Isolation of ERp29, a novel endoplasmic reticulum protein, from rat enamel cells evidence for a unique role in secretory-protein synthesis.

Recently we cloned and described ERp29, a novel 29-kDa endoplasmic reticulum (ER) protein that is widely expressed in rat tissues. Here we report our original isolation of ERp29 from dental enamel cells, and the comprehensive sequence analysis that correlated ERp29 with its cognate cDNA, both in enamel cells and liver. Fractionation of enamel cells using a new freeze-thaw procedure showed that ERp29 partitioned with known reticuloplasmins, and not with soluble proteins from mitochondria or cytosol. The absence of ERp29 in secreted enamel matrix indicated that the C-terminal tetrapeptide (KEEL motif) confers effective ER-retention in enamel cells. ERp29 behaved as a single species (approximately 40 kDa) during size-exclusion chromatography of liver reticuloplasm, suggesting that most ERp29 is not stably associated with other proteins. Immunoblot analysis showed that ERp29 was up-regulated during enamel secretion and expressed most highly in secretory tissues, indicative of a role in secretory-protein synthesis. Unlike other reticuloplasmins, ERp29 was down-regulated during enamel mineralization and thereby dissociated from a calcium-handling role. Tissue-specific variations in ERp29 molecular abundance were revealed by quantification of reticuloplasmin mole ratios. In conclusion: (a) ERp29 is a novel reticuloplasmin of general functional importance; (b) a unique role in protein processing is implicit from the distinctive expression patterns and molecular structure; (c) ERp29 is primarily involved in normal protein secretory events, not the ER stress response; (d) a major role is likely in tissues where ERp29 was equimolar with established molecular chaperones and foldases. This study implicates ERp29 as a new member of the ER protein-processing machinery, and identifies tissues where the physiological role of ERp29 is most likely to be clearly manifested.     

Mesencephalic astrocyte-derived neurotrophic factor protects the heart from ischemic damage and is selectively secreted upon sarco/endoplasmic reticulum calcium depletion

The endoplasmic reticulum (ER) stress protein mesencephalic astrocyte-derived neurotrophic factor (MANF) has been reported to protect cells from stress-induced cell death before and after its secretion; however, the conditions under which it is secreted are not known. Accordingly, we examined the mechanism of MANF release from cultured ventricular myocytes and HeLa cells, both of which secrete proteins via the constitutive pathway. Although the secretion of proteins via the constitutive pathway is not known to increase upon changes in intracellular calcium, MANF secretion was increased within 30 min of treating cells with compounds that deplete sarcoplasmic reticulum (SR)/ER calcium. In contrast, secretion of atrial natriuretic factor from ventricular myocytes was not increased by SR/ER calcium depletion, suggesting that not all secreted proteins exhibit the same characteristics as MANF. We postulated that SR/ER calcium depletion triggered MANF secretion by decreasing its retention. Consistent with this were co-immunoprecipitation and live cell, zero distance, photo affinity cross-linking, demonstrating that, in part, MANF was retained in the SR/ER via its calcium-dependent interaction with the SR/ER-resident protein, GRP78 (glucose-regulated protein 78 kDa). This unusual mechanism of regulating secretion from the constitutive secretory pathway provides a potentially missing link in the mechanism by which extracellular MANF protects cells from stresses that deplete SR/ER calcium. Consistent with this was our finding that administration of recombinant MANF to mice decreased tissue damage in an in vivo model of myocardial infarction, a condition during which ER calcium is known to be dysregulated, and MANF expression is induced.     

The making and breaking of the endoplasmic reticulum

The endoplasmic reticulum (ER) is a dynamic organelle central to many essential cellular functions. It is an important calcium store, which functions in cellular signal transduction cascades. It is also the site of entry for secreted proteins into the secretory pathway. Lumenal enzymes will fold and glycosylate these proteins, and if a protein is destined to be secreted, it will be packaged into membrane vesicles that bud off from the ER. The ER is also the site where most cellular lipids are synthesized. It is contiguous with the nuclear envelope, which serves as a diffusion barrier to control entry into and out of the nucleus. In the life cycle of a cell, the ER is in a constant flux of membrane traffic. What maintains the ER in the shape of an intact reticulum among this constant flux of material? We discuss the mechanisms that contribute to the biogenesis of the ER, the maintenance of the organelle, as well as processes that give the ER its characteristic shape and pattern of inheritance.     

The prodomain of a secreted hydrophobic mini-protein facilitates its export from the endoplasmic reticulum by hitchhiking on sorting receptors

Misfolded secretory proteins are retained in the endoplasmic reticulum (ER) by quality control mechanisms targeted to exposed hydrophobic surfaces. Paradoxically, certain conotoxins expose extensive hydrophobic surfaces upon folding to their bioactive structures. How then can such secreted mini-proteins traverse the secretory pathway? Here we show that secretion of the hydrophobic conotoxin-TxVI is strongly dependent on its propeptide domain, which enhances TxVI export from the ER. The propeptide domain interacts with sorting receptors from the sortilin Vps10p domain family. The sortilin-TxVI interaction occurs in the ER, and sortilin facilitates export of TxVI from the ER to the Golgi. Thus, the prodomain in a secreted hydrophobic protein acts as a tag that can facilitate its ER export by a hitchhiking mechanism.     

The endoplasmic reticulum and calcium storage

Calcium storage is one of the functions commonly attributed to the endoplasmic reticulum (ER) in nonmuscle cells. Several recent studies have added support to this concept. Analysis of reticuloplasm, the luminal ER content, has shown that it contains several proteins (reticuloplasmins) which are prospective calcium storage proteins. One of these, calreticulin, is also present in the sarcoplasmic reticulum (SR). In sea urchin eggs, a calsequestrin-like protein has been clearly localised to the ER. The recent demonstration that the IP3 receptor, which has similarities with the calcium release channel in the SR is also localised in the ER membrane suggests that calcium stored in the ER is important for intracellular signalling. The alternative view, that the physiologically important calcium store is a specialised organelle, the calciosome, is not supported by these observations. Recent evidence also suggests that ER calcium might be important in ER structure and in the retention of the luminal ER proteins.     

Cholesterol is required for efficient endoplasmic reticulum-to-Golgi transport of secretory membrane proteins

Although cholesterol is synthesized in the endoplasmic reticulum (ER), compared with other cellular membranes, ER membrane has low cholesterol (3-6%). Most of the molecular machinery that regulates cellular cholesterol homeostasis also resides in the ER. Little is known about how cholesterol itself affects the ER membrane. Here, we demonstrate that acute cholesterol depletion in ER membranes impairs ER-to-Golgi transport of secretory membrane proteins. Cholesterol depletion is achieved by a brief inhibition of cholesterol synthesis with statins in cells grown in cholesterol-depleted medium. We provide evidence that secretory membrane proteins vesicular stomatitis virus glycoprotein and scavenger receptor A failed to be efficiently transported from the ER upon cholesterol depletion. Fluorescence photobleaching recovery experiments indicated that cholesterol depletion by statins leads to a severe loss of lateral mobility on the ER membrane of these transmembrane proteins, but not loss of mobility of proteins in the ER lumen. This impaired lateral mobility is correlated with impaired ER-to-Golgi transport. These results provide evidence for the first time that cholesterol is required in the ER membrane to maintain mobility of membrane proteins and thus protein secretion.     

Native and Artificial Reticuloplasmins Co-Accumulate in Distinct Domains of the Endoplasmic Reticulum and in Post-Endoplasmic Reticulum Compartments

We compared the subcellular distribution of native and artificial reticuloplasmins in endosperm, callus, and leaf tissues of transgenic rice (Oryza sativa) to determine the distribution of these proteins among endoplasmic reticulum (ER) and post-ER compartments. The native reticuloplasmin was calreticulin. The artificial reticuloplasmin was a recombinant single-chain antibody (scFv), expressed with an N-terminal signal peptide and the C-terminal KDEL sequence for retrieval to the ER (scFvT84.66-KDEL). We found that both molecules were distributed in the same manner. In endosperm, each accumulated in ER-derived prolamine protein bodies, but also in glutelin protein storage vacuoles, even though glutelins are known to pass through the Golgi apparatus en route to these organelles. This finding may suggest that similar mechanisms are involved in the sorting of reticuloplasmins and rice seed storage proteins. However, the presence of reticuloplasmins in protein storage vacuoles could also be due to simple dispersal into these compartments during protein storage vacuole biogenesis, before glutelin deposition. In callus and leaf mesophyll cells, both reticuloplasmins accumulated in ribosome-coated vesicles probably derived directly from the rough ER.     

Secretory cargo composition affects polarized secretion in MDCK epithelial cells

Polarized epithelial cells secrete proteins at either the apical or basolateral cell surface. A number of non-epithelial secretory proteins also exhibit polarized secretion when they are expressed in polarized epithelial cells but it is difficult to predict where foreign proteins will be secreted in epithelial cells. The question is of interest since secretory epithelia are considered as target tissues for gene therapy protocols that aim to express therapeutic secretory proteins. In the parathyroid gland, parathyroid hormone is processed by furin and co-stored with chromogranin A in secretory granules. To test the secretion of these proteins in epithelial cells, they were expressed in MDCK cells. Chromogranin A and a secreted form of furin were secreted apically while parathyroid hormone was secreted 60% basolaterally. However, in the presence of chromogranin A, the secretion of parathyroid hormone was 65% apical, suggesting that chromogranin can act as a “sorting escort” (sorting chaperone) for parathyroid hormone. Conversely, apically secreted furin did not affect the sorting of parathyroid hormone. The apical secretion of chromogranin A was dependent on cholesterol, suggesting that this protein uses an established cellular sorting mechanism for apical secretion. However, this sorting does not involve the N-terminal membrane-binding domain of chromogranin A. These results suggest that foreign secretory proteins can be used as “sorting escorts” to direct secretory proteins to the apical secretory pathway without altering the primary structure of the secreted protein. Such a system may be of use in the targeted expression of secretory proteins from epithelial cells. David V. Cohn—Deceased.     

Calnexin family members as modulators of genetic diseases

The endoplasmic reticulum (ER) is an intracellular compartment devoted to the synthesis, segregation and folding of soluble and membrane secretory proteins. Some mutations in these proteins lead to their incorrect or incomplete folding in the ER. The ER has a quality control system which detects misfolded proteins and then specifies their fate. Some mutated proteins are retained in the ER wherein they accumulate (Russell bodies for misfolded immunoglobulin heavy chains, the PiZZ for alpha 1-antitrypsin), others are retrotranslocated from the ER and degraded by the cytosolic proteasomal system, and yet other proteins are eventually secreted (in AZC-treated cells). In this review we summarize the role of ER resident proteins in quality control of mutated secretory proteins.     

A C-terminal signal prevents secretion of luminal ER proteins

Proteins that permanently reside in the lumen of the endoplasmic reticulum (ER) must somehow be distinguished from newly synthesized secretory proteins, which pass through this compartment on their way out of the cell. Three luminal ER proteins whose sequence is known, grp78 ("BiP"), grp94, and protein disulphide isomerase, share the carboxy-terminal sequence Lys-Asp-Glu-Leu (KDEL). We show that deletion (or extension) of the carboxyl terminus of grp78 results in secretion of this protein when it is expressed in COS cells. Conversely, a derivative of chicken lysozyme containing the last six amino acids of grp78 fails to be secreted and instead accumulates in the ER. We propose that the KDEL sequence marks proteins that are to be retained in the ER and discuss possible retention mechanisms.     

The reticulons: guardians of the structure and function of the endoplasmic reticulum

The endoplasmic reticulum (ER) consists of the nuclear envelope and a peripheral network of tubules and membrane sheets. The tubules are shaped by a specific class of curvature stabilizing proteins, the reticulons and DP1; however it is still unclear how the sheets are assembled. The ER is the cellular compartment responsible for secretory and membrane protein synthesis. The reducing conditions of ER lead to the intra/inter-chain formation of new disulphide bonds into polypeptides during protein folding assessed by enzymatic or spontaneous reactions. Moreover, ER represents the main intracellular calcium storage site and it plays an important role in calcium signaling that impacts many cellular processes. Accordingly, the maintenance of ER function represents an essential condition for the cell, and ER morphology constitutes an important prerogative of it. Furthermore, it is well known that ER undergoes prominent shape transitions during events such as cell division and differentiation. Thus, maintaining the correct ER structure is an essential feature for cellular physiology. Now, it is known that proper ER-associated proteins play a fundamental role in ER tubules formation. Among these ER-shaping proteins are the reticulons (RTN), which are acquiring a relevant position. In fact, beyond the structural role of reticulons, in very recent years new and deeper functional implications of these proteins are emerging in relation to their involvement in several cellular processes.     

SPRED: A machine learning approach for the identification of classical and non-classical secretory proteins in mammalian genomes

Eukaryotic protein secretion generally occurs via the classical secretory pathway that traverses the ER and Golgi apparatus. Secreted proteins usually contain a signal sequence with all the essential information required to target them for secretion. However, some proteins like fibroblast growth factors (FGF-1, FGF-2), interleukins (IL-1 alpha, IL-1 beta), galectins and thioredoxin are exported by an alternative pathway. This is known as leaderless or non-classical secretion and works without a signal sequence. Most computational methods for the identification of secretory proteins use the signal peptide as indicator and are therefore not able to identify substrates of non-classical secretion. In this work, we report a random forest method, SPRED, to identify secretory proteins from protein sequences irrespective of N-terminal signal peptides, thus allowing also correct classification of non-classical secretory proteins. Training was performed on a dataset containing 600 extracellular proteins and 600 cytoplasmic and/or nuclear proteins. The algorithm was tested on 180 extracellular proteins and 1380 cytoplasmic and/or nuclear proteins. We obtained 85.92% accuracy from training and 82.18% accuracy from testing. Since SPRED does not use N-terminal signals, it can detect non-classical secreted proteins by filtering those secreted proteins with an N-terminal signal by using SignalP. SPRED predicted 15 out of 19 experimentally verified non-classical secretory proteins. By scanning the entire human proteome we identified 566 protein sequences potentially undergoing non-classical secretion. The dataset and standalone version of the SPRED software is available at http://www.inb.uni-luebeck.de/tools-demos/spred/spred.     

Characterization of V-ATPase inhibitor-induced secretion of cysteine-rich with EGF-like domains 2

We previously demonstrated that cysteine-rich with EGF-like domains 2 (CRELD2), a novel ER stress-inducible factor, is a secretory glycoprotein; however, the stimuli that induce CRELD2 secretion have not yet been characterized. In this study, we found that the perturbation of intravesicular acidification of cytoplasmic organelles in HEK293 cells stably expressing wild-type (wt) CRELD2 induced its secretion. In particular, Concanamycin A (CMA) and Bafilomycin A1 (Baf), inhibitors of vacuolar ATPase (V-ATPase), increased the secretion of CRELD2 without relying on its C-terminal structure. The levels of secretion of EGFP-fused CRELD2 (SP-EGFP-CRELD2), which consists of EGFP following the putative signal peptide (SP) sequence of CRELD2, from COS7 cells transiently transfected with this construct were also increased after each of the treatments, but their intracellular localization was barely affected by CMA treatment. Transient overexpression of 78-kDa glucose-regulated protein (GRP78) and protein disulfide isomerase (PDI) also increased the secretion of CRELD2 from HEK293 cells expressing wt CRELD2, whereas the perturbation of intravesicular acidification did not alter the expression of GRP78 and PDI in the HEK293 cells. We further studied the roles of intracellular calcium ions and the Golgi apparatus in the secretion of CRELD2 from HEK293 cells in which intravesicular acidification was perturbed. The treatment with calcium ionophore increased the secretion of wt CRELD2, while that with BAPTA-AM, an intracellular calcium chelator, did not reduce the CMA-induced CRELD2 secretion. By contrast, treatment with brefeldin A (BFA), which inhibits the transportation of proteins from the ER to the Golgi apparatus, almost completely abolished the secretion of wt CRELD2 from the HEK293 cells. In conclusion, we demonstrated that the intravesicular acidification by V-ATPase regulates the secretion of CRELD2 without relying on the balance of intracellular calcium ions and the expression of ER chaperones such as GRP78 and PDI. These findings concerning the role of V-ATPases in modulating the secretion of CRELD2, a novel ER stress-inducible secretory factor, may provide new insights into the prevention and treatment of certain ER stress-related diseases.     

The intracellular transport and secretion of calumenin-1/2 in living cells

Calumenin isoforms 1 and 2 (calu-1/2), encoded by the CALU gene, belong to the CREC protein family. Calu-1/2 proteins are secreted into the extracellular space, but the secretory process and regulatory mechanism are largely unknown. Here, using a time-lapse imaging system, we visualized the intracellular transport and secretory process of calu-1/2-EGFP after their translocation into the ER lumen. Interestingly, we observed that an abundance of calu-1/2-EGFP accumulated in cellular processes before being released into the extracellular space, while only part of calu-1/2-EGFP proteins were secreted directly after attaching to the cell periphery. Moreover, we found the secretion of calu-1/2-EGFP required microtubule integrity, and that calu-1/2-EGFP-containing vesicles were transported by the motor proteins Kif5b and cytoplasmic dynein. Finally, we determined the export signal of calu-1/2-EGFP (amino acid positions 20-46) and provided evidence that the asparagine at site 131 was indispensable for calu-1/2-EGFP stabilization. Taken together, we provide a detailed picture of the intracellular transport of calu-1/2-EGFP, which facilitates our understanding of the secretory mechanism of calu-1/2.     

Inhibition of cellular protein secretion by norwalk virus nonstructural protein p22 requires a mimic of an endoplasmic reticulum export signal

Protein trafficking between the endoplasmic reticulum (ER) and Golgi apparatus is central to cellular homeostasis. ER export signals are utilized by a subset of proteins to rapidly exit the ER by direct uptake into COPII vesicles for transport to the Golgi. Norwalk virus nonstructural protein p22 contains a YXΦESDG motif that mimics a di-acidic ER export signal in both sequence and function. However, unlike normal ER export signals, the ER export signal mimic of p22 is necessary for apparent inhibition of normal COPII vesicle trafficking, which leads to Golgi disassembly and antagonism of Golgi-dependent cellular protein secretion. This is the first reported function for p22. Disassembly of the Golgi apparatus was also observed in cells replicating Norwalk virus, which may contribute to pathogenesis by interfering with cellular processes that are dependent on an intact secretory pathway. These results indicate that the ER export signal mimic is critical to the antagonistic function of p22, shown herein to be a novel antagonist of ER/Golgi trafficking. This unique and well-conserved human norovirus motif is therefore an appealing target for antiviral drug development.     

In vitro biosynthesis and secretion of rabbit epididymal secretory proteins: regulation by androgens

The biosynthesis and secretion of epididymal proteins were studied in an in vitro system using explants from rabbit epididymis cultured in a defined medium. Epididymal explants actively incorporated [35S]methionine into cellular proteins, about 7% of them being secreted into the medium. SDS-PAGE of the labeled proteins secreted to the medium showed regional differences in their synthesis and secretion along the epididymal tract. Castration resulted in the inhibition of the synthesis and secretion of at least two polypeptides of Mr 150,000 and 21,000, but at the same time induced the appearance of other polypeptides. Immunoprecipitations with a specific antibody indicated that the variations in the amounts of the secreted 21 kDa component were associated with differences in its rate of synthesis. Epididymis from immature rabbits synthesized some polypeptides that are repressed in the adult state. The results suggest a dual effect of testosterone on rabbit epididymal secretory proteins.     

Endoplasmic reticulum stress and fungal pathogenesis

The gateway to the secretory pathway is the endoplasmic reticulum (ER), an organelle that is responsible for the accurate folding, post-translational modification and final assembly of up to a third of the cellular proteome. When secretion levels are high, errors in protein biogenesis can lead to the accumulation of abnormally folded proteins, which threaten ER homeostasis. The unfolded protein response (UPR) is an adaptive signaling pathway that counters a buildup in misfolded and unfolded proteins by increasing the expression of genes that support ER protein folding capacity. Fungi, like other eukaryotic cells that are specialized for secretion, rely upon the UPR to buffer ER stress caused by fluctuations in secretory demand. However, emerging evidence is also implicating the UPR as a central regulator of fungal pathogenesis. In this review, we discuss how diverse fungal pathogens have adapted ER stress response pathways to support the expression of virulence-related traits that are necessary in the host environment.     

Location of sequences within rotavirus SA11 glycoprotein VP7 which direct it to the endoplasmic reticulum.

The Simian 11 rotavirus glycoprotein VP7 is directed to the endoplasmic reticulum (ER) of the cell and retained as an integral membrane protein. The gene coding for VP7 predicts two potential initiation codons, each of which precedes a hydrophobic region of amino acids (H1 and H2) with the characteristics of a signal peptide. Using the techniques of gene mutagenesis and expression, we have determined that either hydrophobic domain alone can direct VP7 to the ER. A protein lacking both hydrophobic regions was not transported to the ER. Some polypeptides were directed across the ER membrane and then into the secretory pathway of the cell. For a variant retaining only the H1 domain, secretion was cleavage dependent, since an amino acid change which prevented cleavage also stopped secretion. However, secretion of two other deletion mutants lacking H1 and expressing truncated H2 domains was unaffected by this mutation, suggesting that these proteins were secreted without cleavage of their NH2-terminal hydrophobic regions or secreted after cleavage at a site(s) not predicted by current knowledge.