Selenoprotein T is a novel OST subunit that regulates UPR signaling and hormone secretion

Selenoprotein T (SelT) is a recently characterized thioredoxin‐like protein whose expression is very high during development, but is confined to endocrine tissues in adulthood where its function is unknown. We report here that SelT is required for adaptation to the stressful conditions of high hormone level production in endocrine cells. Using immunofluorescence and TEM immunogold approaches, we find that SelT is expressed at the endoplasmic reticulum membrane in all hormone‐producing pituitary cell types. SelT knockdown in corticotrope cells promotes unfolded protein response (UPR) and ER stress and lowers endoplasmic reticulum‐associated protein degradation (ERAD) and hormone production. Using a screen in yeast for SelT‐membrane protein interactions, we sort keratinocyte‐associated protein 2 (KCP2), a subunit of the protein complex oligosaccharyltransferase (OST). In fact, SelT interacts not only with KCP2 but also with other subunits of the A‐type OST complex which are depleted after SelT knockdown leading to POMC N‐glycosylation defects. This study identifies SelT as a novel subunit of the A‐type OST complex, indispensable for its integrity and for ER homeostasis, and exerting a pivotal adaptive function that allows endocrine cells to properly achieve the maturation and secretion of hormones.     

A genome-wide RNAi screen identifies regulators of cholesterol-modified hedgehog secretion in Drosophila

Hedgehog (Hh) proteins are secreted molecules that function as organizers in animal development. In addition to being palmitoylated, Hh is the only metazoan protein known to possess a covalently-linked cholesterol moiety. The absence of either modification severely disrupts the organization of numerous tissues during development. It is currently not known how lipid-modified Hh is secreted and released from producing cells. We have performed a genome-wide RNAi screen in Drosophila melanogaster cells to identify regulators of Hh secretion. We found that cholesterol-modified Hh secretion is strongly dependent on coat protein complex I (COPI) but not COPII vesicles, suggesting that cholesterol modification alters the movement of Hh through the early secretory pathway. We provide evidence that both proteolysis and cholesterol modification are necessary for the efficient trafficking of Hh through the ER and Golgi. Finally, we identified several putative regulators of protein secretion and demonstrate a role for some of these genes in Hh and Wingless (Wg) morphogen secretion in vivo. These data open new perspectives for studying how morphogen secretion is regulated, as well as provide insight into regulation of lipid-modified protein secretion.     

Overexpression of GRP78 mitigates stress induction of glucose regulated proteins and blocks secretion of selective proteins in Chinese hamster ovary cells.

GRP78 is a resident protein of the endoplasmic reticulum (ER) and a member of the glucose regulated protein (GRP) family. Many secretion incompetent proteins are found in stable association with GRP78 and are retained in the ER. Some proteins which are destined for secretion transiently associate with GRP78. To further increase our understanding of the role of GRP78 in secretion, we have stably overexpressed GRP78 in Chinese hamster ovary (CHO) cells and examined the effect on protein secretion and the stress response. GRP78 overexpressing cells treated with tunicamycin or A23187 exhibited a reduced induction of endogenous GRP78 and GRP94 mRNAs compared to wild-type CHO cells. This suggests that GRP78 overexpression either alleviates the stress or is directly involved in signaling stress-induced expression of GRPs. Transient expression of secreted proteins was used to measure secretion efficiency in the GRP78 overexpressing cells. Secretion of von Willebrand factor and a mutant form of factor VIII, two proteins which transiently associate with GRP78, was reduced by GRP78 overexpression. In contrast, secretion of M-CSF, which was not detected in association with GRP78, was unaffected. This indicates that elevated levels of GRP78 may increase stable association and decrease the secretion efficiency of proteins which normally transiently associate with GRP78. These results indicate that one function of GRP78 is selective protein retention in the ER.     

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.     

Exiting the endoplasmic reticulum

Vesicular transport from the endoplasmic reticulum (ER) to the Golgi complex constitutes the initial step in protein secretion. COPII-coated vesicles mediate the export of newly synthesized proteins from the ER, and this transport step is coupled with COPI-mediated retrograde traffic to form a transport circuit that supports the compositional asymmetry of the ER-Golgi system. Biochemical and structural studies have advanced our understanding of the mechanisms that control vesicle formation and cargo-protein capture. Recent work has highlighted the function of transitional ER regions in specifying the location of COPII budding.     

Estrogen-modulated estrogen receptor x Pit-1 protein complex formation and prolactin gene activation require novel protein synthesis

Both estrogen receptor (ER) and Pit-1 proteins are essential for the estrogen-activated expression of the rat prolactin gene. Our results show that ER.Pit-1 protein complex formation is reduced by estrogen in GH3 and PR1 rat pituitary tumor cells. In the latter, this decrease was blocked by cycloheximide, a protein synthesis inhibitor. On the other hand, the direct addition of estrogen to PR1 cell lysates had no effect on the formation of ER.Pit-1 complexes. Estrogen-activated prolactin gene expression was also inhibited by cycloheximide, suggesting that some form of protein synthesis is involved in ER.Pit-1 complex formation and subsequent prolactin gene activation. In support of this notion, we showed that estrogen-induced regulation of ER.Pit-1 complex formation could be transferred from cell lysates prepared from estrogen-treated PR1 cells to control cell lysates. This is not true for GH3 cells; instead, direct administration of estrogen to GH3 cell lysates readily abolished ER.Pit-1 protein complex formation in a dose-dependent manner, and such estrogen-induced regulation was blocked by the antiestrogen ICI 182,780. These findings thus indicate that 1) interaction between ER and Pit-1 proteins is estrogen-regulated in ways specific to different cell types, and 2) auxiliary protein factor synthesis may be involved in this process.     

Lysyl hydroxylase 3 is secreted from cells by two pathways

Lysyl hydroxylase 3 (LH3) is a post-translational modification enzyme with lysyl hydroxylase (LH), collagen galactosyltransferase (GT), and glucosyltransferase (GGT) activities. The active sites responsible for LH and GT/GGT activities of LH3 are localized separately in the carboxy- and the amino-terminal parts of the molecule, respectively. LH3 is found both intracellularly in the ER, as well as extracellularly in serum, the extracellular space and on cell surfaces, and is the only secreted LH isoform. In order to determine whether the activities of LH3 play a role in the secretion, we created various LH3 and mutant expression constructs and over-expressed the proteins in COS-7 and HT-1080 cells. Our data indicate that while the LH active site mediates retention of LH3 in the ER, the GGT active site is required for the secretion of LH3 into the extracellular space. Moreover, Brefeldin A treatment and cholesterol depletion of the cells revealed that the secretion of LH3 from the ER to the extracellular space occurs via two secretory pathways, which generate two glycoforms. LH3 molecules found in the cell medium are secreted through the Golgi complex, and the secretion is dependent on LH3 glycosyltransferase activity. LH3 found on the cell surface bypasses the Golgi complex.     

COPII-dependent ER export in animal cells: adaptation and control for diverse cargo

The export of newly synthesized proteins from the endoplasmic reticulum is fundamental to the ongoing maintenance of cell and tissue structure and function. After co-translational translocation into the ER, proteins destined for downstream intracellular compartments or secretion from the cell are sorted and packaged into transport vesicles by the COPII coat protein complex. The fundamental discovery and characterization of the pathway has now been augmented by a greater understanding of the role of COPII in diverse aspects of cell function. We now have a deep understanding of how COPII contributes to the trafficking of diverse cargoes including extracellular matrix molecules, developmental signalling proteins, and key metabolic factors such as lipoproteins. Structural and functional studies have shown that the COPII coat is both highly flexible and subject to multiple modes of regulation. This has led to new discoveries defining roles of COPII in development, autophagy, and tissue organization. Many of these newly emerging features of the canonical COPII pathway are placed in a context of procollagen secretion because of the fundamental interest in how a coat complex that typically generates 80-nm transport vesicles can package a cargo reported to be over 300 nm. Here we review the current understanding of COPII and assess the current consensus on its role in packaging diverse cargo proteins.     

Differential effect of combined lipase deficiency (cld/cld) on human hepatic lipase and lipoprotein lipase secretion.

Combined lipase deficiency (cld) is a recessively inherited disorder in mice associated with a deficiency of LPL and hepatic lipase (HL) activity. LPL is synthesized in cld tissues but is retained in the endoplasmic reticulum (ER), whereas mouse HL (mHL) is secreted but inactive. In this study we investigated the effect of cld on the secretion of human HL (hHL) protein mass and activity. Differentiated liver cell lines were derived from cld mice and their normal heterozygous (het) littermates by transformation of hepatocytes with SV40 large T antigen. After transient transfection with lipase expression constructs, secretion of hLPL activity from cld cells was only 12% of that from het cells. In contrast, the rate of secretion of hHL activity and protein mass per unit of expressed hHL mRNA was identical for the two cell lines. An intermediate effect was observed for mHL, with a 46% reduction in secretion of activity from cld cells. The ER glucosidase inhibitor, castanospermine, decreased secretion of both hLPL and hHL from het cells by approximately 70%, but by only approximately 45% from cld cells. This is consistent with data suggesting that cld may result from a reduced concentration of the ER chaperone calnexin. In conclusion, our results demonstrate a differential effect of cld on hLPL, mHL, and hHL secretion, suggesting differential requirements for activation and exit of the enzymes from the ER.     

Intracellular interactome of secreted antibody Fab fragment in <Emphasis Type="Italic">Pichia pastoris</Emphasis> reveals its routes of secretion and degradation

Protein translation, translocation, folding, processing, and secretion in eukaryotic cells are complex and not always straightforward processes, e.g., different routes of secretion and degradation exist. Formation of malfolded proteins in the endoplasmic reticulum (ER) can be one of the major bottlenecks for recombinant protein production. In this regard, an in-depth analysis of the interactions of a secreted protein during its pathway through the cell may be beneficial, as realized in this study for the methylotrophic yeast Pichia pastoris. The antibody fragment Fab3H6 used here is the anti-idiotype to the HIV neutralizing antibody 2F5 and is known to be intracellularly degraded in significant amounts when expressed in P. pastoris. The interactome of Fab3H6 was analyzed by using a pull-down mass spectrometry approach, and 23 proteins were found to bind specifically to the antibody fragment. Those allowed concluding that Fab3H6 is post-translationally translocated into the ER and degraded via the proteasome as well as the vacuole. In line with this, the expression of Fab3H6 increased the proteasomal activities by over 20%. Partial inhibition of the proteasome resulted in a significant increase of extracellular Fab3H6. Thus, it seems that ER quality control overshoots its requirements for the recombinant protein expressed and that more than just terminally malfolded protein is degraded by ER-associated degradation. This work will further facilitate our understanding how recombinant proteins behave in the secretory pathway.     

The mystery of nonclassical protein secretion. A current view on cargo proteins and potential export routes.

Most of the examples of protein translocation across a membrane (such as the import of classical secretory proteins into the endoplasmic reticulum, import of proteins into mitochondria and peroxisomes, as well as protein import into and export from the nucleus), are understood in great detail. In striking contrast, the phenomenon of unconventional protein secretion (also known as nonclassical protein export or ER/Golgi-independent protein secretion) from eukaryotic cells was discovered more than 10 years ago and yet the molecular mechanism and the molecular identity of machinery components that mediate this process remain elusive. This problem appears to be even more complex as several lines of evidence indicate that various kinds of mechanistically distinct nonclassical export routes may exist. In most cases these secretory mechanisms are gated in a tightly controlled fashion. This review aims to provide a comprehensive overview of our current knowledge as a basis for the development of new experimental strategies designed to unravel the molecular machineries mediating ER/Golgi-independent protein secretion. Beyond solving a fundamental problem in current cell biology, the molecular analysis of these processes is of major biomedical importance as these export routes are taken by proteins such as angiogenic growth factors, inflammatory cytokines, components of the extracellular matrix which regulate cell differentiation, proliferation and apoptosis, viral proteins, and parasite surface proteins potentially involved in host infection.     

Unconventional protein secretion: an evolving mechanism

The process by which proteins are secreted without entering the classical endoplasmic reticulum (ER)–Golgi complex pathway, in eukaryotic cells, is conveniently called unconventional protein secretion. Recent studies on one such protein called Acb1 have revealed a number of components involved in its secretion. Interestingly, conditions that promote the secretion of Acb1 trigger the biogenesis of a new compartment called CUPS (Compartment for Unconventional Protein Secretion). CUPS form near the ER exit site but lack ER‐specific proteins. Other proteins that share some of the features common with the secretion of Acb1 are interleukin‐1β and tissue transglutaminase. Here I will review recent advances made in the field and propose a new model for unconventional protein secretion.     

Role of N-terminal 28-amino-acid region of <Emphasis Type="Italic">Rhizopus oryzae</Emphasis> lipase in directing proteins to secretory pathway of <Emphasis Type="Italic">Aspergillus oryzae</Emphasis>

To develop a new approach for improving heterologous protein production in Aspergillus oryzae, we focused on the functional role of the N-terminal region of Rhizopus oryzae lipase (ROL). Several N-terminal deletion variants of ROL were expressed in A. oryzae. Interestingly, a segment of 28 amino acids from the C-terminal region of the propeptide (N28) was found to be critical for secretion of ROL into the culture medium. To further investigate the role of N28, the ROL secretory process was visualized in vivo using ROL-green fluorescent protein (GFP) fusion proteins. In cells producing ROL with N28, fluorescence observations showed that the fusion proteins are transported through endoplasmic reticulum (ER), Golgi, and cell wall, which is one of the typical secretory processes in a eukaryotic cell. Because the expression of the mature ROL-GFP fusion protein induced fluorescence accumulation without its translocation into the ER, N28 is considered to play a crucial role in protein transport. When N28 was inserted between the secretion signal and GFP, fluorescence observations showed that GFP, which is originally a cytoplasmic protein, was efficiently translocated into the ER of A. oryzae, resulting in an enhanced secretion of mature GFP after proteolytic cleavage of N28. These findings suggest that N28 facilitates protein translocation into ER and can be a promising candidate for improving heterologous protein production in A. oryzae.     

Cell-specific distributions of estrogen receptor alpha (ERα) and androgen receptor (AR) in anterior pituitary glands from adult cockerels as revealed by immunohistochemistry

Estrogens and androgens play important roles in regulating the hormone-secreting functions of the pituitary gland by binding to their corresponding receptors. However, the expression of estrogen receptors (ERs) and the androgen receptor (AR) and the cell types containing ERs and AR in the anterior pituitary gland of adult chickens have not been well-studied. In this study, the distribution of ERα, AR and their corresponding cell types in the anterior pituitary gland of adult cockerels was detected by immunohistochemistry. The results showed that ERα was expressed in 68.63 % of luteinizing hormone (LH) producing cells but was not found in thyrotropes, lactotropes, somatotropes, corticotropes and folliculo-stellate (FS) cells. Pituitary hormone and AR double labeling results showed that about 37 % of LH cells and 50 % of thyroid-stimulating hormone (TSH) producing cells expressed AR, respectively. In contrast, less than 1 % of the somatotropes had an AR positive signal and AR signals were not detected in lactotropes, corticotropes or FS cells. In addition, there were only a few AR and ERα dual-labeled cells observed. These novel results provide evidence for a cell-specific distribution of ERα and AR in the anterior pituitary from adult cockerels by immunohistochemistry. The different distributions of ERα and AR in the LH cells suggest that the feedback-regulating mechanisms of estrogen and androgen on the pituitary hormones secretion are different. The functions and related mechanisms still need to be elucidated further.     

The Florey Lecture, 1992. The secretion of proteins by cells.

In eukaryotic cells, protein secretion provides a complex organizational problem. Secretory proteins are first transported, in an unfolded state, across the membrane of the endoplasmic reticulum (ER), and are then carried in small vesicles to the Golgi apparatus and finally to the cell membrane. The ER contains soluble proteins which catalyse the folding of newly synthesized polypeptides. These proteins are sorted from secretory proteins in the Golgi complex: they carry a sorting signal (the tetrapeptide KDEL or a related sequence) that allows them to be selectively retrieved and returned to the ER. This retrieval process also appears to be used by some bacterial toxins to aid their invasion of the cell: these toxins contain KDEL-like sequences and may, in effect, follow the secretory pathway in reverse. The membrane-bound receptor responsible for sorting luminal ER proteins has been identified in yeast by genetic means, and related receptors are found in mammalian cells. Unexpectedly, this receptor has a second role: in yeast it is required to maintain the normal size and function of the Golgi apparatus. By helping to maintain the composition of both ER and Golgi compartments, the KDEL receptor has an important role in the organization of the secretory pathway.     

Coupling of ER exit to microtubules through direct interaction of COPII with dynactin

Transport of proteins from the endoplasmic reticulum (ER) to the Golgi is mediated by the sequential action of two coat complexes: COPII concentrates cargo for secretion at ER export sites, then COPI is subsequently recruited to nascent carriers and retrieves recycling proteins back to the ER. These carriers then move towards the Golgi along microtubules, driven by the dynein/dynactin complexes. Here we show that the Sec23p component of the COPII complex directly interacts with the dynactin complex through the carboxy-terminal cargo-binding domain of p150(Glued). Functional assays, including measurements of the rate of recycling of COPII on the ER membrane and quantitative analyses of secretion, indicate that this interaction underlies functional coupling of ER export to microtubules. Together, our data suggest a mechanism by which membranes of the early secretory pathway can be linked to motors and microtubules for subsequent organization and movement to the Golgi apparatus.     

The unfolded protein response and its role in intestinal homeostasis and inflammation

The unfolded protein response (UPR) is a signaling pathway from the endoplasmic reticulum (ER) to the nucleus that protects cells from the stress caused by misfolded or unfolded proteins [1, 2]. As such, ER stress is an ongoing challenge for all cells given the central biologic importance of secretion as part of normal physiologic functions. This is especially the case for cells that are highly dependent upon secretory function as part of their major duties. Within mucosal tissues, the intestinal epithelium is especially dependent upon an intact UPR for its normal activities [3]. This review will discuss the UPR and the special role that it provides in the functioning of the intestinal epithelium and, when dysfunctional, its implications for understanding mucosal homeostasis and intestinal inflammation, as occurs in inflammatory bowel disease (IBD).     

Phosphatidylserine is a critical modulator for Akt activation

The endoplasmic reticulum (ER)-Golgi-independent, unconventional secretion of Acb1 requires many different proteins. They include proteins necessary for the formation of autophagosomes, proteins necessary for the fusion of membranes with the endosomes, proteins of the multivesicular body pathway, and the cell surface target membrane SNARE Sso1, thereby raising the question of what achieves the connection between these diverse proteins and Acb1 secretion. In the present study, we now report that, upon starvation in Saccharomyces cerevisiae, Grh1 is collected into unique membrane structures near Sec13-containing ER exit sites. Phosphatidylinositol 3 phosphate, the ESCRT (endosomal sorting complex required for transport) protein Vps23, and the autophagy-related proteins Atg8 and Atg9 are recruited to these Grh1-containing membranes, which lack components of the Golgi apparatus and the endosomes, and which we call a novel compartment for unconventional protein secretion (CUPS). We describe the cellular proteins required for the biogenesis of CUPS, which we believe is the sorting station for Acb1's release from the cells.