Autophagy delivers misfolded secretory proteins accumulated in endoplasmic reticulum to vacuoles in the filamentous fungus Aspergillus oryzae

Autophagy is a conserved intracellular degradation process of eukaryotic cells. In filamentous fungi, although autophagy has been reported to have multiple physiological roles, it is not clear whether autophagy is involved in the degradation of misfolded proteins. Here, we investigated the role of autophagy in the degradation of misfolded secretory proteins accumulated in endoplasmic reticulum (ER) in the filamentous fungus Aspergillus oryzae. In late-phase cultures, a disulfide bond-deleted mutant of the secretory protein α-amylase AmyB fused with mDsRed that had accumulated in the ER was subsequently delivered to vacuoles, whereas wild-type AmyB-mDsRed was predominantly located at cell walls and septa. To examine the involvement of autophagy in the delivery of mutant AmyB to vacuoles, mutant AmyB-EGFP was expressed in an A. oryzae autophagy-deficient strain (ΔAoatg8). Microscopic examination revealed that the protein delivery to vacuoles did not occur in the absence of autophagic activity, with mutant AmyB-mDsRed forming large spherical structures surrounded by ER membrane. Hence, we conclude that autophagy is responsible for the delivery of misfolded secretory proteins accumulated in the ER to vacuoles for degradation during late-growth phase in A. oryzae. This is the first study to provide evidence that autophagy plays a role in the degradation of misfolded secretory proteins in filamentous fungi.     

Vacuolar granules in Chlamydomonas reinhardtii: polyphosphate and a 70-kDa polypeptide as major components

The alga Chlamydomonas reinhardtii contains cytoplasmic vacuoles that are often filled with a dense granule that is released from the cell by exocytosis. Purified granules contained polyphosphate, complexed with calcium and magnesium, as the predominant inorganic components. Antiserum was raised against the major 70-kDa protein in granules purified from wall-deficient (cw15) mutants, which reacted on immunoblots with larger glycoprotein complexes in purified cell wall fractions from wild-type cells. Confocal fluorescence microscopy detected binding of these antibodies predominantly at the periphery of wall-containing C. reinhardtiiy1 cells but primarily to loci in the interior of cells of the cw15 strain. Immunoelectron microscopy demonstrated that the 70-kDa protein was localized in vacuolar granules and the trans-Golgi network in sections of cw15 cells but not in the cytosol or chloroplast. Treatment of cells with a dye, fluorescent in its protonated form, indicated that the pH within vacuoles was lower than that in the cytosol, which suggested that the vacuoles are similar to lysosomes. Thus, the vacuoles may serve a dual function to provide an environment for degradation within the cell and also serve as a vehicle for secretion of specific proteins. Received: 29 September 1999 / Accepted: 20 November 1999     

Eukaryotic Oligosaccharyltransferase Generates Free Oligosaccharides during N-Glycosylation

Asparagine (N)-linked glycosylation regulates numerous cellular activities, such as glycoprotein quality control, intracellular trafficking, and cell-cell communications. In eukaryotes, the glycosylation reaction is catalyzed by oligosaccharyltransferase (OST), a multimembrane protein complex that is localized in the endoplasmic reticulum (ER). During N-glycosylation in the ER, the protein-unbound form of oligosaccharides (free oligosaccharides; fOSs), which is structurally related to N-glycan, is released into the ER lumen. However, the enzyme responsible for this process remains unidentified. Here, we demonstrate that eukaryotic OST generates fOSs. Biochemical and genetic analyses using mutant strains of Saccharomyces cerevisiae revealed that the generation of fOSs is tightly correlated with the N-glycosylation activity of OST. Furthermore, we present evidence that the purified OST complex can generate fOSs by hydrolyzing dolichol-linked oligosaccharide, the glycan donor substrate for N-glycosylation. The heterologous expression of a single subunit of OST from the protozoan Leishmania major in S. cerevisiae demonstrated that this enzyme functions both in N-glycosylation and generation of fOSs. This study provides insight into the mechanism of PNGase-independent formation of fOSs.     

The Evolution of the Conserved ATPase Domain (CAD): Reconstructing the History of an Ancient Protein Module

The AAA proteins (ATPases Associated with a variety of cellular Activities) are found in eubacterial, archaebacterial, and eukaryotic species and participate in a large number of cellular processes, including protein degradation, vesicle fusion, cell cycle control, and cellular secretory processes. The AAA proteins are characterized by the presence of a 230 to 250-amino acid ATPase domain referred to as the Conserved ATPase Domain or CAD. Phylogenetic analysis of 133 CAD sequences from 38 species reveal that AAA CADs are organized into discrete groups that are related not only in structure but in cellular function. Evolutionary analyses also indicate that the CAD was present in the last common ancestor of eubacteria, archaebacteria, and eukaryotes. The eubacterial CADs are found in metalloproteases, while CAD-containing proteins in the archaebacterial and eukaryotic lineages appear to have diversified by a series of gene duplication events that lead to the establishment of different functional AAA proteins, including proteasomal regulatory, NSF/Sec, and Pas proteins. The phylogeny of the CADs provides the basis for establishing the patterns of evolutionary change that characterize the AAA proteins. Received: 28 January 1997 / Accepted: 8 May 1997     

Sequence elements essential for the rapid turnover of <Emphasis Type="Italic">Crithidia fasciculata</Emphasis> ornithine decarboxylase

Summary. Ornithine decarboxylase (ODC) has a very fast turnover in mammalian cells, but is a stable enzyme in T. brucei and other trypanosmatid parasites like Leishmania donovani. However, Crithidia fasciculata, which is a phylogenetically closely related trypanosomatid to L. donovani, has an ODC with a rapid turnover. Interestingly, C. fasciculata ODC, but not L. donovani ODC, is rapidly degraded also in mammalian systems. In order to obtain information on what sequences are important for the rapid degradation of C. fasciculata ODC, we produced a variety of C. fasciculata/L. donovani ODC hybrid proteins and characterized their turnover using two different mammalian expression systems. The results obtained indicate that C. fasciculata ODC contains several sequence elements essential for the rapid turnover of the protein and that these regions are mainly located in the central part of the enzyme. Present address: Department of Microbiology, Moyne Institute of Preventive Medicine, University of Dublin – Trinity College, Dublin, Ireland Authors’ address: Lo Persson, Department of Experimental Medical Science, Lund University, BMC D-12, S-221 84 Lund, Sweden     

Non-lysosomal degradation pathway for N-linked glycans and dolichol-linked oligosaccharides

There is growing evidence that asparagine (N)-linked glycans play pivotal roles in protein folding and intra- or intercellular trafficking of N-glycosylated proteins. During the N-glycosylation of proteins, significant amounts of free oligosaccharides (fOSs) and phosphorylated oligosaccharides (POSs) are generated at the endoplasmic reticulum (ER) membrane by unclarified mechanisms. fOSs are also formed in the cytosol by the enzymatic deglycosylation of misfolded glycoproteins destined for proteasomal degradation. This article summarizes the current knowledge of the molecular and regulatory mechanisms underlying the formation of fOSs and POSs in mammalian cells and Saccharomyces cerevisiae.     

Localization of mRNA for pea legumin:In situ hybridization using a biotinylated cDNA probe

Summary In situ hybridization has been used to locate mRNA, for the storage protein legumin, in cotyledon storage parenchyma tissue of developing pea (Pisum sativum L.) seeds. The mRNA was hybridized with a biotinylated probe of cDNA in pBR 322 and subsequently located by avidin conjugates. Avidin-rhodamine was used for fluorescence microscopy localization at a tissue/cellular level and avidin-peroxidase (with DAB) and avidin-ferritin compared for localization at an ultrastructural level. Specific fluorescence associated with avidin-rhodamine was distributed unevenly throughout the cytosol but the cell walls, starch grains, vacuoles and protein deposits were unstained. The sizes and distribution of the regions of higher labeling within the cytosol suggest an association with elements of the endomembrane system. Following DAB reaction of the specifically localized avidin-peroxidase most, although not all, stain product was associated with the endoplasmic reticulum. The ER-associated reaction product was also accumulated within the ER lumen.Avidin-ferritin was also localized both in the cytosol and in association with the endoplasmic reticulum, although was less readily visualized in cells with a conventional ultrastructural appearance.Localization of avidin-ferritin was more readily visualized in cells which had undergone some limited structural damage during specimen preparation. In such cases ferritin was also shown to be specifically associated with the transition vesicles and trans-face peripheral vesicles of some dictyosomes.     

Scanning N-glycosylation mutagenesis of membrane proteins

N-Glycosylation of eukaryotic membrane proteins is a co-translational event that occurs in the lumen of the endoplasmic reticulum (ER). This process is catalyzed by a membrane-associated oligosaccharyl transferase (OST) complex that transfers a preformed oligosaccharide (Glc₃Man₉GlcNAc₂-) to an asparagine (Asn) side-chain acceptor located within the sequon (-Asn-X-Ser/Thr-). Scanning N-glycosylation mutagenesis experiments, where novel acceptor sites are introduced at unique sites within membrane proteins, have shown that the acceptor sites must be located a minimum distance (12–14 amino acids) away from the luminal membrane surface of the ER in order to be efficiently N-glycosylated. Scanning N-glycosylation mutagenesis can therefore be used to determine membrane protein topology and it can also serve as a molecular ruler to define the ends of transmembrane (TM) segments. Furthermore, since N-glycosylation is a co-translational event, N-glycosylation mutagenesis can be used to identify folding intermediates in membrane proteins that may expose segments to the ER lumen transiently during biosynthesis.     

Impact of Geometrical Characteristics of the Organellar Store and Organelle-Free Cytosol on Intracellular Calcium Dynamics in the Dendrite: a Simulation Study

The dependence of intracellular calcium dynamics on geometrical size relations between calcium-exchanging parts of the intracellular space was studied in mathematical models corresponding to a thin fragment of the Purkinje neuron spiny dendrite. The plasma membrane contained ion channels typical of this cell type, including channels that conduct an excitatory synaptic current, and ion pumps. The model equations took into account calcium exchange between the cytosol, extracellular medium, intracellular store (a cistern of the endoplasmic reticulum, ER), endogenous calcium buffers, and an exogenous buffer (fluorescent dye used in the experiments). The ER membrane contained the calcium pump and channels of calcium-dependent and inositol-3-phosphate-dependent calcium release, as well as leakage channels. With the compartment size fixed, the ER cistern diameter was varied so that the proportion of the organelle in the total volume changed from 1 to 36%. Under these conditions, identical synaptic excitation caused similar electrical reactions (calcium spikes) but different concentration responses. Equal increments in the ER diameter led to unequal, more pronounced at thicker diameters, increments of the peak cytosolic concentrations of Са²⁺ ([Ca²⁺] _i ) and of a Са²⁺-fluorescent dye complex [CaD], as well as those of the Са²⁺ concentration in the dendrite ER (characterized by a shift from the basal level, Δ[Ca²⁺]_ER). The changes in [Ca²⁺] _i and [CaD] followed more adequately those in the volume of the organelle-free cytosol, while Δ[Ca²⁺]_ER changes were more similar to those in the ER membrane area. Therefore, the relative occupancy of the intracellular volume by organellar calcium stores and their sizes in a dendritic compartment are important structural factors that essentially modulate the calcium dynamics, and this structural dependence can be adequately reflected in the experiments using fluorophores. Neirofiziologiya/Neurophysiology, Vol. 41, No. 1, pp. 19–31, January–February, 2009.     

A bacterial toxin and a nonenveloped virus hijack ER-to-cytosol membrane translocation pathways to cause disease

Abstract

A dedicated network of cellular factors ensures that proteins translocated into the endoplasmic reticulum (ER) are folded correctly before they exit this compartment en route to other cellular destinations or for secretion. When proteins misfold, selective ER-resident enzymes and chaperones are recruited to rectify the protein-misfolding problem in order to maintain cellular proteostasis. However, when a protein becomes terminally misfolded, it is ejected into the cytosol and degraded by the proteasome via a pathway called ER-associated degradation (ERAD). Strikingly, toxins and viruses can hijack elements of the ERAD pathway to access the host cytosol and cause infection. This review focuses on emerging data illuminating the molecular mechanisms by which these toxic agents co-opt the ER-to-cytosol translocation process to cause disease.     

Immunohistochemical localization of estrogen receptors ERα and ERβ in the spiny mouse (<Emphasis Type="Italic">Acomys cahirinus</Emphasis>) ovary during postnatal development

This study was designed to determine the expression pattern of estrogen receptor (ER) subtypes in the Acomys cahirinus ovarian cells during its postnatal development. Immunohistochemical studies revealed the presence of ERα and ERβ in germinal epithelium cells and interstitial tissue. Both these ER subtypes were also seen in granulosa cells and oocytes of growing follicles, however, the level of ERβ expression was higher in comparison with ERα. In contrast to ERβ, ERα protein was also present in theca cells. The expression of ERs increased with animals’ age, but it decreased during follicular maturation. Moreover, the immunolocalization of ER subtypes in luteal cells showed that not ERβ, but ERα expression is up-regulated throughout corpus luteum development. These immunohistochemical studies demonstrate, for the first time, that ERα is also expressed in the mouse granulosa cells and it may be a mediator of estrogen action in granulosa cells proliferation and differentiation.     

Structural investigation of glycan recognition by the ERAD quality control lectin Yos9

Yos9 is an essential component of the endoplasmic reticulum associated protein degradation (ERAD) system that is responsible for removing terminally misfolded proteins from the ER lumen and mediating proteasomal degradation in the cytosol. Glycoproteins that fail to attain their native conformation in the ER expose a distinct oligosaccharide structure, a terminal α1,6-linked mannose residue, that is specifically recognized by the mannose 6-phoshate receptor homology (MRH) domain of Yos9. We have determined the structure of the MRH domain of Yos9 in its free form and complexed with 3α, 6α-mannopentaose. We show that binding is achieved by loops between β-strands performing an inward movement and that this movement also affects the entire β-barrel leading to a twist. These rearrangements may facilitate the processing of client proteins by downstream acting factors. In contrast, other oligosaccharides such as 2α-mannobiose bind weakly with only locally occurring chemical shift changes underscoring the specificity of this substrate selection process within ERAD.     

High capacity of the endoplasmic reticulum to prevent secretion and aggregation of amyloidogenic proteins

Protein aggregation is associated with neurodegeneration and various other pathologies. How specific cellular environments modulate the aggregation of disease proteins is not well understood. Here, we investigated how the endoplasmic reticulum (ER) quality control system handles β‐sheet proteins that were designed de novo to form amyloid‐like fibrils. While these proteins undergo toxic aggregation in the cytosol, we find that targeting them to the ER (ER‐β) strongly reduces their toxicity. ER‐β is retained within the ER in a soluble, polymeric state, despite reaching very high concentrations exceeding those of ER‐resident molecular chaperones. ER‐β is not removed by ER‐associated degradation (ERAD) but interferes with ERAD of other proteins. These findings demonstrate a remarkable capacity of the ER to prevent the formation of insoluble β‐aggregates and the secretion of potentially toxic protein species. Our results also suggest a generic mechanism by which proteins with exposed β‐sheet structure in the ER interfere with proteostasis.     

Sec61p mediates export of a misfolded secretory protein from the endoplasmic reticulum to the cytosol for degradation.

Degradation of misfolded secretory proteins has long been assumed to occur in the lumen of the endoplasmic reticulum (ER). Recent evidence, however, suggests that such proteins are instead degraded by proteasomes in the cytosol, although it remains unclear how the proteins are transported out of the ER. Here we provide the first genetic evidence that Sec61p, the pore-forming subunit of the protein translocation channel in the ER membrane, is directly involved in the export of misfolded secretory proteins. We describe two novel mutants in yeast Sec61p that are cold-sensitive for import into the ER in both intact yeast cells and a cell-free system. Microsomes derived from these mutants are defective in exporting misfolded secretory proteins. These proteins become trapped in the ER and are associated with Sec61p. We conclude that misfolded secretory proteins are exported for degradation from the ER to the cytosol via channels formed by Sec61p.     

Detection of ER stress in vivo by Raman spectroscopy

The endoplasmic reticulum (ER) is an organelle in which most membrane and secretory proteins are synthesized. If these proteins are not folded correctly, unfolded proteins accumulate in the ER lumen, causing a cellular situation known as ER stress. Recently, many studies on the relationship between ER stress and diseases have been reported. Thus, studies of ER stress in vivo should yield information that is useful in pathology. Model mice have been developed as a powerful tool to visualize ER stress in vivo, but this approach depends on transgenic technology. Here, we report on a method of detecting ER stress in vivo by Raman spectroscopy. Our experiments revealed that two specific Raman bands were reduced in both cultured cells and animal tissues in an ER stress dependent manner. This suggests that Raman spectroscopy could be a useful tool to detect ER stress in vivo without transgenic technology.     

The PH domain of Ras-GAP is sufficient forin Vitro binding toβγ subunits of heterotrimeric G proteins

Summary 1. The noncatalytic domain of Ras-GAP can affect signaling through G protein-coupled receptors by a poorly understood mechanism. 2. In this study, fusion proteins containing elements of the noncatalytic domain ofras-GAP were examined for their ability to bindβγ subunits of heterotrimeric G proteins and phosphotyrosine-containing polypeptides. 3. Our results demonstrate that purifiedβγ dimers associated with bacterially expressed GAP proteins and that this association does not require SH2 or SH3 domains but is dependent on the presence of the GAP pleckstrin-homology (PH) domain. In contrast, only the SH2 domains are necessary for binding to tyrosine phosphorylated proteins. 4. These findings raise the possibility that heterotrimeric G proteins might affect functioning ofras-like proteins throughβγ subunits acting on their regulatory molecules.     

Analysis of zeins' ER retention in Xenopus oocytes

Zeins, maize storage proteins, are retained in the endoplasmic reticulum (ER) during the intracellular protein targeting process. Hydrophobic interaction has been postulated as the driving force of zeins' aggregation and retention in the ER. Recently, a class of zein (the 27K zein) has been proposed to facilitate zeins' ER retention by anchoring to the ER membrane. This study investigated the significance of the two proposed mechanisms toward zeins' ER retention using Xenopus oocyte. Following injection of the total or 27K zein mRNA, zein's movement within the ER was analyzed based upon the extent of diffusion to the non-injected oocyte half. This study indicates that the total zeins freely move within the lumen of the ER, thus, suggesting that the intermolecular aggregation, leading to insolubility and exclusion from the ER lumenal fluid, may not be essential for zeins' ER retention. This study also suggests that the 27K zein may not facilitate zeins' ER retention by virtue of an anchor to the ER membrane based on its free movement in the ER. Free movement of the total and 27K zeins, under conditions where zein aggregates should form, necessitates a reevaluation of the mechanisms responsible for zein polypeptides' ER retention and protein body formation.     

Proteotoxic stress and ageing triggers the loss of redox homeostasis across cellular compartments

The cellular proteostasis network integrates the protein folding and clearance machineries in multiple sub‐cellular compartments of the eukaryotic cell. The endoplasmic reticulum (ER) is the site of synthesis and folding of membrane and secretory proteins. A distinctive feature of the ER is its tightly controlled redox homeostasis necessary for the formation of inter‐ and intra‐molecular disulphide bonds. Employing genetically encoded in vivo sensors reporting on the redox state in an organelle‐specific manner, we show in the nematode Caenorhabditis elegans that the redox state of the ER is subject to profound changes during worm lifetime. In young animals, the ER is oxidizing and this shifts towards reducing conditions during ageing, whereas in the cytosol the redox state becomes more oxidizing with age. Likewise, the redox state in the cytosol and the ER change in an opposing manner in response to proteotoxic challenges in C. elegans and in HeLa cells revealing conservation of redox homeostasis. Moreover, we show that organelle redox homeostasis is regulated across tissues within C. elegans providing a new measure for organismal fitness.     

PDI reductase acts on Akita mutant proinsulin to initiate retrotranslocation along the Hrd1/Sel1L-p97 axis

In mutant INS gene–induced diabetes of youth (MIDY), characterized by insulin deficiency, MIDY proinsulin mutants misfold and fail to exit the endoplasmic reticulum (ER). Moreover, these mutants bind and block ER exit of wild-type (WT) proinsulin, inhibiting insulin production. The ultimate fate of ER-entrapped MIDY mutants is unclear, but previous studies implicated ER-associated degradation (ERAD), a pathway that retrotranslocates misfolded ER proteins to the cytosol for proteasomal degradation. Here we establish key ERAD machinery components used to triage the Akita proinsulin mutant, including the Hrd1-Sel1L membrane complex, which conducts Akita proinsulin from the ER lumen to the cytosol, and the p97 ATPase, which couples the cytosolic arrival of proinsulin with its proteasomal degradation. Surprisingly, we find that protein disulfide isomerase (PDI), the major protein oxidase of the ER lumen, engages Akita proinsulin in a novel way, reducing proinsulin disulfide bonds and priming the Akita protein for ERAD. Efficient PDI engagement of Akita proinsulin appears linked to the availability of Hrd1, suggesting that retrotranslocation is coordinated on the lumenal side of the ER membrane. We believe that, in principle, this form of diabetes could be alleviated by enhancing the targeting of MIDY mutants for ERAD to restore WT insulin production.