plasticity is essential for an early step in RNA replication. The strong unsaturated fatty acid dependence also demonstrates that modulating fatty acid balance can be an effective antiviral strategy.

Intercellular calcium waves in HeLa cells expressing GFP-labeled connexin 43, 32, or 26.

This study was undertaken to obtain direct evidence for the involvement of gap junctions in the propagation of intercellular Ca(2+) waves. Gap junction-deficient HeLa cells were transfected with plasmids encoding for green fluorescent protein (GFP) fused to the cytoplasmic carboxyl termini of connexin 43 (Cx43), 32 (Cx32), or 26 (Cx26). The subsequently expressed GFP-labeled gap junctions rendered the cells dye- and electrically coupled and were detected at the plasma membranes at points of contact between adjacent cells. To correlate the distribution of gap junctions with the changes in [Ca(2+)](i) associated with Ca(2+) waves and the distribution of the endoplasmic reticulum (ER), cells were loaded with fluorescent Ca(2+)-sensitive (fluo-3 and fura-2) and ER membrane (ER-Tracker) dyes. Digital high-speed microscopy was used to collect a series of image slices from which the three-dimensional distribution of the gap junctions and ER were reconstructed. Subsequently, intercellular Ca(2+) waves were induced in these cells by mechanical stimulation with or without extracellular apyrase, an ATP-degrading enzyme. In untransfected HeLa cells and in the absence of apyrase, cell-to-cell propagating [Ca(2+)](i) changes were characterized by initiating Ca(2+) puffs associated with the perinuclear ER. By contrast, in Cx-GFP-transfected cells and in the presence of apyrase, [Ca(2+)](i) changes were propagated without initiating perinuclear Ca(2+) puffs and were communicated between cells at the sites of the Cx-GFP gap junctions. The efficiency of Cx expression determined the extent of Ca(2+) wave propagation. These results demonstrate that intercellular Ca(2+) waves may be propagated simultaneously via an extracellular pathway and an intracellular pathway through gap junctions and that one form of communication may mask the other.

Degradation of tobacco mosaic virus movement protein by the 26S proteasome.

Cell-to-cell spread of tobacco mosaic virus is facilitated by the virus-encoded 30-kDa movement protein (MP). This process involves interaction of viral proteins with host components, including the cytoskeleton and the endoplasmic reticulum (ER). During virus infection, high-molecular-weight forms of MP were detected in tobacco BY-2 protoplasts. Inhibition of the 26S proteasome by MG115 and clasto-lactacystin-beta-lactone enhanced the accumulation of high-molecular-weight forms of MP and led to increased stability of the MP. Such treatment also increased the apparent accumulation of polyubiquitinated host proteins. By fusion of MP with the jellyfish green fluorescent protein (GFP), we demonstrated that inhibition of the 26S proteasome led to accumulation of the MP-GFP fusion preferentially on the ER, particularly the perinuclear ER. We suggest that polyubiquitination of MP and subsequent degradation by the 26S proteasome may play a substantial role in regulation of virus spread by reducing the damage caused by the MP on the structure of cortical ER.