A Cluster of Thin Tubular Structures Mediates Transformation of the Endoplasmic Reticulum to Autophagic Isolation Membrane

Recent findings have suggested that the autophagic isolation membrane (IM) might originate from a domain of the endoplasmic reticulum (ER) called the omegasome. However, the morphological relationships between ER, omegasome, and IM remain unclear. In the present study, we found that hybrid structures composed of a double FYVE domain-containing protein 1 (DFCP1)-positive omegasome and the IM accumulated in Atg3-deficient mouse embryonic fibroblasts (MEFs). Moreover, correlative light and electron microscopy and immunoelectron microscopy revealed that green fluorescent protein (GFP)-tagged DFCP1 was localized on tubular or vesicular elements adjacent to the IM rims. Through detailed morphological analyses, including optimization of a fixation method and electron tomography, we observed a cluster of thin tubular structures between the IM edges and ER, part of which were continuous with IM and/or ER. The formation of these thin tubular clusters was observed in several cell lines and MEFs deficient for Atg5, Atg7, or Atg16L1 but not in FIP200-deficient cells, suggesting that they were relevant to the earlier events in autophagosome formation. Taken together, our findings indicate that these tubular profiles represent a part of the omegasome that links the ER with the IM.     

Phospholipid Transport via Mitochondria

In eukaryotic cells, complex membrane structures called organelles are highly developed to exert specialized functions. Mitochondria are one of such organelles consisting of the outer and inner membranes (OM and IM) with characteristic protein and phospholipid compositions. Maintaining proper phospholipid compositions of the membranes is crucial for mitochondrial integrity, thereby contributing to normal cell activities. As cellular locations for phospholipid synthesis are restricted to specific compartments such as the endoplasmic reticulum (ER) membrane and the mitochondrial inner membrane, newly synthesized phospholipids have to be transported and distributed properly from the ER or mitochondria to other cellular membranes. Although understanding of molecular mechanisms of phospholipid transport are much behind those of protein transport, recent studies using yeast as a model system began to provide intriguing insights into phospholipid exchange between the ER and mitochondria as well as between the mitochondrial OM and IM. In this review, we summarize the latest findings of phospholipid transport via mitochondria and discuss the implicated molecular mechanisms.      

Atlastin 2/3 regulate ER targeting of the ULK1 complex to initiate autophagy

Dynamic targeting of the ULK1 complex to the ER is crucial for initiating autophagosome formation and for subsequent formation of ER–isolation membrane (IM; autophagosomal precursor) contact during IM expansion. Little is known about how the ULK1 complex, which comprises FIP200, ULK1, ATG13, and ATG101 and does not exist as a constitutively coassembled complex, is recruited and stabilized on the ER. Here, we demonstrate that the ER-localized transmembrane proteins Atlastin 2 and 3 (ATL2/3) contribute to recruitment and stabilization of ULK1 and ATG101 at the FIP200-ATG13–specified autophagosome formation sites on the ER. In ATL2/3 KO cells, formation of FIP200 and ATG13 puncta is unaffected, while targeting of ULK1 and ATG101 is severely impaired. Consequently, IM initiation is compromised and slowed. ATL2/3 directly interact with ULK1 and ATG13 and facilitate the ATG13-mediated recruitment/stabilization of ULK1 and ATG101. ATL2/3 also participate in forming ER–IM tethering complexes. Our study provides insights into the dynamic assembly of the ULK1 complex on the ER for autophagosome formation.     

THE STRUCTURE AND FORMATION OF PROTEIN GRANULES IN THE FAT BODY OF AN INSECT

In the larva of the butterfly Calpodes ethlius, the fat body begins to store protein in the form of granules at about 30 to 35 hours before pupation, at a time when the endocuticle is being resorbed. At least two sorts of granule can be distinguished. The first granules to arise are those within vesicles of the Golgi complex. These may increase in size by incorporating material from microvesicles at their surface and by coalescence with one another. Later, at about 10 hours before pupation, another sort of granule arises by the isolation of regions of the endoplasmic reticulum (ER) within paired membranes derived from Golgi vesicles. Several of these ER isolation bodies coalesce, with fusion of their outer isolating membranes. The ribosomes and membranes may then disappear and the granules become indistinguishable from the protein granules formed from Golgi vesicles, or the ribosomes may remain and be embedded in dense crystalline protein, forming a storage body for both protein and RNA. Mitochondria are isolated within paired membranes in the same way as regions of the ER. The isolated mitochondria also coalesce in a similar manner. When the inner membranes are lost, the structure of a group of isolation bodies is indistinguishable from that of a cytolysome. Isolation within paired membranes, as described here, may be of general importance in segregating regions of massive lysis or massive sequestration.     

Absence of cytochrome P-450 and presence of autolysosomal membrane antigens on the isolation membranes and autophagosomal membranes in rat hepatocytes

We wished to determine if phenobarbital (PB)-inducible cytochrome P-450 [P-450(PB)] and autolysosomal membrane antigens could be localized immunocytochemically on the isolation membranes and the limiting membranes of autophagosomes in rat hepatocytes by the post-embedding protein A-gold method. P-450(PB) was maximally induced by PB treatment; then formation of autophagosomes and accumulation of autolysosomes were induced by cessation of PB treatment and by injection of leupeptin, respectively. P-450(PB) was detected neither on the isolation membranes nor on the limiting membranes of autophagosomes and autolysosomes. Autolysosomal membrane antigens, which were localized by the immunogold technique exclusively in post-Golgi compartments such as lysosomes, endosomes, and plasma membrane but were not found in pre-Golgi compartments such as endoplasmic reticulum (ER) and nuclear envelope, were detected in large amounts on the isolation membranes. These results suggest that the isolation membranes originate not from ER membranes but from post-Golgi membranes. We also present direct immunoelectron microscopic evidence that P-450(PB) is indeed degraded in the autolysosomes: when rats were treated with leupeptin, P-450(PB) was detected not only within the autophagosomes but also within the autolysosomes, whereas without leupeptin treatment, P-450(PB) was detectable only within the autophagosomes.     

Remodeling the endoplasmic reticulum by poliovirus infection and by individual viral proteins: an autophagy-like origin for virus-induced vesicles

All positive-strand RNA viruses of eukaryotes studied assemble RNA replication complexes on the surfaces of cytoplasmic membranes. Infection of mammalian cells with poliovirus and other picornaviruses results in the accumulation of dramatically rearranged and vesiculated membranes. Poliovirus-induced membranes did not cofractionate with endoplasmic reticulum (ER), lysosomes, mitochondria, or the majority of Golgi-derived or endosomal membranes in buoyant density gradients, although changes in ionic strength affected ER and virus-induced vesicles, but not other cellular organelles, similarly. When expressed in isolation, two viral proteins of the poliovirus RNA replication complex, 3A and 2C, cofractionated with ER membranes. However, in cells that expressed 2BC, a proteolytic precursor of the 2B and 2C proteins, membranes identical in buoyant density to those observed during poliovirus infection were formed. When coexpressed with 2BC, viral protein 3A was quantitatively incorporated into these fractions, and the membranes formed were ultrastructurally similar to those in poliovirus-infected cells. These data argue that poliovirus-induced vesicles derive from the ER by the action of viral proteins 2BC and 3A by a mechanism that excludes resident host proteins. The double-membraned morphology, cytosolic content, and apparent ER origin of poliovirus-induced membranes are all consistent with an autophagic origin for these membranes.     

How complement kills E. coli. I. Location of the lethal lesion

We have studied the action of human complement (C) on E. coli membranes. We find, as have others, that C disrupts the outer membrane (OM), allowing the release of periplasmic proteins. In addition, we have found 1) that in the complete absence of lysozyme, C damages the inner membrane (IM), 2) IM damage is different from OM damage in that only small molecules traverse a damaged IM whereas macromolecules traverse damaged OM, 3) IM damage and OM damage occur with identical kinetics and dose response, suggesting that IM and OM damage are closely coupled events, and 4) upon the addition of purified C8 and C9 to the washed cellular intermediate, E. coli C 1-7, both IM and OM are damaged coordinately. These results, taken together, suggest that C damages E. coli membranes by acting at a site contiguous with both membranes. We speculate that C may simultaneously gain access to both membranes by acting at the junctions between IM and OM.     

Effects of cyclic nucleotide dependent protein kinases on the endoplasmic reticulum Ca2+ pump of bovine pulmonary artery

This paper describes the stimulation by cyclic nucleotide dependent protein kinases on the Ca2+ uptake by isolated endoplasmic reticulum (ER) vesicles from the bovine main pulmonary artery. This ER fraction has previously been shown to be highly enriched in phospholamban, a protein kinase substrate that has been well characterized in cardiac sarcoplasmic reticulum (SR), where its phosphorylation is accompanied by an increased rate of Ca2+ uptake. As previously observed for the phosphorylation of phospholamban, the stimulation of the rate of Ca uptake was as high with cGMP dependent protein kinase as with cAMP dependent protein kinase. The effect of phosphorylation of the ER membranes from smooth muscle on the Ca2+ uptake was smaller than that seen in cardiac SR, and it was only observed if albumin was included during the isolation of the membranes. This relatively small effect is probably not due to a lower ratio of phospholamban to Ca2(+)-transport enzyme in the ER membranes as compared to cardiac SR. Several alternative explanations are discussed.     

Atg2: A novel phospholipid transfer protein that mediates de novo autophagosome biogenesis

The degradation of cytoplasmic components via autophagy is crucial for intracellular homeostasis. In the process of autophagy, a newly synthesized isolation membrane (IM) is developed to sequester degradation targets and eventually the IM seals, forming an autophagosome. One of the most poorly understood autophagy‐related proteins is Atg2, which is known to localize to a contact site between the edge of the expanding IM and the exit site of the endoplasmic reticulum (ERES). Recent advances in structural and biochemical analyses have been applied to Atg2 and have revealed it to be a novel multifunctional protein that tethers membranes and transfers phospholipids between them. Considering that Atg2 is essential for the expansion of the IM that requires phospholipids as building blocks, it is suggested that Atg2 transfers phospholipids from the ERES to the IM during the process of autophagosome formation, suggesting that lipid transfer proteins can mediate de novo organelle biogenesis.     

The isolation of endoplasmic reticulum from barley aleurone layers

Techniques for the isolation and purification of endoplasmic reticulum (ER) from aleurone layers of barley (Hordeum vulgare L.) were assessed. Neither differential centrifugation nor density gradient centrifugation of a homogenate separate the ER or other organelles of this tissue from the lipidcontaining spherosomes. Isopycnic sucrose gradient centrifugation of organelles first purified by molecular sieve chromatography on Sepharose 4B, however, results in separation of the organelles based on their differing buoyant densities. Manipulation of the magnesium concentration of the isolation media and density-gradient solutions affords isolation of ER at a density of 1.13–1.14 g cc^-1 and 1.17–1.18 g cc^-1. Electron microscopy shows that the membranes sedimenting at 1.13–1.14 g cc^-1 are devoid of ribosomes and are characteristic of smooth ER, while those sedimenting at 1.17–1.18 g cc^-1 are studded with ribosomes and have the features of rough ER. Endoplasmic reticulum isolated by isopycnic density gradient centrifugation can be further purified by rate-zonal centrifugation.Abbreviations EDTA ethylenediaminetetraacetic acid - ER endoplasmic reticulum - GA gibberellin - GA₃ gibberellic acid - Trizma tris(hydroxymethyl)aminomethane     

Myometrial effects of selective estrogen receptor modulators on estradiol-responsive gene expression are gene and cell-specific

We examined in vivo effects of selective estrogen receptor modulators (SERMs) 4-OH-tamoxifen (Tam), GW 5638 (GW) and EM-800 (EM) on myometrial gene expression. The uteri of ovariectomized ewes were infused with 10⁻⁷ M of one SERM via indwelling catheters for 24 h preceding hysterectomy. Half of the ewes in each SERM group received an intramuscular injection of 50 μg 17β-estradiol (E2) 18 h prior to hysterectomy. Northern blot analysis and in situ hybridization demonstrated that E2 increased estrogen receptor (ER), progesterone receptor (PR) and cyclophilin (CYC) gene expression in the cells of both inner layer of myometrium (IM) and outer layer of myometrium (OM) as well as glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene expression in OM. Tam also increased ER mRNA levels in OM. EM appeared to increase ER gene expression, but antagonized E2’s up-regulation of PR and CYC gene expression in both IM and OM. Tam and GW also antagonized E2 up-regulation of PR gene expression in OM but not IM. No SERM affected GAPDH gene expression with or without E2. Immunohistochemistry indicated that E2 increased nuclear ER and PR protein levels in both IM and OM. EM was unique in up-regulating ER protein levels, opposite to its effects in endometrial cells. All SERMs tested antagonized this increase in PR immunostaining preferentially in OM compared to the IM layer. These results illustrate gene and cell layer-specific effects of SERMs in sheep myometrium.     

Cytochalasin B binding and actin content of endoplasmic reticulum subfractions isolated from L-929 cells

Heavy rough (HR) endoplasmic reticulum (ER) membranes and a dense fraction of light rough (LR) membranes (LR I) of L-929 cells bind H-cytochalasin B extremely poorly in comparison to smooth (S) membranes and a fraction of LR membranes of low density (LR II). The LR and S subfractions of ER are apparently heterogeneous membrane populations with respect to cytochalasin B binding. The separation of proteins in HR and LR subfractions by electrophoresis followed by immunoblotting with monoclonal antibodies against actin showed that actin was not present in the former membranes while there were large amounts in the LR subfraction. It is concluded that membranes in the LR II fraction of ER are associated with actin-containing microfilaments of the cytoskeleton, but that HR membranes are not.     

Endoplasmic reticulum marker enzymes in Golgi fractions--what does this mean?

NADPH cytochrome c (cyt c) reductase and glucose-6-phosphatase, two enzymes thought to be restricted to the endoplasmic reticulum (ER) and widely used as ER markers, are present in isolated Golgi fractions assayed immediately after their isolation. Both enzymes are rapidly inactivated in fractions stored at 0 degrees C in 0.25 M sucrose, conditions which do not affect the activity of other enzymes in the same preparation. The inactivation process was shown to be dependent on time and protein concentration and could be prevented by EDTA and catalase. Morphological evidence shows that extensive membrane damage occurs parallel with the inactivation. Taken together with the immunological data in the companion paper, the findings indicate that the enzymes NADPH cyt c reductase and probably glucose-6-phosphate are indigenous components of Golgi membranes.     

Use of fluorescent probes that form intramolecular excimers to monitor structural changes in model and biological membranes.

1,3-dipyrenylpropane (PC3P) and bis(4-biphenylmethyl)ether, two molecules that form intramolecular excimers, were embedded in phospholipid vesicles and biological membranes to monitor dynamic properties of membrane lipids. Excimer formation was evaluated from determinations of excimer to monomer emission intensity ratios (ID/IM). ID/IM values of PC3P and bis(4-biphenylmethyl)ether were reduced when cholesterol was added to egg lecithin vesicles. PC3P was sensitive to the temperature-induced crystalline to liquid-crystalline phase transition in dimyristoyl phosphatidylcholine vesicles. For studies of cellular membranes, membranes, PC3P was used exclusively, because of the fluorescence of tryptophan residues of membrane proteins interferes with the responses bis(4-biphenylmethyl)ether. Microviscosities of membrane interiors were calculated from standard curves of IM/ID plotted against solvent viscosity. Microviscosity values of egg lecithin vesicles and biological membranes, especially those obtained with PC3P, were more than an order of magnitude lower than values obtained by other techniques. We concluded that the intramolecular process leading to the formation of the excimer is influenced differently in isotropic solvents than in anisotropic environments, such as lipid bilayers. Although distinguishable ID/IM ratios can be obtained for different biological membranes (mitochondrial, microsomal, and plasma membranes were studied), this parameter may be phenomenological and not simply related to membrane microviscosity. As such, fluorescent probes that form intramolecular excimers are of value in making qualitative comparisons of different membranes and in studying the relative effects of physical changes and chemical agents on membrane structure. These probes may also be valuable for studying structural anisotropy of biological membranes.     

Dynamics and retention of misfolded proteins in native ER membranes

When co-translationally inserted into endoplasmic reticulum (ER) membranes, newly synthesized proteins encounter the lumenal environment of the ER, which contains chaperone proteins that facilitate the folding reactions necessary for protein oligomerization, maturation and export from the ER. Here we show, using a temperature-sensitive variant of vesicular stomatitis virus G protein tagged with green fluorescent protein (VSVG-GFP), and fluorescence recovery after photobleaching (FRAP), the dynamics of association of folded and misfolded VSVG complexes with ER chaperones. We also investigate the potential mechanisms underlying protein retention in the ER. Misfolded VSVG-GFP complexes at 40 degrees C are highly mobile in ER membranes and do not reside in post-ER compartments, indicating that they are not retained in the ER by immobilization or retrieval mechanisms. These complexes are immobilized in ATP-depleted or tunicamycin-treated cells, in which VSVG-chaperone interactions are no longer dynamic. These results provide insight into the mechanisms of protein retention in the ER and the dynamics of protein-folding complexes in native ER membranes.     

ENDOMEMBRANE STRUCTURE AND THE CHLOROPLAST PROTEIN TARGETING PATHWAY IN HETEROSIGMA AKASHIWO (RAPHIDOPHYCEAE, CHROMISTA)

Chloroplasts in heterokont algae are surrounded by four membranes and probably originated from a red algal endosymbiont that was engulfed and retained by eukaryotic host. Understanding how nuclear-encoded chloroplast proteins are translocated from the cytoplasm into the chloroplast across these membranes could give us some insights about how the endosymbiont was integrated into the host cell in the process of secondary symbiogenesis. In multiplastid heterokont algae such as raphidophytes, it has been unclear if the outermost of the four membranes surrounding the chloroplast (the chloroplast endoplasmic reticulum [CER] membrane) is continuous with the nuclear envelope and rough endoplasmic reticulum (ER). Here, we report detailed ultrastructural observations of the raphidophyte Heterosigma akashiwo (Hada) Hada ex Y. Hara et Chihara that show that the CER membranes were continuous with ER membranes that had attached ribosomes, implying that the chloroplast with three envelope membranes is located within the ER lumen, that is, topologically the same structure as that of monoplastid heterokont algae. However, the CER membrane of H. akashiwo had very few, if any, ribosomes attached, unlike the CER membranes in other heterokont algae. To verify that proteins are first targeted to the ER, we assayed protein import into canine microsomes using a precursor for a nuclear-encoded chloroplast protein, the fucoxanthin-chlorophyll a / c protein of H. akashiwo. This demonstrated that the precursor has a functional signal sequence for ER targeting and is cotranslationally translocated into the ER, where a signal sequence of about 17 amino acids is removed. Based on these data, we hypothesize that in H. akashiwo , nuclear-encoded chloroplast protein precursors that have been cotranslationally transported into the ER lumen are sorted in the ER and transported to the chloroplasts through the ER lumen.     

Isolation and Characterization of Golgi Membranes from Suspension-cultured Cells of Rice (Oryza sativa L.)

The isolation of Golgi membranes from suspension-cultured cellsof rice (Oryza sativa L.) was attempted by linear glycerol densitygradient centrifugation. When "burst" membranes in the pelletobtaind after differential centrifugation at 100,000 ? g weresuspended in 20% (w/w) glycerol in 50 mM malate-NaOH (pH 6.0)and loaded onto a linear density gradient of glycerol, whichextended from 30 to 80% (w/w) in 1 mM EDTA in 50 mM glycylglycine-NaOH(pH 7.5), IDPase, a marker enzyme for Golgi membranes, was separatedfrom other membrane markers on the glycerol gradient. In addition,UDPase and GDPase activities overlapped with the peak fractionof IDPase activity. Furthermore, membrane glycoproteins in eachfraction were characterized by lectin-peroxidase staining. ConcanavalinA and lentil lectin, which have the ability to bind to the high-mannosetype of oligosaccharide, bound to glycoproteins distributedin ER membrane fractions, while wheat germ lectin, castor beanlectin, peanut lectin, and Ulex europaeus lectin-I which recognizethe complex type and/or the mucin type of oligosaccharides interactedwith glycoproteins in the Golgi membrane fractions but not withthose in the ER membrane. These results strongly suggest thatthe oligosaccharide structures of glycoproteins in the ER membraneare of the high-mannose type, whereas glycoproteins in the Golgimembrane have modified N-linked and/or O-linked oligosaccharidechains. (Received November 9, 1988; Accepted October 17, 1989)     

Reconstitution of protein targeting to the inner envelope membrane of chloroplasts

The chloroplast envelope plays critical roles in the synthesis and regulated transport of key metabolites, including intermediates in photosynthesis and lipid metabolism. Despite this importance, the biogenesis of the envelope membranes has not been investigated in detail. To identify the determinants of protein targeting to the inner envelope membrane (IM), we investigated the targeting of the nucleus-encoded integral IM protein, atTic40. We found that pre-atTic40 is imported into chloroplasts and processed to an intermediate size (int-atTic40) before insertion into the IM. Int-atTic40 is soluble and inserts into the IM from the internal stromal compartment. We also show that atTic40 and a second IM protein, atTic110, can target and insert into isolated IM vesicles in vitro. Collectively, our experiments are consistent with a "postimport" mechanism in which the IM proteins are first imported from the cytoplasm and subsequently inserted into the IM from the stroma.     

Structural organization and dynamics of the endoplasmic reticulum during spermatogenesis of Drosophila melanogaster: Studies using PDI-GFP chimera protein

An important property of the endoplasmic reticulum (ER) is its ability to change morphology and intracellular localization during the cell cycle and differentiation. Visualization of the ER membranes using the protein disulphide isomerase (PDI) GFP chimeric protein makes it possible to trace the dynamics of all these processes and expose transitional forms and intermediate configurations. In this article the results of the study of the ER morphology during spermatogenesis of D. melanogaster are presented. It was shown that ER membranes retain high level of GFP-fluorescence through all stages of spermatogenesis, so that revealing of the stage-specific features of the ER organization was possible. The ER network has distinctive reticular morphology during the interphase and early prophase. Right before the cell division, this morphology changes and ER forms a system of branchless filamentous membranes. In prometaphase, these membranes form concentric circles adjacent to the nuclear membrane; from metaphase to telophase, they lengthen along the axis of cell division and resemble a spindle. Later, in the next interphase, this configuration transforms to a reticular structure seen previously. At the beginning of spermatid differentiation, the ER encompasses nebenkern and nucleus and elongates adjacent to them. During the latest stages of spermatogenesis, the ER network dissociates into separate membranous granules that are eliminated from the cyst with the individualization complex. Possible mechanisms of the ER dynamics and reorganization are discussed.     

Genes that allow yeast cells to grow in the absence of the HDEL receptor.

The ERD2 gene of Saccharomyces cerevisiae encodes the HDEL receptor that sorts ER proteins; it is essential for growth. In the absence of Erd2p the Golgi apparatus is both functionally and morphologically perturbed. Here we describe the isolation of four SED genes (suppressors of the erd2-deletion) which, when present in multiple copies, allow cells to grow in the absence of ERD2. The suppressed strains secrete the ER protein BiP and their internal membranes show a variety of morphological abnormalities. Sequence analysis indicates that all these SED genes encode membrane proteins: SED1 encodes a probable cell surface glycoprotein; SED2 is identical to SEC12, a gene required for the formation of ER-derived transport vesicles; SED4 encodes a protein whose cytoplasmic domain is 45% identical to that of Sec12p; SED3 is DPM1, the structural gene for dolichol-P-mannose synthase. We suggest that the absence of ERD2 causes an imbalance between membrane flow into and out of the Golgi apparatus, and that the SED gene products can compensate for this either by slowing transport from the ER or by stimulating vesicle budding from Golgi membranes.