Fusion Of Endoplasmic Reticulum Membranes Research Articles

MoSey1 regulates the unfolded protein response, appressorium development, and pathogenicity of Magnaporthe oryzae

Endoplasmic reticulum (ER) is an enclosed three-dimensional eukaryotic membrane network composed of flattened sacs. Fusion of homologous membranes to the ER membrane is essential for the maintenance of this network structure. In yeast, ER membrane fusion is mediated by Sey1p, whose paralogues function distinctly in different species. In this study, we investigated the biological functions of MoSEY1 in the devastating rice blast fungus Magnaporthe oryzae by functional genomic approach. Compared to wild type, deletion of MoSEY1 considerably decreased the growth and conidia production of M. oryzae. Additionally, the absence of MoSEY1 delayed appressorium formation and invasive hyphae growth. The appressorium function was also impaired in ΔMosey1 mutant. Subcellular localization analysis revealed that MoSey1 is localized at the ER. The ΔMosey1 mutant showed augmented sensitivity to ER stress. Additionally, we found that MoSey1 regulated the unfolded protein response, autophagy, and protein secretion in M. oryzae. In conclusion, our study unveiled the involvement of MoSey1 in the development, pathogenesis, and ER functions in M. oryzae.

Yeast lunapark regulates the formation of trans-Sey1p complexes for homotypic ER membrane fusion

The endoplasmic reticulum (ER) consists of the nuclear envelope and a connected peripheral network of tubules and interspersed sheets. The structure of ER tubules is generated and maintained by various proteins, including reticulons, DP1/Yop1p, atlastins, and lunapark. Reticulons and DP1/Yop1p stabilize the high membrane curvature of ER tubules, and atlastins mediate homotypic membrane fusion between ER tubules; however, the exact role of lunapark remains poorly characterized. Here, using isolated yeast ER microsomes and reconstituted proteoliposomes, we directly examined the function of the yeast lunapark Lnp1p for yeast atlastin Sey1p-mediated ER fusion and found that Lnp1p inhibits Sey1p-driven membrane fusion. Furthermore, by using a newly developed assay for monitoring trans-Sey1p complex assembly, a prerequisite for ER fusion, we found that assembly of trans-Sey1p complexes was increased by the deletion of LNP1 and decreased by the overexpression of Lnp1p, indicating that Lnp1p inhibits Sey1p-mediated fusion by interfering with assembly of trans-Sey1p complexes.

Human atlastin-3 is a constitutive ER membrane fusion catalyst.

Homotypic membrane fusion catalyzed by the atlastin (ATL) GTPase sustains the branched endoplasmic reticulum (ER) network in metazoans. Our recent discovery that two of the three human ATL paralogs (ATL1/2) are C-terminally autoinhibited implied that relief of autoinhibition would be integral to the ATL fusion mechanism. An alternative hypothesis is that the third paralog ATL3 promotes constitutive ER fusion with relief of ATL1/2 autoinhibition used conditionally. However, published studies suggest ATL3 is a weak fusogen at best. Contrary to expectations, we demonstrate here that purified human ATL3 catalyzes efficient membrane fusion in vitro and is sufficient to sustain the ER network in triple knockout cells. Strikingly, ATL3 lacks any detectable C-terminal autoinhibition, like the invertebrate Drosophila ATL ortholog. Phylogenetic analysis of ATL C-termini indicates that C-terminal autoinhibition is a recent evolutionary innovation. We suggest that ATL3 is a constitutive ER fusion catalyst and that ATL1/2 autoinhibition likely evolved in vertebrates as a means of upregulating ER fusion activity on demand.

Atlastin Endoplasmic Reticulum-Shaping Proteins Facilitate Zika Virus Replication.

The endoplasmic reticulum (ER) is the site for Zika virus (ZIKV) replication and is central to the cytopathic effects observed in infected cells. ZIKV induces the formation of ER-derived large cytoplasmic vacuoles followed by "implosive" cell death. Little is known about the nature of the ER factors that regulate flavivirus replication. Atlastins (ATL1, -2, and -3) are dynamin-related GTPases that control the structure and the dynamics of the ER membrane. We show here that ZIKV replication is significantly decreased in the absence of ATL proteins. The appearance of infected cells is delayed, the levels of intracellular viral proteins and released virus are reduced, and the cytopathic effects are strongly impaired. We further show that ATL3 is recruited to viral replication sites and interacts with the nonstructural viral proteins NS2A and NS2B3. Thus, proteins that shape and maintain the ER tubular network ensure efficient ZIKV replication.IMPORTANCE Zika virus (ZIKV) is an emerging virus associated with Guillain-Barré syndrome, and fetal microcephaly as well as other neurological complications. There is no vaccine or specific antiviral treatment against ZIKV. We found that endoplasmic reticulum (ER)-shaping atlastin proteins (ATL1, -2, and -3), which induce ER membrane fusion, facilitate ZIKV replication. We show that ATL3 is recruited to the viral replication site and colocalize with the viral proteins NS2A and NS2B3. The results provide insights into host factors used by ZIKV to enhance its replication.

Ergosterol interacts with Sey1p to promote atlastin-mediated endoplasmic reticulum membrane fusion in Saccharomyces cerevisiae.

Sterols play critical roles in various membrane fusion events, including soluble NSF attachment protein receptor-mediated membrane fusion, mainly by modulating the physical properties of biologic membranes; however, it remains unclear whether they also function in atlastin-mediated endoplasmic reticulum (ER) membrane fusion. Although ergosterol, the major sterol in yeast, is essential for fusion of Sey1p (yeast atlastin)-containing liposomes with an ER-mimicking lipid composition, fusion of phosphatidylcholine/phosphatidylserine liposomes does not require sterols. Here, we examined whether sterols are important for Sey1p-mediated ER fusion in Saccharomyces cerevisiae using an in vitro ER fusion assay with isolated yeast ER microsomes. Ergosterol-specific ligands inhibited microsome fusion, indicating that ergosterol is critical for ER fusion. However, microsomes isolated from yeast strains lacking genes that encode enzymes involved in synthesis of ergosterol from lanosterol still fused, suggesting that other sterols can replace ergosterol and support Sey1p-mediated ER fusion. Importantly, disruption of sterol-binding motifs in the transmembrane regions of Sey1p markedly reduced ER fusion. Sey1p physically interacted with Erg11p and Erg4p, which function in ergosterol biosynthesis, suggesting that Sey1p recruits ergosterol-synthesizing enzymes to fusion sites and thereby enriches ergosterol, which, in turn, may recruit more Sey1p. This positive feedback loop may facilitate ER membrane fusion by concentrating fusion factors at fusion sites.-Lee, M., Moon, Y., Lee, S., Lee, C., Jun, Y. Ergosterol interacts with Sey1p to promote atlastin-mediated endoplasmic reticulum membrane fusion in Saccharomyces cerevisiae.

Efficient ER Fusion Requires a Dimerization and a C-Terminal Tail Mediated Membrane Anchoring of RHD3.

The endoplasmic reticulum (ER) is a network of tubules and sheets stretching throughout the eukaryotic cells. The formation of the ER requires homotypic membrane fusion, which is mediated by a family of Dynamin-like Atlastin GTPase proteins. The Arabidopsis (Arabidopsis thaliana) member ROOT HAIR DEFECTIVE3 (RHD3) has been demonstrated to mediate ER membrane fusion, but how exactly RHD3 is involved in the process is still unknown. Here we conducted systemic structure-function analyses of roles of different RHD3 domains in mediating ER fusion. We showed that efficient ER membrane fusion mediated by RHD3 requires a proper dimerization of RHD3 through the GTPase domain (GD) and the first and second three helix bundles (3HBs) in the middle domain. RHD3 has a 3HB-enriched middle domain longer than that of Atlastins, and we revealed that the third and fourth 3HBs are required for the stability of RHD3. The transmembrane segments of RHD3 are essential for targeting and retention of RHD3 in the ER and can also facilitate an oligomerization of RHD3. Furthermore, we showed that an amphipathic helix in the C-terminal cytosolic tail of RHD3 has a membrane anchoring ability that is required for efficient ER membrane fusion mediated by RHD3. This work contributes to a better understanding of a coordinated action of RHD3 in the fusion of ER membranes.

Molecular mechanisms of atlastin-mediated ER membrane fusion revealed by a FRET-based single-vesicle fusion assay

Homotypic fusion of endoplasmic reticulum membranes is driven by atlastin GTPases; however, the underlying mechanism remains largely unknown. Here, using a FRET-based single-vesicle fusion assay with liposomes bearing the yeast atlastin Sey1p, we investigated the molecular mechanisms of atlastin-mediated membrane tethering and fusion. Although Sey1p-bearing proteoliposomes frequently underwent membrane tethering in a GTP hydrolysis-dependent manner as reported in studies using bulk assays, only a small fraction of the tethered liposomes proceeded to fusion. Strikingly, the rest of the tethered liposomes failed to fuse or dissociate. This stable tethering, however, did not require continued GTP hydrolysis because GTP omission and magnesium chelation did not disrupt tethering. Interestingly, an increased Sey1p density on the membrane markedly accelerated tethering but barely affected the fusion rate of the tethered liposomes, indicating that Sey1p requires additional factors to support efficient fusion in vivo. Finally, the assay also revealed that Sey1p-mediated liposome fusion occurs through hemifusion, suggesting the mechanistic conservation between biological membrane fusion events despite the existence of diverse fusogens.

The crossover conformational shift of the GTPase atlastin provides the energy driving ER fusion.

The homotypic fusion of endoplasmic reticulum membranes is catalyzed by the atlastin GTPase. The mechanism involves trans-dimerization between GTPase heads and a favorable crossover conformational shift, catalyzed by GTP hydrolysis, that converts the dimer from a "prefusion" to "postfusion" state. However, whether crossover formation actually energizes fusion remains unclear, as do the sequence of events surrounding it. Here, we made mutations in atlastin to selectively destabilize the crossover conformation and used fluorescence-based kinetic assays to analyze the variants. All variants underwent dimerization and crossover concurrently, and at wild-type rates. However, certain variants were unstable once in the crossover dimer conformation, and crossover dimer stability closely paralleled lipid-mixing activity. Tethering, however, appeared to be unimpaired in all mutant variants. The results suggest that tethering and lipid mixing are catalyzed concurrently by GTP hydrolysis but that the energy requirement for lipid mixing exceeds that for tethering, and the full energy released through crossover formation is necessary for fusion.

Physiological lipid composition is vital for homotypic ER membrane fusion mediated by the dynamin-related GTPase Sey1p.

Homotypic fusion of the endoplasmic reticulum (ER) is required for generating and maintaining the characteristic reticular ER membrane structures. This organelle membrane fusion process depends on the ER-bound dynamin-related GTPases, such as atlastins in animals and Sey1p in yeast. Here, to investigate whether specific lipid molecules facilitate GTPase-dependent ER membrane fusion directly, we comprehensively evaluated membrane docking and lipid mixing of reconstituted proteoliposomes bearing purified Sey1p and a set of ER-mimicking lipids, including phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine, phosphatidic acid, and ergosterol. Remarkably, we revealed that each specific lipid species contributed little to membrane docking mediated by Sey1p. Nevertheless, Sey1p-dependent lipid mixing was strongly reduced by omitting three major acidic lipids from the ER-mimicking set and, moreover, was entirely abolished by omitting either phosphatidylethanolamine or ergosterol. Our reconstitution studies thus established that physiological lipid composition is vital for lipid bilayer rearrangements in GTPase-mediated homotypic ER membrane fusion.

The Atlastin C-Terminal Tail is an Amphipathic Helix that Perturbs the Bilayer Structure during Endoplasmic Reticulum Homotypic Fusion

Fusion of tubular membranes is required to form three-way junctions found in reticular subdomains of the endoplasmic reticulum. The large GTPase Atlastin has recently been shown to drive endoplasmic reticulum membrane fusion and three-way junction formation. The mechanism of Atlastin-mediated membrane fusion is distinct from SNARE-mediated membrane fusion, and many details remain unclear. In particular, the role of the amphipathic C-terminal tail of Atlastin is still unknown. We found that a peptide corresponding to the Atlastin C-terminal tail binds to membranes as a parallel α helix, induces bilayer thinning, and increases acyl chain disorder. The function of the C-terminal tail is conserved in human Atlastin. Mutations in the C-terminal tail decrease fusion activity in vitro, but not GTPase activity, and impair Atlastin function in vivo. In the context of unstable lipid bilayers, the requirement for the C-terminal tail is abrogated. These data suggest that the C-terminal tail of Atlastin locally destabilizes bilayers to facilitate membrane fusion.

Phosphorylation of the C Terminus of RHD3 Has a Critical Role in Homotypic ER Membrane Fusion in Arabidopsis.

The endoplasmic reticulum (ER) consists of dynamically changing tubules and cisternae. In animals and yeast, homotypic ER membrane fusion is mediated by fusogens (atlastin and Sey1p, respectively) that are membrane-associated dynamin-like GTPases. In Arabidopsis (Arabidopsis thaliana), another dynamin-like GTPase, ROOT HAIR DEFECTIVE3 (RHD3), has been proposed as an ER membrane fusogen, but direct evidence is lacking. Here, we show that RHD3 has an ER membrane fusion activity that is enhanced by phosphorylation of its C terminus. The ER network was RHD3-dependently reconstituted from the cytosol and microsome fraction of tobacco (Nicotiana tabacum) cultured cells by exogenously adding GTP, ATP, and F-actin. We next established an in vitro assay system of ER tubule formation with Arabidopsis ER vesicles, in which addition of GTP caused ER sac formation from the ER vesicles. Subsequent application of a shearing force to this system triggered the formation of tubules from the ER sacs in an RHD-dependent manner. Unexpectedly, in the absence of a shearing force, Ser/Thr kinase treatment triggered RHD3-dependent tubule formation. Mass spectrometry showed that RHD3 was phosphorylated at multiple Ser and Thr residues in the C terminus. An antibody against the RHD3 C-terminal peptide abolished kinase-triggered tubule formation. When the Ser cluster was deleted or when the Ser residues were replaced with Ala residues, kinase treatment had no effect on tubule formation. Kinase treatment induced the oligomerization of RHD3. Neither phosphorylation-dependent modulation of membrane fusion nor oligomerization has been reported for atlastin or Sey1p. Taken together, we propose that phosphorylation-stimulated oligomerization of RHD3 enhances ER membrane fusion to form the ER network.

Ultra-small superparamagnetic iron oxide mediated magnetic hyperthermia in treatment of neck lymph node metastasis in rabbit pyriform sinus VX2 carcinoma.

Lymph node metastasis of rabbit VX2 pyriform sinus carcinoma can be enhanced by MR scanning after injecting ultra-small superparamagnetic iron oxide (USPIO) into the submucosa beside the tumor. The metastasis lymph node which fit in with the diagnostic criteria will be placed into the alternating magnetic field after MR scanning. Then, magnetic particles can be heated to the effective therapeutic temperature. And it evaluates the possibility of diagnosis together with therapy in cervical metastasis of pyriform sinus carcinoma. Twenty rabbits bearing VX2 tumor in pyriform sinuses were randomly divided into hyperthermia group and control group after USPIO MR scanning; each group contained 10 rabbits. The hyperthermia for the experimental group was conducted by the alternating magnetic field. After hyperthermia, the detection of apoptosis for the two groups was tested by terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL), transmission electron microscopy (TEM), and the expression of Bcl-2 and Bax evaluated by immunohistochemical analysis. The apoptosis rate detected by TUNEL in hyperthermia group was 100 %, while the control group was only 20 % (p < 0.05). TEM observation showed that cell chromatin condensation and clumping, condensed cytoplasm, endoplasmic reticulum membrane fusion with loose change, and the formation of a bubble could be seen in the hyperthermia group. However, the control group showed a more complete cytoplasm and nucleus. Bcl-2 protein expression in the hyperthermia group was lower than the control group, and Bax protein expression in hyperthermia group was higher (p < 0.05). USPIO indirect lymphography could localize the metastatic lymph nodes for hyperthermia. And it could make the metastatic cervical lymph nodes apoptosis when placed into the alternating magnetic field.

The yeast atlastin Sey1p may not be sufficient to drive homotypic ER fusion at its physiological concentration

The endoplasmic reticulum (ER) is one of the most important organelle in the cell. The ER has many functions including cytoplasmic calcium level control, protein quality control, and lipid synthesis. These functions are thought to be closely related with the unique structure of the ER, which forms a network of sheets and tubules that are maintained by homotypic ER membrane fusion. Although the exact mechanism and players of homotypic ER fusion is yet to be unveiled, the dynamin‐like GTPase atlastin is believed to mediate homotypic ER fusion by forming a homo‐dimer in trans between fusing membranes. In this study, we measured the physiological concentration of the yeast atlastin homolog Sey1p from isolated yeast microsomes and performed lipid mixing assays with Sey1p‐proteoliposomes bearing various concentrations of Sey1p. Our study revealed that the physiological ratio of lipids to Sey1p molecules was approximately 1.0 x 105 and that lipid mixing was hardly detectable for proteoliposomes bearing Sey1p at a 1:4000 molar ratio to lipids, which is still 25 fold abundant as compared to isolated microsomes. Thus, this study suggests that the yeast atlastin homolog Sey1p at its physiological concentration is not sufficient to drive homotypic ER fusion by itself and that Sey1p needs additional factors to support ER fusion in vivo.

Human atlastin GTPases mediate differentiated fusion of endoplasmic reticulum membranes.

Human atlastin GTPases mediate differentiated fusion of endoplasmic reticulum membranes.

The Atlastin C-terminal Tail Is an Amphipathic Helix That Perturbs the Bilayer Structure during Endoplasmic Reticulum Homotypic Fusion

Fusion of tubular membranes is required to form three-way junctions found in reticular subdomains of the endoplasmic reticulum. The large GTPase Atlastin has recently been shown to drive endoplasmic reticulum membrane fusion and three-way junction formation. The mechanism of Atlastin-mediated membrane fusion is distinct from SNARE-mediated membrane fusion, and many details remain unclear. In particular, the role of the amphipathic C-terminal tail of Atlastin is still unknown. We found that a peptide corresponding to the Atlastin C-terminal tail binds to membranes as a parallel α helix, induces bilayer thinning, and increases acyl chain disorder. The function of the C-terminal tail is conserved in human Atlastin. Mutations in the C-terminal tail decrease fusion activity in vitro, but not GTPase activity, and impair Atlastin function in vivo. In the context of unstable lipid bilayers, the requirement for the C-terminal tail is abrogated. These data suggest that the C-terminal tail of Atlastin locally destabilizes bilayers to facilitate membrane fusion.

Unique and conserved features of the plant ER-shaping GTPase RHD3.

The architectural integrity of the endoplasmic reticulum (ER) network depends on the function of membrane-associated dynamin-like GTPases that include metazoan atlastins, plant RHD3 and yeast Sey1p. The evidence that these proteins are sufficient to drive membrane fusion of reconstituted proteoliposomes, and that loss-of-function mutations lead to conspicuous ER shape defects indicates that atlastins, RHD3 and Sey1p promote ER membrane fusion. However, complementation experiments in reciprocal loss-of-function backgrounds have also suggested that RHD3 and Sey1p may be not functionally equivalent, supporting that ER fusion mechanisms may be not entirely conserved in eukaryotes. In this Letter, we provide a brief overview of the field as well as evidence that may explain the functional differences of the plant and yeast ER-shaping dynamin-like GTPases.

Multiple mechanisms determine ER network morphology during the cell cycle in Xenopus egg extracts

In metazoans the endoplasmic reticulum (ER) changes during the cell cycle, with the nuclear envelope (NE) disassembling and reassembling during mitosis and the peripheral ER undergoing extensive remodeling. Here we address how ER morphology is generated during the cell cycle using crude and fractionated Xenopus laevis egg extracts. We show that in interphase the ER is concentrated at the microtubule (MT)-organizing center by dynein and is spread by outward extension of ER tubules through their association with plus ends of growing MTs. Fusion of membranes into an ER network is dependent on the guanosine triphosphatase atlastin (ATL). NE assembly requires fusion by both ATL and ER-soluble N-ethyl-maleimide-sensitive factor adaptor protein receptors. In mitotic extracts, the ER converts into a network of sheets connected by ER tubules and loses most of its interactions with MTs. Together, these results indicate that fusion of ER membranes by ATL and interaction of ER with growing MT ends and dynein cooperate to generate distinct ER morphologies during the cell cycle.

ROOT HAIR DEFECTIVE3 Family of Dynamin-Like GTPases Mediates Homotypic Endoplasmic Reticulum Fusion and Is Essential for Arabidopsis Development

In all eukaryotic cells, the endoplasmic reticulum (ER) forms a tubular network whose generation requires the fusion of ER membranes. In Arabidopsis (Arabidopsis thaliana), the membrane-bound GTPase ROOT HAIR DEFECTIVE3 (RHD3) is a potential candidate to mediate ER fusion. In addition, Arabidopsis has two tissue-specific isoforms of RHD3, namely RHD3-like (RL) proteins, and their function is not clear. Here, we show that a null allele of RHD3, rhd3-8, causes growth defects and shortened root hairs. A point mutant, rhd3-1, exhibits a more severe growth phenotype than the null mutant, likely because it exerts a dominant-negative effect on the RL proteins. Genetic analysis reveals that the double deletion of RHD3 and RL1 is lethal and that the rhd3 rl2 plants produce no viable pollen, suggesting that the RL proteins are redundant to RHD3. RHD3 family proteins can replace Sey1p, the homolog of RHD3 in yeast (Saccharomyces cerevisiae), in the maintenance of ER morphology, and they are able to fuse membranes both in vivo and in vitro. Our results suggest that RHD3 proteins mediate ER fusion and are essential for plant development and that the formation of the tubular ER network is of general physiological significance.

A Conserved Role for Atlastin GTPases in Regulating Lipid Droplet Size

Lipid droplets (LDs) are the major fat storage organelles in eukaryotic cells, but how their size is regulatedis unknown. Using genetic screens in C.elegans for LD morphology defects in intestinal cells, we found that mutations in atlastin, a GTPase required for homotypic fusion of endoplasmic reticulum (ER) membranes, cause not only ER morphology defects, but also a reduction in LD size. Similar results were obtained after depletion of atlastin or expression of a dominant-negative mutant, whereas overexpression of atlastin had the opposite effect. Atlastin depletion in Drosophila fat bodies also reduced LD size and decreased triglycerides in whole animals, sensitizing them to starvation. In mammalian cells, co-overexpression of atlastin-1 and REEP1, a paralog of the ER tubule-shaping protein DP1/REEP5, generates large LDs. The effect of atlastin-1 on LD size correlates with its activity to promote membrane fusion invitro. Our results indicate that atlastin-mediated fusion of ER membranes is important for LD size regulation.

Homotypic fusion of endoplasmic reticulum membranes in plant cells

The endoplasmic reticulum (ER) is a membrane-bounded organelle whose membrane comprises a network of tubules and sheets. The formation of these characteristic shapes and maintenance of their continuity through homotypic membrane fusion appears to be critical for the proper functioning of the ER. The atlastins (ATLs), a family of ER-localized dynamin-like GTPases, have been identified as fusogens of the ER membranes in metazoans. Mutations of the ATL proteins in mammalian cells cause morphological defects in the ER, and purified Drosophila ATL mediates membrane fusion in vitro. Plant cells do not possess ATL, but a family of similar GTPases, named root hair defective 3 (RHD3), are likely the functional orthologs of ATLs. In this review, we summarize recent advances in our understanding of how RHD3 proteins play a role in homotypic ER fusion. We also discuss the possible physiological significance of forming a tubular ER network in plant cells.