3 β-Hydroxysteroid-Δ 24 Reductase (DHCR24) Protects Neuronal Cells from Apoptotic Cell Death Induced by Endoplasmic Reticulum (ER) Stress

3β-Hydroxysteroid-Δ24 reductase (DHCR24) is an endoplasmic reticulum (ER)-localized multifunctional enzyme that possesses anti-apoptotic and cholesterol-synthesizing activities. Accumulating evidence suggests that ER stress is involved in the pathogenesis of neurodegenerative disease. In this study, we investigated whether DHCR24 may function as a neuroprotective protein under ER stress. Neuroblastoma N2A cells were infected with adenovirus expressing myc-tagged DHCR24 (Ad-DHCR24) or lacZ (Ad-lacZ, serving as a control) and subjected to ER-stress, induced with Tunicamycin (TM). Cells infected with Ad-DHCR24-myc were resistant to TM-induced apoptosis, and showed weaker level of caspase-12 activity. These cells also exhibited lower levels of Bip and CHOP proteins than Ad-LacZ-infected cells. Moreover, a stronger and rapid activation of PERK, and a prolonged activation of JNK and p38 were observed in Ad-LacZ–infected cells. The generation of intracellular reactive oxygen species from ER stress was also diminished by the overexpression of DHCR24. Additionally, intracellular cholesterol level was also elevated in the Ad-DHCR24-infected cells, accompanied by a well-organized formation of caveolae (cholesterol-rich microdomain) on the plasma membrane, and improved colocalization of caveolin-1 and insulin-like growth factor 1 receptor. These results demonstrated for the first time that DHCR24 could protect neuronal cells from apoptosis induced by ER stress.     

The terminal enzymes of cholesterol synthesis,DHCR24 and DHCR7, interact physically and functionally

Cholesterol is essential to human health, and its levels are tightly regulated by a balance of synthesis, uptake, and efflux. Cholesterol synthesis requires the actions of more than twenty enzymes to reach the final product, through two alternate pathways. Here we describe a physical and functional interaction between the two terminal enzymes. 24-Dehydrocholesterol reductase (DHCR24) and 7-dehydrocholesterol reductase (DHCR7) coimmunoprecipitate, and when the DHCR24 gene is knocked down by siRNA, DHCR7 activity is also ablated. Conversely, overexpression of DHCR24 enhances DHCR7 activity, but only when a functional form of DHCR24 is used. DHCR7 is important for both cholesterol and vitamin D synthesis, and we have identified a novel layer of regulation, whereby its activity is controlled by DHCR24. This suggests the existence of a cholesterol “metabolon”, where enzymes from the same metabolic pathway interact with each other to provide a substrate channeling benefit. We predict that other enzymes in cholesterol synthesis may similarly interact, and this should be explored in future studies.     

Heat Shock Inhibits alpha-Amylase Synthesis in Barley Aleurone without Inhibiting the Activity of Endoplasmic Reticulum Marker Enzymes

The effects of heat shock on the synthesis of α-amylase and on the membranes of the endoplasmic reticulum (ER) of barley (Hordeum vulgare) aleurone were studied. Heat shock, imposed by raising the temperature of incubation from 25°C to 40°C for 3 hours, inhibits the accumulation of α-amylase and other proteins in the incubation medium of barley aleurone layers treated with gibberellic acid and Ca²⁺. When ER is isolated from heat-shocked aleurone layers, less newly synthesized α-amylase is found associated with this membrane system. ER membranes, as indicated by the activities of NADH cytochrome c reductase and ATP-dependent Ca²⁺ transport, are not destroyed by heat stress, however. Although heat shock did not reduce the activity of ER membrane marker enzymes, it altered the buoyant density of these membranes. Whereas ER from control tissue showed a peak of marker enzyme activity at 27% to 28% sucrose (1.113-1.120 grams per cubic centimeter), ER from heat-shocked tissue peaked at 30% to 32% sucrose (1.127-1.137 grams per cubic centimeter). The synthesis of a group of proteins designated as heat-shock proteins (HSPs) was stimulated by heat shock. These HSPs were localized to different compartments of the aleurone cell. Several proteins ranging from 15 to 30 kilodaltons were found in the ER and the mitochondrial/plasma membrane fractions of heat-shocked cells, but none of the HSPs accumulated in the incubation medium of heat-shocked aleurone layers.     

Epitope Mapping of Antibodies Suggests the Novel Membrane Topology of B-Cell Receptor Associated Protein 31 on the Cell Surface of Embryonic Stem Cells: The Novel Membrane Topology of BAP31

When located in the endoplasmic reticulum (ER) membrane, B-cell receptor associated protein 31 (BAP31) is involved in the export of secreted proteins from the ER to the plasma membrane. In a previous study, we generated two monoclonal antibodies (mAbs), 297-D4 and 144-A8, that bound to surface molecules on human embryonic stem cells (hESCs), but not to surface molecules on mouse embryonic stem cells (mESCs). Subsequent studies revealed that the mAbs recognized BAP31 on the surface of hESCs. To investigate the membrane topology of BAP31 on the cell surface, we first examined the epitope specificity of 297-D4 and 144-A8, as well as a polyclonal anti-BAP31 antibody (α-BAP31). We generated a series of GST-fused BAP31 mutant proteins in which BAP31 was serially deleted at the C- terminus. GST-fused BAP31 mutant proteins were then screened to identify the epitopes targeted by the antibodies. Both 297-D4 and 144-A8 recognized C-terminal residues 208–217, while α-BAP31 recognized C-terminal residues 165–246, of BAP31 on hESCs, suggesting that the C-terminal domain of BAP31 is exposed on the cell surface. The polyclonal antibody α-BAP31 bound to mESCs, which confirmed that the C-terminal domain of BAP31 is also exposed on the surface of these cells. Our results show for the first time the novel membrane topology of cell surface-expressed BAP31 as the extracellular exposure of the BAP31 C-terminal domain was not predicted from previous studies.     

A Novel Role of the Yeast CaaX Protease Ste24 in Chitin Synthesis

Ste24 is a membrane-integral CaaX metalloprotease residing in the endoplasmic reticulum (ER). In yeast, the only known substrate of Ste24 is the mating factor a precursor. A global screening for protein–protein interactions indicated that Ste24 interacts with chitin synthesis deficient (Chs)3, an enzyme required for chitin synthesis. We confirmed this interaction by yeast two-hybrid analyses and mapped the interacting cytoplasmic domains. Next, we investigated the influence of Ste24 on chitin synthesis. In sterile (ste)24Δ mutants, we observed resistance to calcofluor white (CFW), which was also apparent when the cells expressed a catalytically inactive version of Ste24. In addition, ste24Δ cells showed a decrease in chitin levels and Chs3-green fluorescent protein localized less frequently at the bud neck. Overexpression of STE24 resulted in hypersensitivity to CFW and a slight increase in chitin levels. The CFW phenotype of ste24Δ cells could be rescued by its human and insect orthologues. Although Chs3 binds to Ste24, it seems not to be a substrate for this protease. Instead, our data suggest that Chs3 and Ste24 form a complex in the ER that facilitates protease action on prenylated Chs4, a known activator of Chs3 with a C-terminal CaaX motif, leading to a more efficient localization of Chs3 at the plasma membrane.     

Cargo Selection by the COPII Budding Machinery during Export from the ER

Cargo is selectively exported from the ER in COPII vesicles. To analyze the role of COPII in selective transport from the ER, we have purified components of the mammalian COPII complex from rat liver cytosol and then analyzed their role in cargo selection and ER export. The purified mammalian Sec23–24 complex is composed of an 85-kD (Sec23) protein and a 120-kD (Sec24) protein. Although the Sec23–24 complex or the monomeric Sec23 subunit were found to be the minimal cytosolic components recruited to membranes after the activation of Sar1, the addition of the mammalian Sec13–31 complex is required to complete budding. To define possible protein interactions between cargo and coat components, we recruited either glutathione-S-transferase (GST)–tagged Sar1 or GST– Sec23 to ER microsomes. Subsequently, we solubilized and reisolated the tagged subunits using glutathione-Sepharose beads to probe for interactions with cargo. We find that activated Sar1 in combination with either Sec23 or the Sec23–24 complex is necessary and sufficient to recover with high efficiency the type 1 transmembrane cargo protein vesicular stomatitis virus glycoprotein in a detergent-soluble prebudding protein complex that excludes ER resident proteins. Supplementing these minimal cargo recruitment conditions with the mammalian Sec13–31 complex leads to export of the selected cargo into COPII vesicles. The ability of cargo to interact with a partial COPII coat demonstrates that these proteins initiate cargo sorting on the ER membrane before budding and establishes the role of GTPase-dependent coat recruitment in cargo selection.     

RETRACTED ARTICLE: Age-dependent Increase in Desmosterol Restores DRM Formation and Membrane-related Functions in Cholesterol-free DHCR24−/− Mice

Cholesterol is a prominent modulator of the integrity and functional activity of physiological membranes and the most abundant sterol in the mammalian brain. DHCR24-knock-out mice lack cholesterol and accumulate desmosterol with age. Here we demonstrate that brain cholesterol deficiency in 3-week-old DHCR24^−/− mice was associated with altered membrane composition including disrupted detergent-resistant membrane domain (DRM) structure. Furthermore, membrane-related functions differed extensively in the brains of these mice, resulting in lower plasmin activity, decreased β-secretase activity and diminished Aβ generation. Age-dependent accumulation and integration of desmosterol in brain membranes of 16-week-old DHCR24^−/− mice led to the formation of desmosterol-containing DRMs and rescued the observed membrane-related functional deficits. Our data provide evidence that an alternate sterol, desmosterol, can facilitate processes that are normally cholesterol-dependent including formation of DRMs from mouse brain extracts, membrane receptor ligand binding and activation, and regulation of membrane protein proteolytic activity. These data indicate that desmosterol can replace cholesterol in membrane-related functions in the DHCR24^−/− mouse. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. An erratum to this article can be found at     

Reorientation of aquaporin-1 topology during maturation in the endoplasmic reticulum

The topology of most eukaryotic polytopic membrane proteins is established cotranslationally in the endoplasmic reticulum (ER) through a series of coordinated translocation and membrane integration events. For the human aquaporin water channel AQP1, however, the initial four-segment-spanning topology at the ER membrane differs from the mature six-segment-spanning topology at the plasma membrane. Here we use epitope-tagged AQP1 constructs to follow the transmembrane (TM) orientation of key internal peptide loops in Xenopus oocyte and cell-free systems. This analysis revealed that AQP1 maturation in the ER involves a novel topological reorientation of three internal TM segments and two peptide loops. After the synthesis of TMs 4-6, TM3 underwent a 180-degree rotation in which TM3 C-terminal flanking residues were translocated from their initial cytosolic location into the ER lumen and N-terminal flanking residues underwent retrograde translocation from the ER lumen to the cytosol. These events convert TM3 from a type I to a type II topology and reposition TM2 and TM4 into transmembrane conformations consistent with the predicted six-segment-spanning AQP1 topology. AQP1 topological reorientation was also associated with maturation from a protease-sensitive conformation to a protease-resistant structure with water channel function. These studies demonstrate that initial protein topology established via cotranslational translocation events in the ER is dynamic and may be modified by subsequent steps of folding and/or maturation.     

Prosurvival effect of DHCR24/Seladin-1 in acute and chronic responses to oxidative stress

DHCR24/seladin-1, a crucial enzyme in sterol synthesis, is of lower abundance in brain areas affected by Alzheimer's disease. While high levels of DHCR24/seladin-1 exert antiapoptotic function by conferring resistance against oxidative stress, the molecular mechanism for this protective effect is not fully understood. Here we show that DHCR24/seladin-1 expression is up-regulated in an acute response and down-regulated in a chronic response to oxidative stress. High levels of DHCR24/seladin-1 were associated with elevated cholesterol concentrations and a general increase in cholesterol biosynthesis upon oxidative stress exposure in neuroblastoma SH-SY5Y cells. DHCR24/seladin-1 overexpression conferred resistance to oxidative stress in a cholesterol-dependent manner. Mutating the reductase activity within DHCR24/seladin-1 abolished this protective effect. Conversely, DHCR24/seladin-1 levels diminished upon chronic exposure to oxidative stress. Low levels of DHCR24/seladin-1 were associated with reduced p53 levels, independent of DHCR24 activity and cholesterol concentrations. Additionally, ablation of DHCR24/seladin-1 prevented apoptosis of primary neurons in a p53-dependent manner and reduced the response of critical p53 targets due to deficient stabilization of p53 and therefore elevated p53 ubiquitination and degradation. Our findings reveal a dual capacity of DHCR24/seladin-1, which appears to be involved in two mechanistically independent prosurvival effects, exerting an acute response and a chronic response to oxidative stress.     

Purified crystalloid endoplasmic reticulum from UT-1 cells contains multiple proteins in addition to 3-hydroxy-3-methylglutaryl coenzyme A reductase

The crystalloid endoplasmic reticulum (ER) of UT-1 cells is a specialized smooth ER that houses 3-hydroxy-3-methylglutaryl-CoA reductase, a membrane protein that regulates endogenous cholesterol synthesis. The biogenesis of this ER is coupled to the over production of 3-hydroxy-3-methylglutaryl-CoA reductase. To understand better this membrane system and the relationship between the synthesis of a membrane protein and the formation of membrane, we have purified the crystalloid ER. Purified crystalloid ER did not contain significant amounts of membrane derived from the Golgi apparatus, mitochondria, or plasma membrane. Approximately 24% of the protein in this organelle corresponded to 3-hydroxy-3-methylglutaryl-CoA reductase; however, at least eight other proteins were detected by gel electrophoresis. One of these proteins (Mr 73,000) was as abundant as reductase. These results suggest that the biogenesis of this ER involves the coordinate synthesis of multiple membrane and content proteins.     

Subcellular localization and membrane topology of the melon ethylene receptor CmERS1

Ethylene receptors are multispanning membrane proteins that negatively regulate ethylene responses via the formation of a signaling complex with downstream elements. To better understand their biochemical functions, we investigated the membrane topology and subcellular localization of CmERS1, a melon (Cucumis melo) ethylene receptor that has three putative transmembrane domains at the N terminus. Analyses using membrane fractionation and green fluorescent protein imaging approaches indicate that CmERS1 is predominantly associated with the endoplasmic reticulum (ER) membrane. Detergent treatments of melon microsomes showed that the receptor protein is integrally bound to the ER membrane. A protease protection assay and N-glycosylation analysis were used to determine membrane topology. The results indicate that CmERS1 spans the membrane three times, with its N terminus facing the luminal space and the large C-terminal portion lying on the cytosolic side of the ER membrane. This orientation provides a platform for interaction with the cytosolic signaling elements. The three N-terminal transmembrane segments were found to function as topogenic sequences to determine the final topology. High conservation of these topogenic sequences in all ethylene receptor homologs identified thus far suggests that these proteins may share the same membrane topology.     

Type II transmembrane domain hydrophobicity dictates the cotranslational dependence for inversion

Membrane insertion by the Sec61 translocon in the endoplasmic reticulum (ER) is highly dependent on hydrophobicity. This places stringent hydrophobicity requirements on transmembrane domains (TMDs) from single-spanning membrane proteins. On examining the single-spanning influenza A membrane proteins, we found that the strict hydrophobicity requirement applies to the N_out-C_in HA and M2 TMDs but not the N_in-C_out TMDs from the type II membrane protein neuraminidase (NA). To investigate this discrepancy, we analyzed NA TMDs of varying hydrophobicity, followed by increasing polypeptide lengths, in mammalian cells and ER microsomes. Our results show that the marginally hydrophobic NA TMDs (ΔG_app > 0 kcal/mol) require the cotranslational insertion process for facilitating their inversion during translocation and a positively charged N-terminal flanking residue and that NA inversion enhances its plasma membrane localization. Overall the cotranslational inversion of marginally hydrophobic NA TMDs initiates once ∼70 amino acids past the TMD are synthesized, and the efficiency reaches 50% by ∼100 amino acids, consistent with the positioning of this TMD class in type II human membrane proteins. Inversion of the M2 TMD, achieved by elongating its C-terminus, underscores the contribution of cotranslational synthesis to TMD inversion.     

Retrograde Transport of Golgi-localized Proteins to the ER

The ER is uniquely enriched in chaperones and folding enzymes that facilitate folding and unfolding reactions and ensure that only correctly folded and assembled proteins leave this compartment. Here we address the extent to which proteins that leave the ER and localize to distal sites in the secretory pathway are able to return to the ER folding environment during their lifetime. Retrieval of proteins back to the ER was studied using an assay based on the capacity of the ER to retain misfolded proteins. The lumenal domain of the temperature-sensitive viral glycoprotein VSVGtsO45 was fused to Golgi or plasma membrane targeting domains. At the nonpermissive temperature, newly synthesized fusion proteins misfolded and were retained in the ER, indicating the VSVGtsO45 ectodomain was sufficient for their retention within the ER. At the permissive temperature, the fusion proteins were correctly delivered to the Golgi complex or plasma membrane, indicating the lumenal epitope of VSVGtsO45 also did not interfere with proper targeting of these molecules. Strikingly, Golgi-localized fusion proteins, but not VSVGtsO45 itself, were found to redistribute back to the ER upon a shift to the nonpermissive temperature, where they misfolded and were retained. This occurred over a time period of 15 min–2 h depending on the chimera, and did not require new protein synthesis. Significantly, recycling did not appear to be induced by misfolding of the chimeras within the Golgi complex. This suggested these proteins normally cycle between the Golgi and ER, and while passing through the ER at 40°C become misfolded and retained. The attachment of the thermosensitive VSVGtsO45 lumenal domain to proteins promises to be a useful tool for studying the molecular mechanisms and specificity of retrograde traffic to the ER.     

Golgi targeting of Drosophila melanogaster β4GalNAcTB requires a DHHC protein family–related protein as a pilot

Drosophila melanogaster β4GalNAcTB mutant flies revealed that this particular N-acetylgalactosaminyltransferase is predominant in the formation of lacdiNAc (GalNAcβ1,4GlcNAc)-modified glycolipids, but enzymatic activity could not be confirmed for the cloned enzyme. Using a heterologous expression cloning approach, we isolated β4GalNAcTB together with β4GalNAcTB pilot (GABPI), a multimembrane-spanning protein related to Asp-His-His-Cys (DHHC) proteins but lacking the DHHC consensus sequence. In the absence of GABPI, inactive β4GalNAcTB is trapped in the endoplasmic reticulum (ER). Coexpression of β4GalNAcTB and GABPI generates the active enzyme that is localized together with GABPI in the Golgi. GABPI associates with β4GalNAcTB and, when expressed with an ER retention signal, holds active β4GalNAcTB in the ER. Importantly, treatment of isolated membrane vesicles with Triton X-100 disturbs β4GalNAcTB activity. This phenomenon occurs with multimembrane-spanning glycosyltransferases but is normally not a property of glycosyltransferases with one membrane anchor. In summary, our data provide evidence that GABPI is required for ER export and activity of β4GalNAcTB.     

Five Arabidopsis Reticulon Isoforms Share Endoplasmic Reticulum Location,Topology, and Membrane-Shaping Properties

The cortical endoplasmic reticulum (ER) in tobacco (Nicotiana tabacum) epidermal cells is a network of tubules and cisternae undergoing dramatic rearrangements. Reticulons are integral membrane proteins involved in shaping ER tubules. Here, we characterized the localization, topology, effect, and interactions of five Arabidopsis thaliana reticulons (RTNs), isoforms 1-4 and 13, in the cortical ER. Our results indicate that RTNLB13 and RTNLB1-4 colocate to and constrict the tubular ER membrane. All five RTNs preferentially accumulate on ER tubules and are excluded from ER cisternae. All isoforms share the same transmembrane topology, with N and C termini facing the cytosol and four transmembrane domains. We show by Förster resonance energy transfer and fluorescence lifetime imaging microscopy that several RTNs have the capacity to interact with themselves and each other, and we suggest that oligomerization is responsible for their residence in the ER membrane. We also show that a complete reticulon homology domain is required for both RTN residence in high-curvature ER membranes and ER tubule constriction, yet it is not necessary for homotypic interactions.