Endoplasmic Reticulum

The endoplasmic reticulum (ER) is involved in several crucial cellular functions, e.g. protein folding and quality control, maintenance of Ca 2+ balance, and cholesterol synthesis. Many genetic and environmental insults can disturb the function of ER and induce ER stress. ER contains three branches of stress sensors, i.e. IRE1, PERK and ATF6 transducers, which recognize the misfolding of proteins in ER and activate a complex signaling network to generate the unfolded protein response (UPR). Alzheimer's disease (AD) is a progressive neurodegenerative disorder involving misfolding and aggregation of proteins in conjunction with prolonged cellular stress, e.g. in redox regulation and Ca 2+ homeostasis. Emerging evidence indicates that the UPR is activated in neurons but not in glial cells in AD brains. Neurons display pPERK, peIF2α and pIRE1α immunostaining along with abundant diffuse staining of phosphorylated tau protein. Recent studies have demonstrated that ER stress can also induce an inflammatory response via different UPR transducers. The most potent pathways are IRE1-TRAF2, PERK-eIF2α, PERK-GSK-3, ATF6-CREBH, as well as inflammatory caspase-induced signaling pathways. We will describe the mechanisms which could link the ER stress of neurons to the activation of the inflammatory response and the evolution of pathological changes in AD.

The B cell antigen receptor (BCR) comprises the membrane-bound immunoglobulin (mIg) molecule and the Ig-a/Ig-b heterodimer. By comparing the stability of the IgD-BCR and IgM-BCR in dierent detergent lysates, we ®nd that the IgD-BCR is more stable than the IgM-BCR. Analysis of chimeric mIgD molecules suggests that the dm transmembrane region is responsible for the more stable association of mIgD with the Ig-a/Ig-b heterodimer. Further, the dierential glycosylation of Iga molecules, in the two dierent BCR complexes, is determined solely by the ectodomains of the mIg molecules. The implications of these ®ndings for the intracellular transport and the signalling capacity of the BCRs are discussed.

three strains of mutant rats with congenital conjugated The workshop covered three major areas: Unconjuhyperbilirubinemia. (2) The roles of the classical and gated bilirubin (UCB) chemistry and physical chemistry; newer molecular biological approaches to identification UCB transport and intracellular trafficking; and evaluaof these transporters were contrasted, and their limitation and therapy of neonatal and congenital hyperbilitions were discussed. (3) The relative roles of the multirubinemias. Findings of studies in the chemistry and ple carriers in UCB transport under different conditions physical chemistry area were as follows. (1) Nuclear and substrate concentrations were discussed. (4) Cytomagnetic resonance (NMR) studies of highly enriched solic UCB-binding proteins (e.g., ligandin) were shown 13 COOH mesobilirubin in water-dimethyl sulfoxide systo promote transcellular movement of UCB by solubiliztems indicated that the pK a values of the carboxyl ing and transporting the pigment in the aqueous phase groups are 4.2 and 4.9, respectively. This finding differs while limiting binding of UCB to the relatively immobile from some reports that suggest that the two pK a values membranes of cell organelles. (5) Mechanisms were prein aqueous systems are near or above pH 7.0. (2) Consented for translocation of UDP-glucuronic acid (UDtrasting views of the hydrophobic interactions of UCB PGA) into the lumenal location of UDPGA transferase with bile salts were presented: one suggested that multiin the endoplasmic reticulum, as well as the enhanceple bile salt monomers bind to one UCB molecule; the ment of this process by N-acetyl-glucosamine. Studies other suggested that UCB binds to the nonpolar surface in the neonatal and congenital jaundice area were as of helical bile salt micelles. (3) Structures were proposed follows. (1) Criteria were reviewed for initiating treatfor the varied calcium and copper bilirubinate salts ment of neonatal jaundice, emphasizing the primacy of formed at various pH values and cation/UCB ratios. (4) serum bilirubin levels, gestational age, and hemolysis Studies of binding of UCB to human serum albumin as risk factors for kernicterus. (2) New methods were (HSA) showed marked diminution of UCB-binding affinpresented for frequent, automated monitoring of serum ity as albumin and chloride concentrations increased. bilirubin levels and breath CO levels as an index of rates (5) A unique UCB derivative, bilirubin-C10-sulfonic acid, of formation of UCB from heme. (3) The current status was identified as the major bile pigment in bullfrog bile. and limitations of new approaches to treatment of se-(6) New methods were presented for removal of impurivere unconjugated hyperbilirubinemia were discussed: ties from preparations of bile salts and UCB. Findings hepatocyte transplantation and gene therapy, still in the of studies in the transport area were as follows. (1) Four stage of development in animal models, have provided putative basolateral and two putative canalicular hepaonly partial and temporary relief of hyperbilirubinemia; tocytic transporters of UCB and related organic anions extracorporeal liver assist devices have had some sucwere described. Special emphasis was given to the adencess in initial human studies; and inhibition of heme osine triphosphate (ATP)-dependent canalicular multioxygenase (HO) with metalloporphyrins, especially tin specific organic anion transporter that is defective in mesoporphyrin, which markedly decreases bilirubin production for prolonged periods, is a new alternative Abbreviations: UCB, unconjugated bilirubin; NMR, nuclear magnetic resonance; NOE, to phototherapy. (4) The ontogeny of the two HO isonuclear Overhauser enhancement; DMSO, dimethyl sulfoxide; TC, taurocholate; CMC, criti-zymes was contrasted in the liver, spleen, kidney, and cal micellar concentration; CD, circular dichroism; Bf, concentration of unbound UCB; Kf,

The recent discovery of three closely related cell surface receptors that bind to progesterone and mediate its actions on various cytoplasmic signalling cascades has been heralded as a major break-through. The reason for this is all too obvious. Progesterone is an essential regulator of all major reproductive events and progestins and antiprogestins are widely used in the treatment of many different gynaecological and obstetrical disorders. The novel membrane progestin receptors (mPRalpha, beta, gamma) reportedly resemble and function as G-protein-coupled receptors and therefore are promising pharmaceutical targets. However, our studies failed to corroborate that mPRs are expressed on the cell surface, that they mediate rapid progesterone signalling events, and even that they are bona fide progestin binding moieties. While the reason for these startling opposing results remains unclear, a critical review of existing data may help to shed some light onto the controversial mPRs. Time has come to talk.

Cytoplasmic lipid droplets (LDs) are found in all types of plant cells; they are derived from the endoplasmic reticulum and function as a repository for neutral lipids, as well as serving in lipid remodelling and signalling. However, the mechanisms underlying the formation, steady-state maintenance and turnover of plant LDs, particularly in non-seed tissues, are relatively unknown. Previously, we showed that the LD-associated proteins (LDAPs) are a family of plant-specific, LD surface-associated coat proteins that are required for proper biogenesis of LDs and neutral lipid homeostasis in vegetative tissues. Here, we screened a yeast two-hybrid library using the Arabidopsis LDAP3 isoform as 'bait' in an effort to identify other novel LD protein constituents. One of the candidate LDAP3-interacting proteins was Arabidopsis At5g16550, which is a plant-specific protein of unknown function that we termed LDIP (LDAP-interacting protein). Using a combination of biochemical and cellu...

Lymph nodes from sheep foetuses and postnatal lambs were examined to determine the participation of different leucocyte populations in primary follicle formation, with special emphasis on the emergence and subsequent development of follicular dendritic cells during late gestation and early postnatal life. A series of immune and enzyme histochemical markers was used. The first 5'-nucleotidase-positive primary follicles were found at 80 days gestational age (gestation in sheep is 150 days) in superficial cervical lymph nodes. In the last month of gestation the primary follicles possessed follicular dendritic cells, macrophages, dendritic cells, and CD5-positive lymphocytes, in addition to IgM-positive cells. Follicular dendritic cells in primary follicles were found to be ultrastructurally immature. These follicular dendritic cells were characterised by a few, coarse surface projections and many ribosomes attached to the endoplasmic reticulum. A final differentiation to mature follicular dendritic cells was coincident with the postnatal germinal centre reaction. Computer-assisted morphometric analysis demonstrated that the size of 5'-nucleotidase-positive primary follicles in the distal jejunal lymph node, but not in the superficial cervical lymph node, increased significantly during late gestation. It was concluded that stroreal cells in primary follicles of foetal sheep lymph nodes were a continuously developing population but that ultrastructural maturity was only achieved in the germinal centres of postnatal lambs.

Aberrant keratinocyte differentiation is considered to be a key mechanism in the onset of hyperproliferative dermatological diseases, including basal cell carcinoma (BCC). It is, therefore, vital to understand what drives keratinocytes to develop such pathological phenotypes.

aERD2 and aSARl of Arabidopsis are functional homologs of yeast genes encoding proteins essential for endoplasmic reticulum (ER)-to-Golgi transport. The regulation of these secretory pathway genes in yeast, mammals, and plants is not known. High levels of expression of aERD2 and aSARl were observed in roots, flowers, and inflorescence stems, with the highest levels being detected in roots. The aSARl transcript levels were highest in young leaves and declined during leaf maturation. Low levels of aERD2 were detected in both young and fully mature leaves when compared with roots. In situ hybridization showed that trichomes accumulate more aERD2 transcript as the leaf expands, whereas aSARl is expressed equally in ali leaf cell types. Teating plants with tunicamycln, a drug that blocks N-glycosylation in the ER, or with cold shock, known to block secretory protein transport, led to a marked accumulation of aERD2 and aSARl transcripts. The Arabidopsis ARF gene, which encodes a GTPase probably involved in Golgi vesicle traffic, was not affected by these treatments. This study is an essential first step toward understanding the regulation of genes that encode proteins involved in vesicular trafficking.

Matrix metalloproteinase-2 (MMP-2) is best understood for its biological actions outside the cell. However, MMP-2 also localizes to intracellular compartments and the cytosol where it has several substrates, including troponin I (TnI). Despite a growing list of cytosolic substrates, we currently do not know the mechanism(s) that give rise to the equilibrium between intracellular and secreted MMP-2 moieties. Therefore, we explored how cells achieve the unique distribution of this protease. Our data show that endogenous MMP-2 targets inefficiently to the endoplasmic reticulum (ER) and shows significant amounts in the cytosol. Transfection of canonical MMP-2 essentially reproduces this targeting pattern, suggesting it is the quality of the MMP-2 signal sequence that predominantly determines MMP-2 targeting. However, we also found that human cardiomyocytes express an MMP-2 splice variant which entirely lacks the signal sequence. Like the fraction of ER-excluded, full-length MMP-2, this variant MMP-2 is restricted to the cytosol and specifically enhances TnI cleavage upon hypoxia-reoxygenation injury in cardiomyocytes. Together, our findings describe for the first time a set of mechanisms that cells utilize to equilibrate MMP-2 both in the extracellular milieu and intracellular, cytosolic locations. Our results also suggest approaches to specifically investigate the overlooked intracellular biology of MMP-2.

Gaucher disease is a lysosomal storage disorder caused by deficiency in lysosomal acid ␤-glucosidase (GlcCerase), the enzyme responsible for the catabolism of glucosylceramide. One of the most prevalent disease-causing mutations, N370S, results in an enzyme with lower catalytic activity and impaired exit from the endoplasmic reticulum. Here, we report that the iminosugar isofagomine (IFG), an active-site inhibitor, increases GlcCerase activity 3.0 ؎ 0.6-fold in N370S fibroblasts by several mechanisms. A major effect of IFG is to facilitate the folding and transport of newly synthesized GlcCerase in the endoplasmic reticulum, thereby increasing the lysosomal pool of the enzyme. In addition, N370S GlcCerase synthesized in the presence of IFG exhibits a shift in pH optimum from 6.4 to 5.2 and altered sensitivity to SDS. Although IFG fully inhibits GlcCerase in the lysosome in an in situ assay, washout of the drug leads to partial recovery of GlcCerase activity within 4 h and full recovery by 24 h. These findings provide support for the possible use of active-site inhibitors in the treatment of some forms of Gaucher disease.

Untreated cystathionine ␤-synthase (CBS) deficiency in humans is characterized by extremely elevated plasma total homocysteine (tHcy>200 M), with thrombosis as the major cause of morbidity. Treatment with vitamins and diet leads to a dramatic reduction in thrombotic events, even though patients often still have severe elevations in tHcy (>80 M). To understand the difference between extreme and severe hyperhomocysteinemia, we have examined two mouse models of CBS deficiency: Tg-hCBS Cbs Ϫ/Ϫ mice, with a mean serum tHcy of 169 M, and Tg-I278T Cbs Ϫ/Ϫ mice, with a mean tHcy of 296 M. Only Tg-I278T Cbs Ϫ/Ϫ animals exhibited strong biological phenotypes, including facial alopecia, osteoporosis, endoplasmic reticulum (ER) stress in the liver and kidney, and a 20% reduction in mean survival time. Metabolic profiling of serum and liver reveals that Tg-I278T Cbs Ϫ/Ϫ mice have significantly elevated levels of free oxidized homocysteine but not protein-bound homocysteine in serum and elevation of all forms of homocysteine and S-adenosylhomocysteine in the liver compared to Tg-hCBS Cbs Ϫ/Ϫ mice. RNA profiling of livers indicate that Tg-I278T Cbs Ϫ/Ϫ and Tg-hCBS Cbs Ϫ/Ϫ mice have unique gene signatures, with minimal overlap. Our results indicate that there is a clear pathogenic threshold effect for tHcy and bring into question the idea that mild elevations in tHcy are directly pathogenic. Mouse models of cystathionine ␤-synthase deficiency reveal significant threshold effects of hyperhomocysteinemia. FASEB J. 23, 883-893 (2009) Key Words: osteoporosis ⅐ ER stress ⅐ inborn error of metabolism ⅐ sulfur metabolism Cystathionine ␤-synthase (CBS) deficiency is the most common inborn error of sulfur metabolism and is the cause of classical homocystinuria, a condition char-

A series of 10 1,4-bis(3-aminopropyl)piperazine compounds was found to display antiplasmodial activity with 50% growth inhibition between 30 and 250 nM, on three Plasmodium falciparum strains differently sensitive to chloroquine. By affinity chromatography using one of these compounds, a 52-kDa protein was isolated from P. falciparum, microsequenced and cloned. It corresponded to a single copy gene encoding a 453 amino acid protein displaying the typical features of protein disulfide isomerases, a thiol metabolizing enzyme belonging to the thiol : disulfide oxidoreductase superfamily, which was not previously described in malarial species. ß

Darier's disease (DD) is an autosomal dominant genodermatology. Mutations in the ATP2A2 gene encoding sarco-endoplasmic reticulum calcium pumping ATPase type 2 (SERCA2) have been identiWed as the molecular basis of DD. The aim of this study was to report two Chinese pedigree of DD and to explore the genetic mutations. Polymerase chain reaction was carried out to amplify the exons and Xanking intron boundaries of the ATP2A2 gene followed by direct sequencing. Two novel missense mutations were identiWed, a change of C203 to A (A68E) in exon 3 was found in one family and a change of C2759 to T (S920F) in exon 19 in the other, which were located within the transmembrane domain of SERCA2, highly conserved during evolution. The A68E and S920F mutations might be regarded as the causes of the disease in two Chinese families, but these were not tested functionally. Additional functional experiments are necessary to verify the relevance and suitability of these Wndings for future use in genetic counseling and prenatal diagnosis.

Celastrol, a natural substance isolated from plant extracts used in traditional Chinese medicine, has been extensively investigated as a possible drug for treatment of cancer, autoimmune diseases, and protein misfolding disorders. Although studies focusing on celastrol's effects in specific cellular pathways have revealed a considerable number of targets in a diverse array of in vitro models there is an essential need for investigations that can provide a global view of its effects. To assess cellular effects of celastrol and to identify target proteins as biomarkers for monitoring treatment regimes, we performed large-scale quantitative proteomics in cultured human lymphoblastoid cells, a cell type that can be readily prepared from human blood samples. Celastrol substantially modified the proteome composition and 158 of the close to 1800 proteins with robust quantitation showed at least a 1.5 fold change in protein levels. Up-regulated proteins play key roles in cytoprotection with a prominent group involved in quality control and processing of proteins traversing the endoplasmic reticulum. Increased levels of proteins essential for the cellular protection against oxidative stress including heme oxygenase 1, several peroxiredoxins and thioredoxins as well as proteins involved in the control of iron homeostasis were also observed. Specific analysis of the mitochondrial proteome strongly indicated that the mitochondrial association of certain antioxidant defense and apoptosis-regulating proteins increased in cells exposed to celastrol. Analysis of selected mRNA transcripts showed that celastrol activated several different stress response pathways and dose response studies furthermore showed that continuous exposure to sub-micromolar concentrations of celastrol is associated with reduced cellular viability and proliferation. The extensive catalog of regulated proteins presented here identifies numerous cellular effects of celastrol and constitutes a valuable biomarker tool for the development and monitoration of disease treatment strategies.

Antigen recognition by ab T lymphocytes is mediated via the multisubunit TCR complex consisting of invariant CD3g,d,o and z chains associated with clonotypic TCRa and b molecules. Charged amino acids located centrally within the TCRa transmembrane region are necessary and sufficient for assembly with the CD3do heterodimer. Previously, we have shown that deletion of 6-12 amino acids from the carboxy terminus of the TCRa-chain dramatically abrogates surface TCR expression, suggesting that the distal portion of the TCRa transmembrane region contains information that regulates the assembly and/or intracellular transport of TCR complexes. We have examined in more detail the molecular basis for reduced TCR expression in T cells bearing truncated TCRa chains. We found that in contrast to wild-type (wt), variant TCRa proteins missing the last nine C-terminal amino acids did not associate with core CD3g,d,o chains and were not assembled into disulphide-linked ab heterodimers. The stability of newly synthesised wt and variant TCRa molecules was similar, showing that the abrogated surface TCR expression was not a consequence of impaired protein survival. Nevertheless, truncated TCRa chains still assembled with the chaperon protein calnexin in the endoplasmic reticulum, indicating that the distal portion of the TCRa transmembrane region is not essential for calnexin interaction. These data document a role for the distal portion of the TCRa transmembrane region in the assembly of TCR complexes and provide a molecular basis for reduced TCR expression in cells bearing truncated TCRa chains.

Mitochondria promptly respond to Ca 2+ -mediated cell stimulations with a rapid accumulation of the cation into the matrix. In this article, we review (i) the basic principles of mitochondrial Ca 2+ transport, (ii) the physiological/pathological role of mitochondrial Ca 2+ uptake, (iii) the regulatory mechanisms that may operate in vivo, and (iv) the new targeted Ca 2+ probes that allowed the brediscoveryQ of these organelles in calcium signalling. D

Hypersensitivity pneumonitis (HP) is a lung inflammatory disease caused by the inhalation of a variety of antigens. Previous studies support the role of the major histocompatibility complex (MHC) class II genes in the susceptibility to develop HP. However, the putative role of other MHC loci has not been elucidated. Transporters associated with antigen processing (TAP) genes are located within the MHC class II region and play an important role transporting peptides across the endoplasmic reticulum membrane for MHC class I molecules assembly. The distribution of single nucleotide polymorphisms (SNPs) in TAP1 genes was analyzed in 73 hypersensitivity pneumonitis (HP) patients and 58 normal subjects. We found a significant association of the allele Gly-637 GGC) ( p = 0.00004, OR = 27.30, CI = 3.87-548.04) and the genotypes Asp-637/Gly-637 ( p = 0.01, OR = 16.0, CI = 2.19-631.21), Pro-661/Pro-661 ( p = 0.006, OR = 11.30, CI = 2.28-75.77) with HP. A significant decrease in the frequency of the allele Pro-661 (CCA) ( p = 0.008, OR = 0.06, CI = 0-0.45), the genotype Asp-637/Asp-637 ( p = 0.01, OR = 0.17, 95% CI = 0.05-0.58) and the haplotype [Val-333 (GTC), Val-458 (GTG), Gly-637 (GGC), Pro-661 (CCA)] was detected in HP patients compared with controls ( p = 0.002, OR = 0.07, CI = 0.0-0.57). These findings suggest that TAP1 gene polymorphisms are related to HP risk, and highlight the importance of the MHC in the development of this disease.

The Sec61 complex is the major protein translocation channel of the endoplasmic reticulum (ER), where it plays a central role in the biogenesis of membrane and secretory proteins. Whilst Sec61-mediated protein translocation is typically coupled to polypeptide synthesis, suggestive of significant complexity, an obvious characteristic of this core translocation machinery is its surprising simplicity. Over thirty years after its initial discovery, we now understand that the Sec61 complex is in fact the central piece of an elaborate jigsaw puzzle which can be partly solved using new research findings. We propose that the Sec61 complex acts as a dynamic hub for co-translational protein translocation at the ER, proactively recruiting a range of accessory complexes that enhance and regulate its function in response to different protein clients. It is now clear that the Sec61 complex does not have a monopoly on co-translational insertion, with some transmembrane proteins preferentially utilising the ER membrane complex instead. We also have a better understanding of post-insertion events, where at least one membrane-embedded chaperone complex can capture the newly inserted transmembrane domains of multi-span proteins and co-ordinate their assembly into a native structure. Having discovered this array of Sec61-associated components and competitors, our next challenge is to understand how they act together in order to expand the range and complexity of the membrane proteins that can be synthesised at the ER. Furthermore, this diversity of components and pathways may open up new opportunities for targeted therapeutic interventions designed to selectively modulate protein biogenesis at the ER.

The assembly of atherogenic lipoproteins requires the formation in the endoplasmic reticulum of a complex between apolipoprotein (apo)B, a microsomal triglyceride transfer protein (MTP) and protein disulphide isomerase (PDI). Here we show by molecular modelling and mutagenesis that the globular amino-terminal regions of apoB and MTP are closely related in structure to the ancient egg yolk storage protein, vitellogenin (VTG). In the MTP complex, conserved structural motifs that form the reciprocal homodimerization interfaces in VTG are re-utilized by MTP to form a stable heterodimer with PDI, which anchors MTP at the site of apoB translocation, and to associate with apoB and initiate lipid transfer. The structural and functional evolution of the VTGs provides a unifying scheme for the invertebrate origins of the major vertebrate lipid transport system.

As a first step in developing compartment-specific markers for protein trafficking within Theileria par6a, we have isolated cDNAs encoding homologues of the small GTP binding proteins Rab1 and Rab4. The T. par6a homologue of Rab1 (TpRab1), a protein which regulates vesicular transport between the endoplasmic reticulum and cis golgi in other organisms, was unusual in that it contained a unique 17 amino acid C-terminal extension. The C-terminal motif sequence KCT (XCX) contrasted with the CXC or XCC motifs which act as as signals for isoprenylation by geranylgeranyl in most Rab proteins, including all known Rab1 homologues, in containing only a single cysteine. [C 14 ]mevalonic acid lactone and [H 3 ]geranylgeranyl pyrophosphate were specifically incorporated into recombinant TpRab1 in vitro, demonstrating that the novel motif was functional for isoprenylation. Recombinant TpRab1 bound radiolabeled GTP, and this binding was inhibited by excess unlabeled GTP and GDP and also partially by ATP. The TpRab1 gene contained four short (34-67 bp) introns with a distinct pattern of occurrence within the protein sequence as compared to the introns of other lower eukaryote Rab1 genes. Immunofluorescence microscopy using antiserum specific for the novel C-terminal peptide in combination with labelling of cells using the nucleic acid-staining dye DAPI, indicated that TpRab1 was located in the vicinity of the schizont nucleus within the infected lymphocyte.

Cholera toxin, secreted by Vibrio cholerae, consists of A and B subunits. The latter binds to G M1 -ganglioside receptors as a pentamer (∼55 kDa). Tomato plants were transformed with the gene encoding cholera toxin B subunit (ctxB) along with an endoplasmic reticulum retention signal (SEKDEL) under the control of the CaMV 35S promoter via Agrobacterium-mediated transformation. PCR and Southern analysis confirmed the presence of the ctxB gene in transformed tomato plants. Northern analysis showed the presence of the ctxB-specific transcript. Immunoblot assays of the plant-derived protein extract showed the presence of cholera toxin subunit B (CTB) with mobility similar to purified CTB from V. cholerae. Both tomato leaves and fruits expressed CTB at levels up to 0.02 and 0.04% of total soluble protein, respectively. The G M1 -ELISA showed that the plant-derived CTB bound specifically to G M1 -ganglioside receptor, suggesting that it retained its native pentameric form. This study forms a basis for exploring the utility of CTB to develop tomato-based edible vaccines against cholera.

Polypeptide growth factors are a diverse group of hormone-like agents that regulate growth and differentiation through cell surface receptors. They are generally represented by homologous families containing several members with distinct overlapping receptor interactions and hence, responsive h u e specificities. Similarly, their receptors are also clustered in family groups of sequence-related proteins.

The multifunctional AAA-ATPase p97 is one of the most abundant and conserved proteins in eukaryotic cells. The p97/Npl4/Ufd1 complex dislocates proteins that fail the protein quality control in the endoplasmic reticulum to the cytosol where they are subject to degradation by the ubiquitin/proteasome system. Substrate dislocation depends on the unfoldase activity of p97. Interestingly, p97 is also involved in the degradation of specific soluble proteasome substrates but the exact mode of action of p97 in this process is unclear. Here, we show that both the central pore and ATPase activity of p97 are necessary for the degradation of cytosolic ubiquitin-fusion substrates. Addition of a flexible extended Cterminal peptide to the substrate relieves the requirement for p97. Deletion mapping reveals a conserved length dependency of 20 residues for the peptide, which allows p97-independent degradation to occur. Our results suggest that initiation of unfolding may be more complex than previously anticipated and that the 19S regulatory complex of the proteasome can require preprocessing of highly folded, ubiquitylated substrates by the p97 Ufd1/Npl4 complex. Our data provide an explanation for the observation that p97 is only essential for a subpopulation of soluble substrates and predict that a common characteristic of soluble p97-dependent substrates is the lack of an initiation site to facilitate unfolding by the 26S proteasome.

In the fatal neurodegenerative disease amyotrophic lateral sclerosis (ALS), motor neurons degenerate with signs of organelle fragmentation, free radical damage, mitochondrial Ca 2+ overload, impaired axonal transport and accumulation of proteins in intracellular inclusion bodies. Subgroups of motor neurons of the brainstem and the spinal cord expressing low amounts of Ca 2+ buffering proteins are particularly vulnerable. In ALS, chronic excitotoxicity mediated by Ca 2+ -permeable AMPA type glutamate receptors seems to initiate a self-perpetuating process of intracellular Ca 2+ dysregulation with consecutive endoplasmic reticulum Ca 2+ depletion and mitochondrial Ca 2+ overload. The only known effective treatment, riluzole, seems to reduce glutamatergic input. This review introduces the hypothesis of a "toxic shift of Ca 2+ " within the endoplasmic reticulum-mitochondria Ca 2+ cycle (ERMCC) as a key mechanism in motor neuron degeneration, and discusses molecular targets which may be of interest for future ERMCC modulating neuroprotective therapies.

Each stage of preimplantation development in the mouse from the fertilized egg to the blastocyst stage (including the unfertilized egg) was studied cytologically and ultrastructurally. Observations were made on the appearance and elaboration of several cellular organelles, inclusions and cell surface specilizations. The fertilized egg exhibits many intranuclear annulate lamellae, an increase in cytoplasmic vesicle number when compared to the unfertilized egg, and small amounts of crystalloids; mitochondria are vacuolated and small. The 2cell stage is very similar to the fertilized egg but shows an increase in the number of cytoplasmic vesicles.

To elucidate the mechanism underlying transport and processing defects from the viewpoint of enzyme folding, we constructed three-dimensional models of human acid a-glucosidase encompassing 27 relevant amino acid substitutions by means of homology modeling. Then, we determined in each separate case the number of affected atoms, the root-mean-square distance value and the solvent-accessible surface area value. The analysis revealed that the amino acid substitutions causing a processing or transport defect responsible for Pompe disease were widely spread over all of the five domains comprising the acid a-glucosidase. They were distributed from the core to the surface of the enzyme molecule, and the predicted structural changes varied from large to very small. Among the structural changes, we paid particular attention to G377R and G483R. These two substitutions are predicted to cause electrostatic changes in neighboring small regions on the molecular surface. The quality control system of the endoplasmic reticulum apparently detects these very small structural changes and degrades the mutant enzyme precursor (G377R), but also the cellular sorting system might be misled by these minor changes whereby the precursor is secreted instead of being transported to lysosomes (G483R).

δ-Helices are marginally hydrophobic α-helical segments in soluble proteins that exhibit certain sequence characteristics of transmembrane (TM) helices [Cunningham, F., Rath, A., Johnson, R. M. & Deber, C. M. (2009). Distinctions between hydrophobic helices in globular proteins and TM segments as factors in protein sorting. J. Biol. Chem., 284, 5395–402]. In order to better understand the difference between δ-helices and TM helices, we have studied the insertion of five TM-like δ-helices into dog pancreas microsomal membranes. Using model constructs in which an isolated δ-helix is engineered into a bona fide membrane protein, we find that, for two δ-helices originating from secreted proteins, at least three single-nucleotide mutations are necessary to obtain efficient membrane insertion, whereas one mutation is sufficient in a δ-helix from the cytosolic protein P450BM-3. We further find that only when the entire upstream region of the mutated δ-helix in the intact cytochrome P450BM-3 is deleted does a small fraction of the truncated protein insert into microsomes. Our results suggest that upstream portions of the polypeptide, as well as embedded charged residues, protect δ-helices in globular proteins from being recognized by the signal recognition particle–Sec61 endoplasmic-reticulum-targeting machinery and that δ-helices in secreted proteins are mutationally more distant from TM helices than δ-helices in cytosolic proteins.► δ-Helices are marginally hydrophobic α-helical segments in soluble proteins that exhibit certain sequence characteristics of TM helices. ► We have studied the membrane integration potential of δ-helices selected from secreted and cytosolic proteins. ► Three mutations can turn δ-helices from secreted proteins into TM helices. ► A single mutation turns a δ-helix from a cytosolic protein into a TM helix. ► Upstream secondary structure protects hydrophobic helices from membrane integration.

Although plant plastidial ω3-desaturases are closely related to microsomal desaturases, heterologous expression in yeast of the Helianthus annuus FAD7 ω3-desaturase showed low activity in contrast to similar expression of microsomal FAD3 ω3-desaturases. However, the removal of the plastidial transit peptide and the incorporation of a KKNL motif to the C-terminus of HaFAD7 increased the activity by 10-fold compared to the native protein. N-terminal fusion of transmembrane-domains from either the yeast microsomal ELO3, (a type III signal anchor domain), or FAE1, an endoplasmic reticulum membrane anchoring domain, resulted in moderate increases in enzyme activity (5- and 7-fold, respectively), suggesting that the first, most hydrophobic transmembrane domain of HaFAD7 is sufficient to direct targeting to, and insertion into, the endoplasmic reticulum membrane. Furthermore, fusing a hemagglutinin (HA) epitope tag upstream of an endogenous C-terminal KEK motif resulted in a significant loss of activity compared to the un-tagged construct, indicating that the endogenous KEK C-terminal di-lysine motif is capable of directing in yeast the ER-retention of this normally plastidial-located protein. Western blotting analysis of constructs with internal HA epitope revealed that in whole cell extracts, with the exception of the one bound to C-terminal, it did not display a reduced level of protein accumulation. Whilst ferredoxin was shown to be required for HaFAD7 activity in yeast, it appears not necessary for protein stability and accumulation of this plastidial desaturase in the endoplasmic reticulum.Metabolic engineering is focus in the production of a huge diversity of polyunsaturated fatty acids with an economical interest. In this study, we characterized factors contributing to improve the activity of sunflower plastidial ω3-desaturase, HaFAD7, expressed in yeast.

Membrane-enveloped vesicles travel among the compartments of the cytoplasm of eukaryotic cells, delivering their specific cargo to programmed locations by membrane fusion. The pairing of vesicle v-SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) with target membrane t-SNAREs has a central role in intracellular membrane fusion. We have tested all of the potential v-SNAREs encoded in the yeast genome for their capacity to trigger fusion by partnering with t-SNAREs that mark the Golgi, the vacuole and the plasma membrane. Here we find that, to a marked degree, the pattern of membrane flow in the cell is encoded and recapitulated by its isolated SNARE proteins, as predicted by the SNARE hypothesis.

The soybean binding protein (BiP) gene family consists of at least four members designated soyBiPA, soyBiPB, soyBiPC and soyBiPD. We have performed immunoblotting of two-dimensional (2D) gels and RT-PCR assays with gene-specific primers to analyze the differential expression of this gene family in various soybean organs. The 2D gel profiles of the BiP forms from different organs were distinct and suggested that the BiP genes are under organ-specific regulation. In fact, while all four BiP transcripts were detected in leaves by gene-specific reverse transcriptase-polymerase chain reaction (RT-PCR) assays, different subsets were detected in the other organs. The soyBiPD was expressed in all organs, whereas the expression of the soyBiPB was restricted to leaves. The soyBiPA transcripts were detected in leaves, roots and seeds and soyBiPC RNA was confined to leaves, seeds and pods. Our data are consistent with organ-specific expression of the soybean BiP gene family.

Xestospongins, a group of macrocyclic bis-1-oxaquinolizidines isolated from the Australian sponge, Xestospongia species, are potent blockers of the inositol 1,4,5-trisphosphate (IP 3 )-induced Ca 2+ release in bi-directional Ca 2+ -flux conditions. We have now studied the effects of xestospongin C on the 45 Ca 2+ uptake and the uni-directional 45 Ca 2+ efflux in permeabilized A7r5 smooth-muscle cells. Xestospongin C not only inhibits the IP 3 -induced Ca 2+ release, but is also an equally potent blocker of the endoplasmic-reticulum Ca 2+ pump, while it has no effect on the passive Ca 2+ leak. The inhibition of the IP 3 receptor did not depend on the IP 3 , Ca 2+ or ATP concentration. Xestospongin C can, therefore, not be considered as a selective blocker of IP 3 receptors.

In this article, we will cover the folding of proteins in the lumen of the endoplasmic reticulum (ER), including the role of three types of covalent modifications: signal peptide removal, Nlinked glycosylation, and disulfide bond formation, as well as the function and importance of resident ER folding factors. These folding factors consist of classical chaperones and their cochaperones, the carbohydrate-binding chaperones, and the folding catalysts of the PDI and proline cis-trans isomerase families. We will conclude with the perspective of the folding protein: a comparison of characteristics and folding and exit rates for proteins that travel through the ER as clients of the ER machinery.

The genomic era facilitates the understanding of how transcriptional networks are interconnected to program seed development and filling. However, to date, little information is available regarding dicot seeds with a transient perisperm and a persistent, copious endosperm. Coffea arabica is the subject of increasing genomic research and is a model for nonorthodox albuminous dicot seeds of tropical origin.The aim of this study was to reconstruct the metabolic pathways involved in the biosynthesis of the main coffee seed storage compounds, namely cell wall polysaccharides, triacylglycerols, sucrose, and chlorogenic acids. For this purpose, we integrated transcriptomic and metabolite analyses, combining real-time RT-PCR performed on 137 selected genes (of which 79 were uncharacterized in Coffea) and metabolite profiling.Our map-drawing approach derived from model plants enabled us to propose a rationale for the peculiar traits of the coffee endosperm, such as its unusual fatty acid composition, remarkable accumulation of chlorogenic acid and cell wall polysaccharides.Comparison with the developmental features of exalbuminous seeds described in the literature revealed that the two seed types share important regulatory mechanisms for reserve biosynthesis, independent of the origin and ploidy level of the storage tissue.

proteins are structurally related transmembrane proteins that have been studied in and Drosophila and yeast. In Drosophila, Cornichon (Cni) is involved in embryo polarization by the TGF␣-related Gurken. In yeast, the Cni-related Erv14 is required for axial budding. A cargo receptor function has been proposed for Erv14 and Cni. Four mammalian Cni-like sequences have been identified. We carried out parallel functional analyses of the human Cni ortholog CNIH and Drosophila Cni in the processing and presentation of TGF␣ family proteins. Human CNIH complements the loss of Erv14 in yeast. Human CNIH and Drosophila Cni are primarily localized in the endoplasmic reticulum and associate with immature TGF␣ family proteins. Alterations of cornichon expression result in changes in transport, processing and secretion of TGF␣ proteins. In particular, increased cornichon expression retains TGF␣ proteins in the endoplasmic reticulum, whereas cornichon is required for their transport and secretion. Thus, cornichon proteins represent a functionally conserved protein family that acts in the selective transport and maturation of TGF␣ family proteins.

This article describes the ultrastructural events associated with the differentiation and liberation of the exogenous gemmae produced in branched acropetal chains along the margins of the leaves in the liverwort Odontoschisma denudatum. Formation of a dorsal protrusion from young leaf cells containing a large central nucleus, small vacuoles, starch-free chloroplasts, scattered cytoplasmic lipid droplets but no oil bodies, signals the onset of the formation of the initial cell of a gemmiferous filament. The protrusion enlarges and the nucleus migrates into its base, therein dividing with the equator of the spindle virtually filling the central isthmus between the leaf surface and the now swollen tip of the initial cell. Subsequent divisions of the initial cell produce a chain of cells in atropetal succession. Transversely orientated microtubules line the cortical cytoplasm along the lateral walls of the terminal cells of the gemmiferous filaments, but are absent from the tips, thus suggesting that these cells elongate by intercalary, rather than by tip, growth. During mitosis microtubules are closely associated with the envelopes of spindle-shaped prophase nuclei, radiate from ill-defined spindle poles surrounded by plastids at metaphase and anaphase and form a dense phragmoplast array during telophase. Pre-prophase bands are absent and it may be that the nuclear equator determines the plane of division in gemmiferous filaments. Chloroplast division, associated with extremely transient plastid-dividing rings, takes place during interphase. Lateral branches of the growing filaments arise from subapical cells by reiteration of the first division mechanism.Immediately following the proliferative divisions, which take place in cells measuring only 5-6 μm in diameter, oil bodies suddenly appear as flat pleomorphic cisternae associated with endoplasmic reticulum and occasional microtubules. Their contents are electron-transparent apart from scattered osmiophilic droplets. Throughout their ontogeny the oil bodies are closely associated with cytoplasmic lipid bodies but there is no evidence of fusion. The nascent oil bodies swell rapidly to their final diameter, become ovoid to spherical in outline and are eventually suspended by fine cytoplasmic bridges within the vacuoles. The latter rapidly increase in size together with an expansion of the cells themselves until these reach their final diameter and length. The final event in gemma maturation is an endogenous division with the formation of a new internal wall along a phragmosome. Separation of the bicellular gemmae proceeds basipetally and involves the appearance of an electron-transparent line along the middle lamella in the cross walls, which often develop convex thickenings, and severing of the plasmodesmata. After their liberation shallow scars are visible on the leaf surface under the SEM. Gemma maturation sees a marked increase in the electron-opacity of the walls and dense staining of these together with Golgi vesicles with the periodic acid/thiocarbohydrazide/silver proteinate test for non-cellulosic carbohydrates. This change in wall chemistry and ultrastructure may be related to the fact that the maturing gemmae become extremely water repellant and are probably dispersed either on the surface of water films or in the air.

As an alternative approach to conventional allergen-specific immunotherapy, transgenic rice seed expressing a major house dust mite (HDM) allergen, Der p 1, was developed as an edible vaccine. The C-terminal KDEL-tagged Der p 1 allergen specifically accumulated in seed endosperm tissue under the control of the endosperm-specific GluB1 promoter. Der p 1 reached a maximum concentration of 58 lg/ grain and was deposited in the endoplasmic reticulum (ER)-derived protein body I (PB-I). Plant-derived Der p 1 was posttranslationally modified with high-mannose-type glycan structures. Glycosylated Der p 1 displayed reduced IgE binding capacity in comparison with its unglycosylated counterpart in vitro. Our results indicate that transgenic Der p 1 rice seeds are a safe, potential oral delivery vaccine for the treatment of HDM allergy.