ORGANELLE MARKERS - Endoplasmic Reticulum

rBet1

rBet1 is a 17 kDa transmembrane protein that mediates protein transport between endoplasmic reticulum (ER) and the Golgi apparatus. rBet1 serves as the vesicle SNAP receptor (v-SNARE) on ER-derived vesicles, which interacts with its partner t-SNARE on the Golgi apparatus during vesicle docking and fusion. rBet1 is found in a complex with syntaxin 5, GS27/membrin, GOS28, rsec22 and rsly1. Antibodies to rBet1 have been shown to inhibit the transfer of vesicular stomatitis virus G-protein from the ER to the Golgi apparatus in a cell free assay, providing evidence for the involvement of rBet1 in ER to Golgi transport.

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Calnexin

Calnexin (CNX), an abundant ~90 Kda molecular chaperone, is a unglycosylated resident ER transmembrane protein. In mammalian cells, calnexin, together with calreticulin (CRT), plays a key role in glycoprotein folding and its control within the ER, by interacting with folding intermediates via their monoglucosylated glycans. Calnexin associates with newly synthesized monomeric glycoproteins and only recognizes glycoproteins when they are incompletely folded. Calnexin associates with numerous oligomeric protein complexes within the ER, including integrins, major histocompatibility class I and class II molecules, the antigen receptors expressed on T and B lymphocytes, human thyroperoxidase (hTPO), and acetylcholine receptor . Recent data also suggest that calnexin might be responsible for the prolonged retention of pro-6 integrin within ER compartment. Furthermore, calnexin has been demonstrated to function as a bonafide molecular chaperone capable of interacting with polypeptide segments of folding glycoproteins.

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Heme oxygenase-1

Heme oxygenase-1 (HO-1) or HSP32 is the inducible isoform of heme oxygenase which catalyzes the NADPH, O2 and cytochrome P450 reductase dependent oxidation of heme to carbon monoxide, iron and biliverdin that is immediately reduced to bilirubin. These products of the HO reaction have important physiological effects: carbon monoxide is a potent vasodilator; biliverdin and its product bilirubin are potent antioxidants; .free. iron increases oxidative stress and regulates the expression of many mRNAs (e.g., DCT-1, ferritin and transferrin receptor) by affecting the conformation of iron regulatory protein (IRP)-1 and its binding to iron regulatory elements (IREs) in the 5.- or 3.-UTRs of the mRNAs. To date, three heme oxygenase isoforms HO-1, HO-2 and HO-3 have been identified. HO-1, also known as Hsp32, a major heat shock/stress response protein, is ubiquitous and its mRNA as well as its activity can be increased several-fold by heme, other metalloporphyrins, transition metals and stimuli that induce cellular stress. The 5.-untranslated region (UTR) of HO-1 has several consensus regulatory elements which include sites for activator protein 1 (AP-1), metal responsive element (MRE), oncogene c-myc/max heterodimer binding site (Myc/Max), antioxidant response element (ARE) and GC box binding (Sp1). HO-1 expression has been shown to increase in benign postatic hyperplasia (BPH) and malignant prostate tissue suggesting a role for this stress protein in the pathogenesis of BPH and prostate cancer. There is recent data which indicates the ability of peroxynitrite (ONOO-) to modulate the expression of HO-1 and suggest that the heme oxygenase pathway contributes to protection against the cytotoxic action of ONOO- which is a potent oxidizing agent generated by the interaction of nitric oxide (NO) and the superoxide anion. ONOO- rapidly decomposes to a highly reactive hydroxyl radical and nitrogen dioxide, both of which cause oxidative damage.

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Heme oxygenase-2

Heme oxygenase-2 (HO-2) is the constitutive isoform of heme oxygenase which catalyzes the NADPH, O2 and cytochrome P450 reductase dependent oxidation of heme to carbon monoxide, iron and biliverdin that is immediately reduced to bilirubin. These products of the HO reaction have important physiological effects: carbon monoxide is a potent vasodilator; biliverdin and its product bilirubin are potent antioxidants; .free. iron increases oxidative stress and regulates the expression of many mRNAs (e.g., DCT-1, ferritin and transferrin receptor) by affecting the conformation of iron regulatory protein (IRP)-1 and its binding to iron regulatory elements (IREs) in the 5.- or 3.- UTRs of the mRNAs. To date, three heme oxygenase isoforms HO-1, HO-2 and HO-3 have been identified. HO-1 or HSP32 , a major heat shock/stress response protein is ubiquitous and its mRNA as well as its activity can be increased several-fold by heme, other metalloporphyrins, transition metals and stimuli that induce cellular stress. In contrast to HO-1, HO-2 is highly concentrated in neurons and testes. Multiple HO-2 transcripts which differ in size and use 3 different 5. untranslated regions (referred to as rHO-2, rHO- 2-1 and rHO-2-2) and 2 poly(A) signals have been identified and a functional glucocorticoid response element (GRE) in the promoter region of rHO-2 has been characterized. In the adult rat testis, there is developmentally regulated expression of two transcripts for HO-2 of ~2.1kb and ~1.45kb which are not present in the brain, kidney, thymus, heart, spleen, liver or in prepubertal 14 day old rat testis. Both of these transcripts exclusively use rHO-2 and they contain all of the coding region exons present in the ~1.3kb and ~1.9kb transcripts which are common to all other organs including the adult and prepubertal rat testis. This data suggests that HO-2 levels in the testis are controlled by glucocorticoids and that developmental and tissue-specific

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Tapasin

Tapasin is a 48 kDa glycoprotein which is a member of the immunoglobulin (Ig) superfamily and it is required for efficient peptide binding to Transporter associated with Antigen Processing (TAP). TAP binds peptides in its cytolic part and subsequently translocates the peptides into the lumen of the endoplasmic reticulum (ER) where assembly of MHC class I and peptide takes place. Assembly of MHC class I-ß2-microglobulin (ß2-m) dimers in the ER involves 2 chaperones, calnexin which interacts with free class I heavy (H) chains and calreticulin which binds human class I-ß2 dimers prior to peptide loading. Calreticulin remains associated with at least a subset of class I molecules when they in turn bind to TAP. Polymorphic differences in MHC class I H chains can result in quantitative as well as qualitative differences in how they interact with peptide, ß2-m, calnexin, calreticulin, Erp57, TAP and Tapasin, a subunit of the TAP complex which binds to both TAP1 and MHC class I . Data obtained with Tapasin deletion mutants revealed that binding to TAP is mediated by the C-terminal region and that the N-terminal region is required to stabilize the MHC class I loading complex . The Tapasin gene is centromeric of HLA-DP locus between the HSET and HKE1.5 genes and within 500 kbp of the transporters associated with antigen processing, TAP1 and TAP2 genes. The localization of these genes within such a short distance of each other on the chromosome implies some regulatory or functional significance.

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Ethanol-inducible cytochrome P450 IIE1 (CYP2E1)

Ethanol-inducible cytochrome P450 IIE1 (CYP2E1) is considered one of the major causes of oxidative stress in the liver following ethanol treatment. Radicals produced by this enzyme are thought to induce lipid peroxidation reactions and other damage to the cells and to be a major cause for ethanol-dependent liver toxicity. The importance of CYP2E1 lies in its ability to metabolize a wide range compounds such as organic solvents, acetaminophen, dimethylnitrosaoamine and aliphatic alcohols, which have relevant toxicological effects in humans. To date, over 75 substrates of CYP2E1 have been identified. CYP2E1 is mainly found in the liver although it has also been detected in a variety of other organs, including the brain, colon and lungs. In the liver it is localized to the centrilobular region, specifically the hepatocyte layers most proximal to the central vein . Cytochrome P450 is considered to be part of the mixed function oxidase (MFO) sytem along with NADPHcytochrome P450 reductase.

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Calreticulin

Calreticulin is the major calcium binding protein found in smooth muscle sarcoplasmic reticulum (SR) and non-muscle endoplasmic reticulum (ER) membranes. This protein was originally identified in SR membranes and plays a minor role in calcium storage in skeletal and cardiac muscle SR. Calreticulin is also known as calregulin, CRP55, CaBP3, calsequestrin-like protein and Ro/SS-A antigen. Calreticulin binds calcium with low affinity and high capacity, however it also exhibits a single high affinity binding site. The highly conserved sequence Lys-Asp-Glu-Leu (KDEL) is present at the C-terminus of calreticulin and other resident ER proteins including glucose regulated protein 78 (GRP78), GRP94 and protein disulfide isomerase (PDI). This sequence is responsible for the retention of newly synthesized proteins within the ER lumen. This retention is reported to be mediated by a KDEL receptor. Recent reports indicate that calreticulin can act as a modulator of the regulation of gene transcription by nuclear hormone receptors and may also act as a molecular chaperone.

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