Dual Localization of Squalene Epoxidase, Erg1p, in Yeast Reflects a Relationship between the Endoplasmic Reticulum and Lipid Particles

Squalene epoxidase, encoded by the ERG1 gene in yeast, is a key enzyme of sterol biosynthesis. Analysis of subcellular fractions revealed that squalene epoxidase was present in the microsomal fraction (30,000 × g) and also cofractionated with lipid particles. A dual localization of Erg1p was confirmed by immunofluorescence microscopy. On the basis of the distribution of marker proteins, 62% of cellular Erg1p could be assigned to the endoplasmic reticulum and 38% to lipid particles in late logarithmic-phase cells. In contrast, sterol Δ²⁴-methyltransferase (Erg6p), an enzyme catalyzing a late step in sterol biosynthesis, was found mainly in lipid particles cofractionating with triacylglycerols and steryl esters. The relative distribution of Erg1p between the endoplasmic reticulum and lipid particles changes during growth. Squalene epoxidase (Erg1p) was absent in an erg1 disruptant strain and was induced fivefold in lipid particles and in the endoplasmic reticulum when the ERG1 gene was overexpressed from a multicopy plasmid. The amount of squalene epoxidase in both compartments was also induced approximately fivefold by treatment of yeast cells with terbinafine, an inhibitor of the fungal squalene epoxidase. In contrast to the distribution of the protein, enzymatic activity of squalene epoxidase was only detectable in the endoplasmic reticulum but was absent from isolated lipid particles. When lipid particles of the wild-type strain and microsomes of an erg1 disruptant were mixed, squalene epoxidase activity was partially restored. These findings suggest that factor(s) present in the endoplasmic reticulum are required for squalene epoxidase activity. Close contact between lipid particles and endoplasmic reticulum may be necessary for a concerted action of these two compartments in sterol biosynthesis.     

植物内质网胁迫研究进展

内质网是蛋白质折叠和蛋白质糖基化修饰的重要场所。在内质网中存在多种调控机制来确保其中的蛋白质被正确地折叠、修饰和组装,以维持内质网稳态,这对于细胞正常的生理活动十分重要。然而,多种物理、化学因素均可使内质网稳态失衡,即在应激条件下,错误折叠和未折叠蛋白质的大量积累将导致内质网胁迫(endoplasmic reticulum stress, ERS),进而会引起未折叠蛋白质响应(unfolded protein response, UPR),极端情况下还会启动细胞程序性死亡(program cell death, PCD)。目前,植物内质网胁迫方面的研究较酵母和动物滞后,因此,从内质网质量控制系统和未折叠蛋白质响应2个方面对植物内质网胁迫现有研究进行了综述,以期为进一步理解内质网胁迫与植物逆境胁迫的关系提供参考。     

Ultrastructural demonstration of thioredoxin and thioredoxin reductase in rat hepatocytes

Electron microscopic immunocytochemistry, in conjunction with the immunogold technique, was used to demonstrate the ultrastructural localization of thioredoxin and thioredoxin reductase in rat liver hepatocytes. Gold particles representing thioredoxin and thioredoxin reductase antigenic sites were found throughout the cell, but particularly densely associated with the granular endoplasmic reticulum and the cisternae of the Golgi complex. Label was also distributed over the cytosol and in the chromatin of the nucleus. We conclude that thioredoxin and thioredoxin reductase are present in several different cellular compartments including the nucleus. In particular, the enrichment of thioredoxin and thioredoxin reductase to the endoplasmic reticulum is consistent with functions in protein processing, secretion and the formation of nascent protein disulfides.     

Isolation of glucanase-containing vesicles from budding yeast

Summary In an investigation of the role of glucanases in modifying yeast cell walls at the location of new buds, vesicles derived from the endoplasmic reticulum, which are secreted locally into the cell wall of growing buds, and may be involved in the secretion of glucanases, have been isolated.In yeast, exo--1,3-glucanase is present both extra- and intracellularly. Exponentially growing cells contain at least 11% of the enzyme activity intracellularly (within the plasmalemma). Most of this intracellular glucanase is sedimentable. Of the three classes of subcellular particles that contain glucanase, one is almost completely absent from stationary phase cells and largely absent from cells of the late budding phase of the cell cycle. These particles were isolated from budding cells by combined differential and density gradient centrifugation. They contain exo- and endo--1,3-glucanases, mannan and protein. The isolate consists mainly of membrane-bounded vesicles with diameters corresponding to those of the secretory vesicles observed in situ. It is concluded that these particles are identical with the vesicles derived from the endoplasmic reticulum.     

Lipid inclusions in cells and their changes during the desiccation and reactivation of yeast organisms

The formation of lipid granules in Saccharomyces cerevisiae cells was shown to begin in the exponential phase of the culture growth and to be associated with the activity of the endoplasmic reticulum. Lipid granules can be formed (1) when the endoplasmic reticulum cisterns extend and (2) when the endoplasmic reticulum membranes separate relatively small regions of the cell cytoplasm. Yeast cells contain spherosomes which differ in their structure. Lipid granules merge together upon dehydration of yeast cells. The authors discuss possible participation of these granules in the reparation of damages of the intracellular membranes.     

Erv14p directs a transmembrane secretory protein into COPII-coated transport vesicles

Erv14p is a conserved integral membrane protein that traffics in COPII-coated vesicles and localizes to the early secretory pathway in yeast. Deletion of ERV14 causes a defect in polarized growth because Axl2p, a transmembrane secretory protein, accumulates in the endoplasmic reticulum and is not delivered to its site of function on the cell surface. Herein, we show that Erv14p is required for selection of Axl2p into COPII vesicles and for efficient formation of these vesicles. Erv14p binds to subunits of the COPII coat and binding depends on conserved residues in a cytoplasmically exposed loop domain of Erv14p. When mutations are introduced into this loop, an Erv14p-Axl2p complex accumulates in the endoplasmic reticulum, suggesting that Erv14p links Axl2p to the COPII coat. Based on these results and further genetic experiments, we propose Erv14p coordinates COPII vesicle formation with incorporation of specific secretory cargo.     

The yeast EUG1 gene encodes an endoplasmic reticulum protein that is functionally related to protein disulfide isomerase.

The product of the EUG1 gene of Saccharomyces cerevisiae is a soluble endoplasmic reticulum protein with homology to both the mammalian protein disulfide isomerase (PDI) and the yeast PDI homolog encoded by the essential PDI1 gene. Deletion or overexpression of EUG1 causes no growth defects under a variety of conditions. EUG1 mRNA and protein levels are dramatically increased in response to the accumulation of native or unglycosylated proteins in the endoplasmic reticulum. Overexpression of the EUG1 gene allows yeast cells to grow in the absence of the PDI1 gene product. Depletion of the PDI1 protein in Saccharomyces cerevisiae causes a soluble vacuolar glycoprotein to accumulate in its endoplasmic reticulum form, and this phenotype is only partially relieved by the overexpression of EUG1. Taken together, our results indicate that PDI1 and EUG1 encode functionally related proteins that are likely to be involved in interacting with nascent polypeptides in the yeast endoplasmic reticulum.     

Formation and transfer of disulphide bonds in living cells

Protein disulphide bonds are formed in the endoplasmic reticulum of eukaryotic cells and the periplasmic space of prokaryotic cells. The main pathways that catalyse the formation of protein disulphide bonds in prokaryotes and eukaryotes are remarkably similar, and they share several mechanistic features. The recent identification of new redox-active proteins in humans and yeast that mechanistically parallel the more established redox-active enzymes indicates that there might be further uncharacterized redox pathways throughout the cell.     

Endoplasmic reticulum (ER)-associated degradation of T cell receptor subunits. Involvement of ER-associated ubiquitin-conjugating enzymes (E2s)

Degradation of proteins from the endoplasmic reticulum is fundamental to quality control within the secretory pathway, serves as a way of regulating levels of crucial proteins, and is utilized by viruses to enhance pathogenesis. In yeast two ubiquitin-conjugating enzymes (E2s), UBC6p and UBC7p are implicated in this process. We now report the characterization of murine homologs of these E2s. MmUBC6 is an integral membrane protein that is anchored via its hydrophobic C-terminal tail to the endoplasmic reticulum. MmUBC7, which is not an integral membrane protein, shows significant endoplasmic reticulum colocalization with MmUBC6. Overexpression of catalytically inactive MmUBC7 significantly delayed degradation from the endoplasmic reticulum of two T cell antigen receptor subunits, alpha and CD3-delta, and suggests a role for the ubiquitin conjugating system at the initiation of retrograde movement from the endoplasmic reticulum. These findings also implicate, for the first time, a specific E2 in degradation from the endoplasmic reticulum in mammalian cells.     

Perturbation of sphingolipid metabolism induces endoplasmic reticulum stress‐mediated mitochondrial apoptosis in budding yeast

Sphingolipids are a class of membrane lipids conserved from yeast to mammals which determine whether a cell dies or survives. Perturbations in sphingolipid metabolism cause apoptotic cell death. Recent studies indicate that reduced sphingolipid levels trigger the cell death, but little is known about the mechanisms. In the budding yeast Saccharomyces cerevisiae, we show that reduction in complex sphingolipid levels causes loss of viability, most likely due to the induction of mitochondria‐dependent apoptotic cell death pathway, accompanied by changes in mitochondrial and endoplasmic reticulum morphology and endoplasmic reticulum stress. Elevated cytosolic free calcium is required for the loss of viability. These results indicate that complex sphingolipids are essential for maintaining endoplasmic reticulum homeostasis and suggest that perturbation in complex sphingolipid levels activates an endoplasmic reticulum stress‐mediated and calcium‐dependent pathway to propagate apoptotic signals to the mitochondria.     

20S proteasome biogenesis

26S proteasomes are multi-subunit protease complexes responsible for the turnover of short-lived proteins. Proteasomal degradation starts with the autocatalytic maturation of the 20S core particle. Here, we summarize different models of proteasome assembly. 20S proteasomes are assembled as precursor complexes containing alpha and unprocessed beta subunits. The propeptides of the beta subunits are thought to prevent premature conversion of the precursor complexes into matured particles and are needed for efficient beta subunit incorporation. The complex biogenesis is tightly regulated which requires additional components such as the maturation factor Ump1/POMP, an ubiquitous protein in eukaryotic cells. Ump1/POMP is associated with precursor intermediates and degraded upon final maturation. Mammalian proteasomes are localized all over the cell, while yeast proteasomes mainly localize to the nuclear envelope/endoplasmic reticulum (ER) membrane network. The major localization of yeast proteasomes may point to the subcellular place of proteasome biogenesis.     

The unfolded protein response: no longer just a special teams player

The endoplasmic reticulum stress pathway known as the unfolded protein response is currently the best understood model of interorganellar signal transduction. Bridging a physical separation, the pathway provides a direct line of communication between the endoplasmic reticulum lumen and the nucleus. With the unfolded protein response, the cell has the means to monitor and respond to the changing needs of the endoplasmic reticulum. Beginning with the discovery of its remarkable signaling mechanism in yeast, the unfolded protein response has not ceased to reveal more of its many secrets. By applying powerful biochemical, genetic, genomic, and cytological approaches, the recent efforts of many groups have buried the long-held notion that the unfolded protein response is simply a regulatory platform for endoplasmic reticulum chaperones. We now know that the unfolded protein response regulates many genes that affect diverse aspects of cellular physiology. In addition, studies in mammals have revealed novel unfolded protein response signaling factors that may contribute to the specialized needs of multicellular organisms. This article focuses on these and other recent developments in the field.     

The yeast inositol polyphosphate 5-phosphatase Inp54p localizes to the endoplasmic reticulum via a C-terminal hydrophobic anchoring tail: regulation of secretion from the endoplasmic reticulum

The budding yeast Saccharomyces cerevisiae has four inositol polyphosphate 5-phosphatase (5-phosphatase) genes, INP51, INP52, INP53, and INP54, all of which hydrolyze phosphatidylinositol (4,5)-bisphosphate. INP54 encodes a protein of 44 kDa which consists of a 5-phosphatase domain and a C-terminal leucine-rich tail, but lacks the N-terminal SacI domain and proline-rich region found in the other three yeast 5-phosphatases. We report that Inp54p belongs to the family of tail-anchored proteins and is localized to the endoplasmic reticulum via a C-terminal hydrophobic tail. The hydrophobic tail comprises the last 13 amino acids of the protein and is sufficient to target green fluorescent protein to the endoplasmic reticulum. Protease protection assays demonstrated that the N terminus of Inp54p is oriented toward the cytoplasm of the cell, with the C terminus of the protein also exposed to the cytosol. Null mutation of INP54 resulted in a 2-fold increase in secretion of a reporter protein, compared with wild-type yeast or cells deleted for any of the SacI domain-containing 5-phosphatases. We propose that Inp54p plays a role in regulating secretion, possibly by modulating the levels of phosphatidylinositol (4,5)-bisphosphate on the cytoplasmic surface of the endoplasmic reticulum membrane.     

Subcellular distribution of phytin in the endosperm of developing castor bean: a possibility for its synthesis in the cytoplasm prior to deposition within protein bodies

Studies using light and electron microscopy, and energy-dispersive X-ray analysis have allowed us to identify phytin particles within the cytoplasm of the developing endosperm of castor bean (Ricinus communis L.). These particles are present at the time of the formation of globoid particles within the protein bodies, but they are absent from mature tissue with fully formed protein bodies. We suggest that phytin is formed initially in the cytoplasm (perhaps in association with the cisternal endoplasmic reticulum) before being transported to the protein bodies, wherein it condenses to form the globoid.Abbreviations AMBB alcoholicmercuric bromophenol blue - ATBO acidic toluidine blue O - CER cisternal endoplasmic reticulum - DAP days after pollination - EDX energy-dispersive X-ray     

Calreticulin and calnexin in the endoplasmic reticulum are important for phagocytosis.

Calreticulin and calnexin are Ca2+-binding proteins with chaperone activity in the endoplasmic reticulum. These proteins have been eliminated by gene replacement in Dictyostelium, the only microorganism known to harbor both proteins; family members in Dictyostelium are located at the base of phylogenetic trees. A dramatic decline in the rate of phagocytosis was observed in double mutants lacking calreticulin and calnexin, whereas only mild changes occurred in single mutants. Dictyostelium cells are professional phagocytes, capable of internalizing particles by a sequence of activities: adhesion of the particle to the cell surface, actin-dependent outgrowth of a phagocytic cup, and separation of the phagosome from the plasma membrane. In the double-null mutants, particles still adhered to the cell surface, but the outgrowth of phagocytic cups was compromised. Green fluorescent protein-tagged calreticulin and calnexin, expressed in wild-type cells, revealed a direct link of the endoplasmic reticulum to the phagocytic cup enclosing a particle, such that the Ca2+ storage capacity of calreticulin and calnexin might directly modulate activities of the actin system during particle uptake.     

Ultrastructural and immunocytochemical evidences of core-particle formation in the methylotrophic Pichia pastoris yeast when expressing HCV structural proteins (core-E1).

Particulate antigens of the Hepatitis C virus (HCV) are reported for the first time by transmission electron microscopy in Pichia pastoris. The yeast was cloned to express the first 339 NH2-terminal amino acids of the HCV polyprotein (C-E1.339 polypeptide). The C-E1.339 polypeptide covers the putative 191 aa of the core protein (aa 1-191) and 148 aa of the E1 envelope antigen (aa 192-339). Virus-like particles (VLP) with diameters ranging from 20 nm to 30 nm were specifically observed in those cells expressing the HCV polyprotein. The VLP appeared along the membrane of the endoplasmic reticulum, but were fundamentally localized in vacuoles, either free or inside autophagic bodies. Clustered particles, chains of particles, high-density reticular structures, and crystalloid bodies were also detected, the last one being an orderly arrangement of particles with 20 nm diameters. The crystal-associated particles are well differentiated from the intracellular VLP because of their uniform size and shape. We argue that membrane components are retained in the architecture of the VLP, conferring to this particle certain heterogeneity. Both kinds of particles, the VLP formed after treatment with NP-40 and the crystal-associated particles, were core protein-positives. Whether they reflect mature HCV nucleocapsid or intermediary states in the viral nucleocapsid morphogenesis remains unknown. We conclude that, like mammalian cell lines, the P. pastoris yeast could be an appropriate host for the analysis of HCV polyprotein processing and, eventually, virus assembly.     

Cell-free analysis of Golgi apparatus membrane traffic in rat liver

 Cell-free systems for the analysis of Golgi apparatus membrane traffic rely either on highly purified cell fractions or analysis by specific trafficking markers or both. Our work has employed a cell-free transfer system from rat liver based on purified fractions. Transfer of any constituent present in the donor fraction that can be labeled (protein, phospholipid, neutral lipid, sterol, or glycoconjugate) may be investigated in a manner not requiring a processing assay. Transition vesicles were purified and Golgi apparatus cisternae were subfractionated by means of preparative free-flow electrophoresis. Using these transition vesicles and Golgi apparatus subfractions, transfer between transitional endoplasmic reticulum and cis Golgi apparatus was investigated and the process subdivided into vesicle formation and vesicle fusion steps. In liver, vesicle formation exhibited both ATP-independent and ATP-dependent components whereas vesicle fusion was ATP-independent. The ATP-dependent component of transfer was donor and acceptor specific and appeared to be largely unidirectional, i.e., ATP-dependent retrograde (cis Golgi apparatus to transitional endoplasmic reticulum) traffic was not observed. ATP-dependent transfer in the liver system and coatomer-driven ATP-independent transfer in more refined yeast and cultured cell systems are compared and discussed in regard to the liver system. A model mechanism developed for ATP-dependent budding is proposed where a retinol-stimulated and brefeldin A-inhibited NADH protein disulfide oxidoreductase (NADH oxidase) with protein disulfide-thiol interchange activity and an ATP-requiring protein capable of driving physical membrane displacement are involved. It has been suggested that this mechanism drives both the cell enlargement and the vesicle budding that may be associated with the dynamic flow of membranes along the endoplasmic reticulum-vesicle-Golgi apparatus-plasma membrane pathway. Accepted: 26 January 1998     

The budding mechanism of spikeless vesicular stomatitis virus particles.

Virus particles, lacking the spike G-glycoproteins, are produced during infection of Vero cells with the vesicular stomatitis virus mutant ts045 at the restrictive temperature 39.5 degrees C. At this temperature the mutated G proteins are blocked in their intracellular transport in the endoplasmic reticulum. We have studied the role of the G proteins in the formation of these spikeless virus particles. The results showed that the spikeless particles contain a full complement of membrane anchors, derived from the carboxy-terminal end of the G protein. Our observations suggest that virus particles are formed at the restrictive temperature with G protein which is later cleaved to produce spikeless particles. We suggest that this is due to a leak of G protein to the cell surface at 39.5 degrees C where budding then takes place, presumably driven by a G protein C-terminal tail--nucleocapsid interaction.     

Four distinct secretory pathways serve protein secretion, cell surface growth, and peroxisome biogenesis in the yeast Yarrowia lipolytica.

We have identified and characterized mutants of the yeast Yarrowia lipolytica that are deficient in protein secretion, in the ability to undergo dimorphic transition from the yeast to the mycelial form, and in peroxisome biogenesis. Mutations in the SEC238, SRP54, PEX1, PEX2, PEX6, and PEX9 genes affect protein secretion, prevent the exit of the precursor form of alkaline extracellular protease from the endoplasmic reticulum, and compromise peroxisome biogenesis. The mutants sec238A, srp54KO, pex2KO, pex6KO, and pex9KO are also deficient in the dimorphic transition from the yeast to the mycelial form and are affected in the export of only plasma membrane and cell wall-associated proteins specific for the mycelial form. Mutations in the SEC238, SRP54, PEX1, and PEX6 genes prevent or significantly delay the exit of two peroxisomal membrane proteins, Pex2p and Pex16p, from the endoplasmic reticulum en route to the peroxisomal membrane. Mutations in the PEX5, PEX16, and PEX17 genes, which have previously been shown to be essential for peroxisome biogenesis, affect the export of plasma membrane and cell wall-associated proteins specific for the mycelial form but do not impair exit from the endoplasmic reticulum of either Pex2p and Pex16p or of proteins destined for secretion. Biochemical analyses of these mutants provide evidence for the existence of four distinct secretory pathways that serve to deliver proteins for secretion, plasma membrane and cell wall synthesis during yeast and mycelial modes of growth, and peroxisome biogenesis. At least two of these secretory pathways, which are involved in the export of proteins to the external medium and in the delivery of proteins for assembly of the peroxisomal membrane, diverge at the level of the endoplasmic reticulum.     

Signal peptide peptidase-catalyzed cleavage of hepatitis C virus core protein is dispensable for virus budding but destabilizes the viral capsid

The capsid of hepatitis C virus (HCV) particles is considered to be composed of the mature form (p21) of core protein. Maturation to p21 involves cleavage of the transmembrane domain of the precursor form (p23) of core protein by signal peptide peptidase (SPP), a cellular protease embedded in the endoplasmic reticulum membrane. Here we have addressed whether SPP-catalyzed maturation to p21 is a prerequisite for HCV particle morphogenesis in the endoplasmic reticulum. HCV structural proteins were expressed by using recombinant Semliki Forest virus replicon in mammalian cells or recombinant baculovirus in insect cells, because these systems have been shown to allow the visualization of HCV budding events and the isolation of HCV-like particles, respectively. Inhibition of SPP-catalyzed cleavage of core protein by either an SPP inhibitor or HCV core mutations not only did not prevent but instead tended to facilitate the observation of viral buds and the recovery of virus-like particles. Remarkably, although maturation to p21 was only partially inhibited by mutations in insect cells, p23 was the only form of core protein found in HCV-like particles. Finally, newly developed assays demonstrated that p23 capsids are more stable than p21 capsids. These results show that SPP-catalyzed cleavage of core protein is dispensable for HCV budding but decreases the stability of the viral capsid. We propose a model in which p23 is the form of HCV core protein committed to virus assembly, and cleavage by SPP occurs during and/or after virus budding to predispose the capsid to subsequent disassembly in a new cell.