Oligomerization properties of ERp29, an endoplasmic reticulum stress protein (vol 431, pg 322, 1998) (original) (raw)
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Oligomerization properties of ERp29, an endoplasmic reticulum stress protein
FEBS Letters, 1998
ERp29, a novel and ubiquitously expressed endoplasmic reticulum (ER) stress-inducible protein, was recently isolated and cDNA cloned in our laboratory. Using size exclusion chromatography and chemical cross-linking we have assessed the oligomerization properties of ERp29. Purified ERp29 in solution as well as in rat hepatoma cells self-associates predominantly into homodimers. Labeling of the cells with [ QS S]methionine with subsequent cross-linking and immunoprecipitation showed that ERp29 interacts with a number of ER proteins, one of which was previously identified as BiP/GRP78. Secondary structure prediction and fold recognition methods indicate that the native conformation of ERp29 resembles the thioredoxin fold, a structural motif characteristic of a number of enzymes with the redox function, including protein disulfide isomerase (with which ERp29 shares limited sequence similarity). Dimerization of the protein is suggested to be advantageous for the protein binding potential of ERp29.
Biochemistry, 1996
GRP94 is an abundant, resident glycoprotein of the mammalian endoplasmic reticulum lumen and member of the hsp90 family of molecular chaperones. To identify the structure/function relationships which define the molecular basis of GRP94 activity, we have performed a structural analysis of native GRP94 and identified a discrete domain, representing amino acids 676-719, which regulates dimerization and displays autonomous oligomerization activity. Velocity sedimentation and gel filtration chromatography were used to identify native GRP94 as a dimer with an extended, rod-like shape. Limited proteolysis resulted in the loss of approximately 16 kDa from the C-terminus and disassembly into monomers, implicating the C-terminus as the site of assembly. An assembly function for the C-terminal domain was established by analysis of the quaternary structure of C-terminal constructs synthesized either in vitro or through recombinant expression. In vitro translation was used to demonstrate that a C-terminal 20 kDa domain was both necessary and sufficient for dimerization. Structural studies of recombinant fusion protein constructs yielded identification of a 44 amino acid domain that displayed autonomous dimerization activity and conferred a highly elongated structure, characteristic of native GRP94, to the fusion protein. These data, combined with molecular dimensions obtained from rotary shadowing electron microscopy, provide a structural model of GRP94 and identify the molecular basis of GRP94 self-assembly.
Journal of Biological Chemistry, 2008
Normally, non-native polypeptides are not transported through the secretory pathway. Rather, they are translocated from the endoplasmic reticulum (ER) lumen into the cytosol where they are degraded by proteasomes. Here we characterize the function in ER quality control of two proteins derived from alternative splicing of the OS-9 gene. OS-9.1 and OS-9.2 are ubiquitously expressed in human tissues and are amplified in tumors. They are transcriptionally induced upon activation of the Ire1/Xbp1 ER-stress pathway. OS-9 variants do not associate with folding-competent proteins. Rather, they selectively bind folding-defective ones thereby inhibiting transport of non-native conformers through the secretory pathway. The intralumenal level of OS-9.1 and OS-9.2 inversely correlates with the fraction of a folding-defective glycoprotein, the Null hong kong (NHK) variant of ␣1-antitrypsin that escapes retention-based ER quality control. OS-9 up-regulation does not affect NHK disposal, but reduction of the intralumenal level of OS-9.1 and OS-9.2 substantially delays disposal of this model substrate. OS-9.1 and OS-9.2 also associate transiently with non-glycosylated foldingdefective proteins, but association is unproductive. Finally, OS-9 activity does not require an intact mannose 6-P homology domain. Thus, OS-9.1 and OS-9.2 play a dual role in mammalian ER quality control: first as crucial retention factors for misfolded conformers, and second as promoters of protein disposal from the ER lumen.
Endoplasmic Reticulum and the Unfolded Protein Response
International Review of Cell and Molecular Biology, 2013
The endoplasmic reticulum (ER) is a dynamic intracellular organelle with multiple functions essential for cellular homeostasis, development, and stress responsiveness. In response to cellular stress, a well-established signaling cascade, the unfolded protein response (UPR), is activated. This intricate mechanism is an important means of reestablishing cellular homeostasis and alleviating the inciting stress. Now, emerging evidence has demonstrated that the UPR influences cellular metabolism through diverse mechanisms, including calcium and lipid transfer, raising the prospect of involvement of these processes in the pathogenesis of disease, including neurodegeneration, cancer, diabetes mellitus and cardiovascular disease. Here, we review the distinct functions of the ER and UPR from a metabolic point of view, highlighting their association with prevalent pathologies.
Regulation of ERGIC-53 Gene Transcription in Response to Endoplasmic Reticulum Stress
Journal of Biological Chemistry, 2007
Accumulation of unfolded proteins within the endoplasmic reticulum (ER) activates the unfolded protein response, also known as the ER stress response. We previously demonstrated that ER stress induces transcription of the ER Golgi intermediate compartment protein ERGIC-53. To investigate the molecular events that regulate unfolded protein response-mediated induction of the gene, we have analyzed the transcriptional regulation of ERGIC-53. We found that the ERGIC-53 promoter contains a single cis-acting element that mediates induction of the gene by thapsigargin and other ER stress-causing agents. This ER stress response element proved to retain a novel structure and to be highly conserved in mammalian ERGIC-53 genes. The ER stress response element identified contains a 5-end CCAAT sequence that constitutively binds NFY/CBF and, 9 nucleotides away, a 3-end region (5-CCCTGTTGGCCATC-3) that is equally important for ER stressmediated induction of the gene. This sequence is the binding site for endogenous YY1 at the 5-CCCTGTTGG-3 part and for undefined factors at the CCATC 3-end. ATF6␣-YY1, but not XBP1, interacted with the ERGIC-53 regulatory region and activated ERGIC-53 ER stress response element-dependent transcription. A molecular model for the transcriptional regulation of the ERGIC-53 gene is proposed.
Journal of Biological Chemistry, 1992
THE JOURNAL OF BIOLOGICAL CHEMISTRY The Endoplasmic Reticulum (ll), ATP-dependent (12) and likely to involve cycles of Stress Protein GRP94, in Addition to BiP, Associates with Unassembled Immunoglobulin L chain remain undefined. By analogy to other molecular Chains* association and dissociation (8). The first constant domain of the H chain as well as the variable domain are involved in binding to BiP (2, 3, 13), whereas the binding site(s) on the chaperones, BiP is thought to maintain the Ig subunits in a state that enables them to complete efficiently their folding (Received for publication, August 5, 1992) and assembly into functional antibody molecules (9, 10). In addition to BiP/GRP78, the ER of higher eukaryotic Jeffrey Sigal and Yair cells contain another stress protein, known alternately as FrOmthe Department of Immu~logY~ Duke University GRP94 (14), endoplasmin (15), ERp99 (16), or gp96 (17). Medical Center, Durham, North Carolina 27710 and the GRpg4 is a member of the HSpgO family and has been &own 7Basel Institute for Immunology, Grenzacherstrasse 487, CH-4005 Basel, Switzerland to be a major Ca2+ and ATP binding component of the ER lumen (18, 19). However, unlike BiP, the function of GRP94 The molecular chaperone BiP/GRP78 associates with various polypeptides in the endoplasmic reticulum, including immunoglobulin chains. We now show, using chemical cross-linking, that another endoplasmic reticulum stress protein, GRP94, associates with newly synthesized immunoglobulin light and heavy chains. We demonstrate the presence of ternary complexes composed of immunoglobulin chains, BiP and GRP94. Because both BiP and GRP94 associate far less with fully assembled immunoglobulin than with unassembled subunits, our data suggest that GRP94, like BiP, functions as a molecular chaperone. The presence of both BiP and GRP94 in the same complex further suggests that the two stress proteins work in concert during the folding and assembly of immunoglobulins. The heavy (H)' and light (L) chains of immunoglobulin (Ig), like other secreted proteins, begin to fold and to associate with each other co-translationally (1). Assembly into antigenbinding Ig occurs in the lumen of the endoplasmic reticulum (ER) and is completed within minutes of synthesis. Prior to assembly, each of the two subunits is found in association with BiP/GRP78 (2-8), the ER chaperone, which is a member of the HSP7O family of stress proteins (9,lO). The interaction between BiP and H or L chains has been shown to be transient
Molecular and Cellular Biology, 2001
When mammalian cells are subjected to stress targeted to the endoplasmic reticulum (ER), such as depletion of the ER Ca 2+ store, the transcription of a family of glucose-regulated protein (GRP) genes encoding ER chaperones is induced. The GRP promoters contain multiple copies of the ER stress response element (ERSE), consisting of a unique tripartite structure, CCAAT(N 9 )CCACG. Within a subset of mammalian ERSEs, N 9 represents a GC-rich sequence of 9 bp that is conserved across species. A novel complex (termed ERSF) exhibits enhanced binding to the ERSE of the grp78 and ERp72 promoters using HeLa nuclear extracts prepared from ER-stressed cells. Optimal binding of ERSF to ERSE and maximal ERSE-mediated stress inducibility require the conserved GGC motif within the 9-bp region. Through chromatographic purification and subsequent microsequencing, we have identified ERSF as TFII-I. Whereas TFII-I remains predominantly nuclear in both nontreated NIH 3T3 cells and cells treated with t...
Computational and structural biotechnology journal, 2013
The endoplasmic reticulum (ER) stress response is a cytoprotective mechanism that maintains homeostasis of the ER by upregulating the capacity of the ER in accordance with cellular demands. If the ER stress response cannot function correctly, because of reasons such as aging, genetic mutation or environmental stress, unfolded proteins accumulate in the ER and cause ER stress-induced apoptosis, resulting in the onset of folding diseases, including Alzheimer's disease and diabetes mellitus. Although the mechanism of the ER stress response has been analyzed extensively by biochemists, cell biologists and molecular biologists, many aspects remain to be elucidated. For example, it is unclear how sensor molecules detect ER stress, or how cells choose the two opposite cell fates (survival or apoptosis) during the ER stress response. To resolve these critical issues, structural and computational approaches will be indispensable, although the mechanism of the ER stress response is compli...