Two hydrophobic segments of the RTN1 family determine the ER localization and retention

Reticulon (RTN) proteins are localized to the endoplasmic reticulum (ER), and are related to intracellular membrane trafficking, apoptosis, inhibiting axonal regeneration, and Alzheimer's disease. The RTN proteins are produced without an N-terminal signal peptide. Their C-terminal domain contains two long hydrophobic segments. We analyzed the ER localization signal of human RTN1-A. Mutant proteins lacking the first (39 residues) or second (36 residues) hydrophobic segment showed ER localization. On the other hand, the mutant lacking both hydrophobic segments was cytosolic. Enhanced green fluorescent protein (EGFP) tagged with the first or second hydrophobic segment of RTN1-A was localized to the ER. These results suggest that each hydrophobic segment determines the ER localization. In addition, EGFP tagged with the truncated form of the first hydrophobic segment exhibited the localization to the Golgi rather than the ER. This suggests that the length of the hydrophobic segment contributes to the ER retention of RTN1-A.     

Arabidopsis thaliana GPAT8 and GPAT9 are localized to the ER and possess distinct ER retrieval signals: Functional divergence of the dilysine ER retrieval motif in plant cells

Glycerol-3-phosphate acyltransferase (GPAT; EC 2.3.1.15) catalyzes the committed step in the production of glycerolipids, which are major components of cellular membranes, seed storage oils, and epicuticular wax coatings. While the biochemical activities of GPATs have been characterized in detail, the cellular features of these enzymes are only beginning to emerge. Here we characterized the phylogenetic relationships and cellular properties of two GPAT enzymes from the relatively large Arabidopsis thaliana GPAT family, including GPAT8, which is involved in cutin biosynthesis, and GPAT9, which is a new putative GPAT that has extensive homology with a GPAT from mammalian cells involved in storage oil formation and, thus, may have a similar role in plants. Immunofluorescence microscopy of transiently-expressed myc-epitope-tagged GPAT8 and GPAT9 revealed that both proteins were localized to the endoplasmic reticulum (ER), and differential permeabilization experiments indicated that their N- and C-termini were oriented towards the cytosol. However, these two proteins contained distinct types of ER retrieval signals, with GPAT8 possessing a divergent type of dilysine motif (–KK–COOH rather than the prototypic –KKXX–COOH or –KXKXX–COOH motif) and GPAT9 possessing a hydrophobic pentapeptide motif (––X–X–K/R/D/E––; where are large hydrophobic amino acid residues). Notably, the divergent dilysine motif in GPAT8 only functioned effectively when additional upstream residues were included to provide the proper protein context. Extensive mutational analyses of the divergent dilysine motif, based upon sequences present in the C-termini of other GPAT8s from various plant species, further expanded the functional definition of this molecular targeting signal, thereby providing insight to the targeting signals in other GPAT family members as well as other ER-resident membrane proteins within plant cells.     

Endoplasmic Reticulum (ER) Localization Is Critical for DsbA-L Protein to Suppress ER Stress and Adiponectin Down-regulation in Adipocytes

Adiponectin is an adipokine with insulin-sensitizing and anti-inflammatory functions. We previously reported that adiponectin multimerization and stability are promoted by the disulfide bond A oxidoreductase-like protein (DsbA-L) in cells and in vivo. However, the precise mechanism by which DsbA-L regulates adiponectin biosynthesis remains elusive. Here we show that DsbA-L is co-localized with the endoplasmic reticulum (ER) marker protein disulfide isomerase and the mitochondrial marker MitoTracker. In addition, DsbA-L interacts with the ER chaperone protein Ero1-Lα in 3T3-L1 adipocytes. In silico analysis and truncation mapping studies revealed that DsbA-L contains an ER targeting signal at its N terminus. Deletion of the first 6 residues at the N terminus greatly impaired DsbA-L localization in the ER. Overexpression of the wild type but not the ER localization-defective mutant of DsbA-L protects against thapsigargin-induced ER stress and adiponectin down-regulation in 3T3-L1 adipocytes. In addition, overexpression of the wild type but not the ER localization-defective mutant of DsbA-L promotes adiponectin multimerization. Together, our results reveal that DsbA-L is localized in both the mitochondria and the ER in adipocytes and that its ER localization plays a critical role in suppressing ER stress and promoting adiponectin biosynthesis and secretion.     

Ca transfer from the ER to mitochondria: When, how and why

The heterogenous subcellular distribution of a wide array of channels, pumps and exchangers allows extracellular stimuli to induce increases in cytoplasmic Ca²⁺ concentration ([Ca²⁺]_c) with highly defined spatial and temporal patterns, that in turn induce specific cellular responses (e.g. contraction, secretion, proliferation or cell death). In this extreme complexity, the role of mitochondria was considered marginal, till the direct measurement with targeted indicators allowed to appreciate that rapid and large increases of the [Ca²⁺] in the mitochondrial matrix ([Ca²⁺]_m) invariably follow the cytosolic rises. Given the low affinity of the mitochondrial Ca²⁺ transporters, the close proximity to the endoplasmic reticulum (ER) Ca²⁺-releasing channels was shown to be responsible for the prompt responsiveness of mitochondria. In this review, we will summarize the current knowledge of: i) the mitochondrial and ER Ca²⁺ channels mediating the ion transfer, ii) the structural and molecular foundations of the signaling contacts between the two organelles, iii) the functional consequences of the [Ca²⁺]_m increases, and iv) the effects of oncogene-mediated signals on mitochondrial Ca²⁺ homeostasis. Despite the rapid progress carried out in the latest years, a deeper molecular understanding is still needed to unlock the secrets of Ca²⁺ signaling machinery.     

Genomic isolation of genes encoding starch branching enzyme II (SBEII) in apple: toward characterization of evolutionary disparity in <Emphasis Type="Italic">Sbe</Emphasis>II genes between monocots and eudicots

Reproductive and storage tissues of many plants produce large amounts of serine proteinase inhibitors (PIs). The ornamental tobacco, Nicotiana alata, produces a series of 6 kDa chymotrypsin and trypsin inhibitors that accumulate to up to 30% of soluble protein in the stigma. These inhibitors are derived by proteolytic processing of two closely related multidomain precursor proteins. Using immunogold electron microscopy, we find that the stigmatic PIs accumulate in both the central vacuole and in the extracellular mucilage. Labelling with antibodies specific for the C-terminal vacuolar targeting peptide (VTS) of each precursor confirms earlier biochemical data showing that the VTS is removed during passage through the secretory pathway. We have isolated and characterised the extracellular population of PIs, which are largely identical to PIs isolated from whole stigmas and are functional inhibitors of serine proteases. Subcellular fractionation of immature stigmas reveals that a sub-population of the PI precursor protein is proteolytically processed within the endoplasmic reticulum. This proteolysis results in the removal of the vacuolar sorting information, causing secretion of this PI population. We propose a novel mechanism whereby a single gene product may be simultaneously trafficked to two separate compartments mediated by proteolysis early in the secretory pathway.     

Beyond KDEL: the role of positions 5 and 6 in determining ER localization

The C-terminal amino acid sequence of a protein plays an important role in determining the endoplasmic reticulum (ER) localization of many soluble proteins that enter the secretory pathway. While it is known that the four amino acids found at the extreme C-terminus of the protein (e.g., KDEL) play a critical role in the interaction with the receptors that mediate retrograde transport back to the ER, other factors within the protein are less well known. Here we show that positions − 5 and − 6 play an important role in determining the ER localization of soluble proteins, with the amino acids at these positions playing an essential role in ER localization of the human protein disulfide isomerase family member, ERp18. Three other naturally occurring C-terminal motifs were also found that work efficiently in ER localization as six-amino-acid variants, but inefficiently as the four-amino-acid variant. Using bimolecular fluorescence complementation, we demonstrate that positions − 5 and − 6 from the C-terminus of the protein play an important role in the recognition of KDEL-like ER retrieval motifs, with the three different human KDEL receptors showing different specificities for changes at these positions for both inefficient and efficient ER localization four-amino-acid motifs.     

Different endoplasmic reticulum trafficking and processing pathways for calsequestrin (CSQ) and epitope-tagged CSQ

Cardiac calsequestrin (CSQ) is a protein that traffics to and concentrates inside sarcoplasmic reticulum (SR) terminal cisternae, a protein secretory compartment of uncertain origin. To investigate trafficking of CSQ within standard ER compartments, we expressed CSQ in nonmuscle cell lines and examined its localization by immunofluorescence and its molecular structure from the mass spectrum of total cellular CSQ. In all cells examined, CSQ was a highly phosphorylated protein with a glycan structure predictive of ER-retained proteins: Man9,8GlcNAc2 lacking terminal GlcNAc. Immunostaining was restricted to polymeric ER cisternae. Secretory pathway disruption by brefeldin A and thapsigargin led to altered CSQ glycosylation and phosphorylation consistent with post-ER trafficking. When epitope-tagged forms of CSQ were expressed in the same cells, mannose trimming of CSQ glycans was far more extensive, and C-terminal phosphorylation sites were nearly devoid of phosphate, in complete contrast to the highly phosphorylated wild-type protein that concentrates in all cells tested. Epitope-tagged CSQ also showed a reduced ER staining compared to wild-type protein, with significant staining in juxta-Golgi compartments. Loss of ER retention due to epitope tags or thapsigargin and resultant changes in protein structure or levels of bound Ca(2+) point to CSQ polymerization as an ER/SR retention mechanism.     

The 14-3-3γ isoform binds to and regulates the localization of endoplasmic reticulum (ER) membrane protein TMCC3 for the reticular network of the ER

The reticular network of the endoplasmic reticulum (ER) is formed by connecting ER tubules through three-way junctions and undergoes constant remodeling through formation and loss of the three-way junctions. Transmembrane and coiled-coil domain family 3 (TMCC3), an ER membrane protein localizing at three-way junctions, has been shown to positively regulate formation of the reticular ER network. However, elements that negatively regulate TMCC3 localization have not been characterized. In this study, we report that 14-3-3γ, a phospho-serine/phospho-threonine-binding protein involved in various signal transduction pathways, is a negative regulator of TMCC3. We demonstrate that overexpression of 14-3-3γ reduced localization of TMCC3 to three-way junctions and decreased the number of three-way junctions. TMCC3 bound to 14-3-3γ through the N terminus and had deduced 14-3-3 binding motifs. Additionally, we determined that a TMCC3 mutant substituting alanine for serine to be phosphorylated in the binding motif reduced binding to 14-3-3γ. The TMCC3 mutant was more prone than wildtype TMCC3 to localize at three-way junctions in the cells overexpressing 14-3-3γ. Furthermore, the TMCC3 mutant rescued the ER sheet expansion caused by TMCC3 knockdown less than wild-type TMCC3. Taken together, these results indicate that 14-3-3γ binding negatively regulates localization of TMCC3 to the three-way junctions for the proper reticular ER network, implying that the negative regulation of TMCC3 by 14-3-3γ would underlie remodeling of the reticular network of the ER.     

Different fatty acids inhibit apoB100 secretion by different pathways: unique roles for ER stress, ceramide, and autophagy

Although short-term incubation of hepatocytes with oleic acid (OA) stimulates secretion of apolipoprotein B100 (apoB100), exposure to higher doses of OA for longer periods inhibits secretion in association with induction of endoplasmic reticulum (ER) stress. Palmitic acid (PA) induces ER stress, but its effects on apoB100 secretion are unclear. Docosahexaenoic acid (DHA) inhibits apoB100 secretion, but its effects on ER stress have not been studied. We compared the effects of each of these fatty acids on ER stress and apoB100 secretion in McArdle RH7777 (McA) cells: OA and PA induced ER stress and inhibited apoB100 secretion at higher doses; PA was more potent because it also increased the synthesis of ceramide. DHA did not induce ER stress but was the most potent inhibitor of apoB100 secretion, acting via stimulation of autophagy. These unique effects of each fatty acid were confirmed when they were infused into C57BL6J mice. Our results suggest that when both increased hepatic secretion of VLDL apoB100 and hepatic steatosis coexist, reducing ER stress might alleviate hepatic steatosis but at the expense of increased VLDL secretion. In contrast, increasing autophagy might reduce VLDL secretion without causing steatosis.     

Regulation of brain-type creatine kinase by AMP-activated protein kinase: Interaction,phosphorylation and ER localization

AMP-activated protein kinase (AMPK) and cytosolic brain-type creatine kinase (BCK) cooperate under energy stress to compensate for loss of adenosine triphosphate (ATP) by either stimulating ATP-generating and inhibiting ATP-consuming pathways, or by direct ATP regeneration from phosphocreatine, respectively. Here we report on AMPK-dependent phosphorylation of BCK from different species identified by in vitro screening for AMPK substrates in mouse brain. Mass spectrometry, protein sequencing, and site-directed mutagenesis identified Ser6 as a relevant residue with one site phosphorylated per BCK dimer. Yeast two-hybrid analysis revealed interaction of active AMPK specifically with non-phosphorylated BCK. Pharmacological activation of AMPK mimicking energy stress led to BCK phosphorylation in astrocytes and fibroblasts, as evidenced with a highly specific phospho-Ser6 antibody. BCK phosphorylation at Ser6 did not affect its enzymatic activity, but led to the appearance of the phosphorylated enzyme at the endoplasmic reticulum (ER), close to the ER calcium pump, a location known for muscle-type cytosolic creatine kinase (CK) to support Ca²⁺-pumping.     

Identification and characterization of novel ERC‐55 interacting proteins: Evidence for the existence of several ERC‐55 splicing variants; including the cytosolic ERC‐55‐C

ERC‐55, encoded from RCN2, is localized in the ER and belongs to the CREC protein family. ERC‐55 is involved in various diseases and abnormal cell behavior, however, the function is not well defined and it has controversially been reported to interact with a cytosolic protein, the vitamin D receptor. We have used a number of proteomic techniques to further our functional understanding of ERC‐55. By affinity purification, we observed interaction with a large variety of proteins, including those secreted and localized outside of the secretory pathway, in the cytosol and also in various organelles. We confirm the existence of several ERC‐55 splicing variants including ERC‐55‐C localized in the cytosol in association with the cytoskeleton. Localization was verified by immunoelectron microscopy and sub‐cellular fractionation. Interaction of lactoferrin, S100P, calcyclin (S100A6), peroxiredoxin‐6, kininogen and lysozyme with ERC‐55 was further studied in vitro by SPR experiments. Interaction of S100P requires [Ca²⁺] of ～10^?7 M or greater, while calcyclin interaction requires [Ca²⁺] of >10^?5 M. Interaction with peroxiredoxin‐6 is independent of Ca²⁺. Co‐localization of lactoferrin, S100P and calcyclin with ERC‐55 in the perinuclear area was analyzed by fluorescence confocal microscopy. The functional variety of the interacting proteins indicates a broad spectrum of ERC‐55 activities such as immunity, redox homeostasis, cell cycle regulation and coagulation.     

Evidence for different functional properties of the neuronal calcium sensor proteins VILIP-1 and VILIP-3: from subcellular localization to cellular function

The visinin-like-proteins VILIP-1 and -3 are EF-hand calcium-binding proteins and belong to the family of neuronal calcium sensor (NCS) proteins. Members of this family are involved in the calcium-dependent regulation of signal transduction cascades mainly in the nervous system. VILIP-1 and VILIP-3 are expressed in different populations of neuronal cells. To gain insights into the different functional characteristics of VILIP-1 and -3, we have compared the localization of the proteins in intact cells and the calcium-dependent membrane association in subcellular fractions. Furthermore, we have investigated the different functional properties of the two proteins in activating cGMP signal pathways and have defined different sets of protein interaction partners. Our data indicate that VILIP-3, which is mainly expressed in Purkinje cells, and VILIP-1, which is expressed in granule cells in the cerebellum, show a different calcium-dependent subcellular localization, may activate different cellular signaling pathways, and thus have signaling functions which seem to be cell-type specific.     

Brain Na+, K+-ATPase isoforms: different hypothalamus and mesencephalon response to acute desipramine treatment

We studied Na(+), K(+)-ATPase activity alpha isoforms by performing ouabain inhibition curves in rat hypothalamus and mesencephalon after acute administration of desipramine to rats. In hypothalamus, Ki values for high, intermediate and low affinity populations were 0.075x10(-9) M, 0.58x10(-6) M and 0.97x10(-3) M, with isoform distribution of 55%, 28% and 17%, respectively. In mesencephalon, Ki values for high, intermediate and low affinity populations were 1.80x10(-9) M, 0.56x10(-6) M and 0.21x10(-3) M, with isoform distribution of 28%, 46% and 21%, respectively. Three hours after acute administration of 10 mg/kg desipramine to rats, Na(+), K(+)-ATPase activity in hypothalamus increased significantly 54%, 39% and 51% as assayed respectively in the absence of ouabain or in the presence of 1x10(-9) M, or 5x10(-6) M ouabain, whereas only a trend was recorded in the presence of 1x10(-3) M ouabain. In such conditions, enzyme activity in mesencephalon increased significantly 73%, 54%, 30% and 271%, respectively. Present results showed that desipramine treatment enhances the activity of Na(+), K(+)-ATPase alpha isoforms in rat hypothalamus and mesencephalon, but the extent of this increase differs according to the isoform and the anatomical area studied, suggesting a differential enzyme regulation in response to noradrenergic stimulation.     

The central role of calcium in the effects of cytokines on beta-cell function: implications for type 1 and type 2 diabetes

The appropriate regulation of intracellular calcium is a requirement for proper cell function and survival. This review focuses on the effects of proinflammatory cytokines on calcium regulation in the insulin-producing pancreatic beta-cell and how normal stimulus-secretion coupling, organelle function, and overall beta-cell viability are impacted. Proinflammatory cytokines are increasingly thought to contribute to beta-cell dysfunction not only in type 1 diabetes (T1D), but also in the progression of type 2 diabetes (T2D). Cytokine-induced disruptions in calcium handling result in reduced insulin release in response to glucose stimulation. Cytokines can alter intracellular calcium levels by depleting calcium from the endoplasmic reticulum (ER) and by increasing calcium influx from the extracellular space. Depleting ER calcium leads to protein misfolding and activation of the ER stress response. Disrupting intracellular calcium may also affect organelles, including the mitochondria and the nucleus. As a chronic condition, cytokine-induced calcium disruptions may lead to beta-cell death in T1D and T2D, although possible protective effects are also discussed. Calcium is thus central to both normal and pathological cell processes. Because the tight regulation of intracellular calcium is crucial to homeostasis, measuring the dynamics of calcium may serve as a good indicator of overall beta-cell function.     

Laminin isoforms and their integrin receptors in glioma cell migration and invasiveness: Evidence for a role of alpha5-laminin(s) and alpha3beta1 integrin

Glioma cell infiltration of brain tissue often occurs along the basement membrane (BM) of blood vessels. In the present study we have investigated the role of laminins, major structural components of BMs and strong promoters of cell migration. Immunohistochemical studies of glioma tumor tissue demonstrated expression of alpha2-, alpha3-, alpha4- and alpha5-, but not alpha1-, laminins by the tumor vasculature. In functional assays, alpha3 (Lm-332/laminin-5)- and alpha5 (Lm-511/laminin-10)-laminins strongly promoted migration of all glioma cell lines tested. alpha1-Laminin (Lm-111/laminin-1) displayed lower activity, whereas alpha2 (Lm-211/laminin-2)- and alpha4 (Lm-411/laminin-8)-laminins were practically inactive. Global integrin phenotyping identified alpha3beta1 as the most abundant integrin in all the glioma cell lines, and this laminin-binding integrin exclusively or largely mediate the cell migration. Moreover, pretreatment of U251 glioma cells with blocking antibodies to alpha3beta1 integrin followed by intracerebral injection into nude mice inhibited invasion of the tumor cells into the brain tissue. The cell lines secreted Lm-211, Lm-411 and Lm-511, at different ratios. The results indicate that glioma cells secrete alpha2-, alpha4- and alpha5-laminins and that alpha3- and alpha5-laminins, found in brain vasculature, selectively promote glioma cell migration. They identify alpha3beta1 as the predominant integrin and laminin receptor in glioma cells, and as a brain invasion-mediating integrin.     

Differential and regulated binding of cAMP-dependent protein kinase and protein kinase C isoenzymes to gravin in human model neurons: Evidence that gravin provides a dynamic platform for the localization for kinases during neuronal development

The membrane cortex has an important role in generating and maintaining spatially and functionally distinct domains in neurons. As a tool to functionally characterize molecules of the membrane cortex, we generated novel monoclonal antibodies against a fraction enriched for components of the neuronal membrane skeleton. We obtained two antibodies against the kinase-anchoring protein gravin. Gravin was strongly up-regulated during differentiation of human model neurons (NT2-N neurons) and was enriched at the inner peripheral cortex in close proximity to the plasma membrane where its localization primarily depended on association with membranes. In differentiated neurons, gravin colocalized in putative signaling complexes with protein kinase C (PKCbetaII) and partially with PKCalpha and cAMP-dependent protein kinase (PKA). Colocalization with PKCepsilon was not observed. PKCbetaII, PKCalpha, and PKA but not PKCepsilon coprecipitated with gravin indicating physical interaction. Binding of gravin to PKCalpha required the presence of Ca2+ and was increased after inhibition of PKC. In contrast, binding of PKCbetaII and PKA were independent of Ca2+ and PKC inhibition. Activation of PKC decreased binding of PKCalpha to gravin, decreased its association with the plasma membrane, and reduced the mean size of gravin particles. Taken together the data suggest that gravin provides a dynamic platform to localize kinases in an isoenzyme-specific and activation-dependent manner at specific sites in neurons.     

Ultrastructural studies on calcitonin gene-related peptide-, tachykinins- and somatostatin-immunoreactive neurones in rat dorsal root ganglia: Evidence for the colocalization of different peptides in single secretory granules

Summary Calcitonin gene-related peptide (CGRP)-, tachykinins- and somatostatin-immunoreactive neurones in rat dorsal root ganglia have been studied by means of single and double immunogold labelling techniques. Peptide-immunoreactive neurones are generally B- or C-type cells of small size, with well developed rough endoplasmic reticulum and scanty neurofilaments. In neurones classifiable as A₂-type cells, i.e. larger neurones with a lighter cytoplasm due to the presence of poorly developed Nissl bodies and numerous neurofilaments, only CGRP immunoreactivity was detected. Immunolabelled structures were identified as large (60–100 nm diameter), electron-dense, membranebounded p-type granules. They were observed only in neuronal cell bodies or in the intraganglionic portions of the axons. No granules immunoreactive to the antisera applied in this study were observed in non-neuronal cells. Immunostaining experiments with different combinations of the antisera revealed, in some cells, the presence of double immunolabelled granules; in particular localization of CGRP and tachykinins, CGRP and somatostatin, and tachykinins and somatostatin to single secretory granules was demonstrated. The finding that more than one peptide is localized to the same secretory granule supports the postulate that peptides are co-released upon nerve stimulation providing morphological support for physiological and pharmacological data demonstrating an interaction between different peptides in the modulation of synaptic activity.     

Fecal corticosteroids in a territorial bird selected for different personalities: daily rhythm and the response to social stress

In this study we tested the hypothesis that in a passerine bird (great tit, Parus major) individuals differing for coping strategies differ in the magnitude of the adrenocortical response to social stress as well. Furthermore, we aimed at characterizing daily rhythms in corticosteroid release before and after social stress. We used 16 males from either of two lines bidirectionally selected for different coping strategies (fast and slow explorers). Social stress was induced by confrontation with an aggressive resident male. Corticosteroid metabolites were analyzed in feces collected at 90-min intervals from 900 to 1630 h on a baseline day, on the day of the social conflict, and on the following day. In both days and in both lines levels varied with time of day in a robust rhythm with a peak in the first sample of the morning and a trough at the end of the light phase. This rhythm correlates with activity (perch hopping). An overall increase in levels relative to baseline day was observed between 30 and 140 min after the challenge. Birds of the less aggressive and more cautious line (slow explorers) showed a trend for a higher response compared to birds of the more aggressive and bolder line (fast explorers), which showed almost no response. On the day after the challenge the birds of the slow line exhibited significantly reduced corticosteroid secretion, probably due to an increased negative feedback. The results provide evidence for a physiological basis of different coping strategies in birds, emerging in response to social stress and with a pattern similar to that in other vertebrates.