Paccaud, J.P. et al. Cloning and functional characterization of mammalian homologues of the COPII component Sec23. Mol. Biol. Cell 7, 1535-1546

Department of Morphology, University of Geneva Medical Center, Switzerland.
Molecular Biology of the Cell (Impact Factor: 4.47). 11/1996; 7(10):1535-46. DOI: 10.1091/mbc.7.10.1535
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We screened a human cDNA library with a probe derived from a partial SEC23 mouse homologue and isolated two different cDNA clones (hSec23A and hSec23B) encoding proteins of a predicted molecular mass of 85 kDa. hSec23Ap and hSec23Bp were 85% identical and shared 48% identity with the yeast Sec23p. Affinity-purified anti-hSec23A recognized a protein of approximately 85 kDa on immunoblots of human, mouse, and rat cell extracts but did not recognize yeast Sec23p. Cytosolic hSec23Ap migrated with an apparent molecular weight of 350 kDa on a gel filtration column, suggesting that it is part of a protein complex. By immunoelectron microscopy, hSec23Ap was found essentially in the ribosome-free transitional face of the endoplasmic reticulum (ER) and associated vesicles. hSec23Ap is a functional homologue of the yeast Sec23p as the hSec23A isoform complemented the temperature sensitivity of the Saccharomyces cerevisiae sec23-1 mutation at a restrictive temperature of 34 degrees C. RNase protection assays indicated that both hSec23 isoforms are coexpressed in various human tissues, although at a variable ratio. Our data demonstrate that hSec23Ap is the functional human counterpart of the yeast COPII component Sec23p and suggest that it plays a similar role in mammalian protein export from the ER. The exact function of hSec23Bp remains to be determined.

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Available from: Walter Reith, Jun 23, 2014
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    • "Therefore, to determine whether a direct 565–567) to DAA disrupted Sec23/24 binding (Wang et al., 2004). Because the human homologue of the yeast COPII component, Sec23A, complements the temperature-sensitive growth of the yeast sec23-1 mutant at the restrictive temperature (37ºC; Paccaud et al., 1996), it is possible that the yeast COPII coat can bind to the diacidic sequence of EGFP-CFTR expressed in yeast cells and may have some role in coordinating the sequestration process. Therefore we examined the ability of the diacidic EGFP-CFTR-D567A mutant to be sequestered into ERACs, but this did not significantly affect its entry into the ERAC (Figure 5). "
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    ABSTRACT: Proteins that fail to fold in the endoplasmic reticulum (ER) are subjected to ER-associated degradation (ERAD). Certain transmembrane ERAD substrates are segregated into specialized ER subdomains, termed ER-associated compartments (ERACs), before targeting to the ubiquitin-proteasome degradation. The traffic-independent function of several proteins involved in COPII-mediated ER-to-Golgi transport have been implicated in the segregation of exogenously expressed human cystic fibrosis transmembrane conductance regulator (CFTR) into ERACs in Saccharomyces cerevisiae. Here, we focused on the properties of COPII components in the sequestration of EGFP-CFTR into ERACs. It has been demonstrated that the temperature-sensitive growth defects in many COPII mutants can be suppressed by overexpressing other genes involved in COPII vesicle formation. However, we show that these suppression abilities are not always correlated with the ability to rescue the ERAC formation defect, suggesting that COPII-mediated EGFP-CFTR entry into ERACs is independent of its ER-to-Golgi trafficking function. In addition to COPII machinery, we identified that the ER-associated Hsp40s are also involved in the sequestration process by directly interacting with EGFP-CFTR. We show that COPII components and ER-associated Hsp40, Hlj1p, act in the same pathway to sequester EGFP-CFTR into ERACs. Our findings point to an as-yet-undefined role of COPII proteins in the formation of ERACs.
    Molecular biology of the cell 01/2013; 24(5). DOI:10.1091/mbc.E12-08-0639 · 4.47 Impact Factor
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    • "ER-to-Golgi transport is mediated by coat protein complex II (COPII) vesicles. Components responsible for COPII vesicle formation are well conserved between yeast and mammals (Kuge et al., 1994; Swaroop et al., 1994; Paccaud et al., 1996; Tang et al., 1999; Tang et al., 2000; Weissman et al., 2001; Bhattacharyya and Glick, 2007) and include COPII coat protein subunits Sec23, Sec24, Sec13, and Sec31, a small GTPase Sar1, and its specific guanine nucleotide exchange factor (GEF) Sec12 (Nakano et al., 1988; Nakano and Muramatsu, 1989; Barlowe and Schekman, 1993; Barlowe et al., 1994). When activated by Sec12, Sar1–GTP initiates COPII vesicle formation on the ER by sequentially recruiting Sec23/24 heterodimers and Sec13/31 heterodimers (Lee et al., 2004). "
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    ABSTRACT: Protein export from the endoplasmic reticulum (ER) to the Golgi apparatus occurs at specialized regions known as the ER exit sites (ERES). In Saccharomyces cerevisiae, ERES appear as numerous scattered puncta throughout the ER. We examined ERES within the peripheral ER, finding that the proteins comprising the ERES localize on high-curvature ER domains where curvature-stabilizing protein Rtn1 is present. Δrtn1 Δrtn2 Δyop1 cells have fewer high-curvature ER domains, but ERES accumulate at the remaining high-curvature ER domains on the edge of expanded ER sheets. We propose that membrane curvature is a key geometric feature for the regulation of ERES localization. We also investigated a spatial relationship between ERES and Golgi cisternae. Golgi cisternae in S. cerevisiae are unstacked, dispersed, and moving in the cytoplasm with cis-cisternae positioned adjacent to ERES, whereas trans-cisternae are not. Morphological changes in the ER of Δrtn1 Δrtn2 Δyop1 cells resulted in aberrant Golgi structures, including cis- and trans-markers, and there was reduced movement at ERES between expanded ER sheets and the plasma membrane.
    Journal of Cell Science 03/2012; 125(Pt 14):3412-20. DOI:10.1242/jcs.100065 · 5.43 Impact Factor
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    • "The SEC23B gene appears to play a pivotal and probably unique function in erythroid precursors [19,20]. Although detailed genetic analyses have been conducted, the effects of the mutations on mRNA content in erythroblast cells have not been documented. "
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    ABSTRACT: Congenital dyserythropoietic anemia type II (CDAII), the most common form of CDA, is an autosomal recessive condition. CDAII diagnosis is based on invasive, expensive, and time consuming tests that are available only in specialized laboratories. The recent identification of SEC23B mutations as the cause of CDAII opens new possibilities for the molecular diagnosis of the disease. The aim of this study was to characterize molecular genomic SEC23B defects in 16 unrelated patients affected by CDAII and correlate the identified genetic alterations with SEC23B transcript and protein levels in erythroid precursors. SEC23B was sequenced in 16 patients, their relatives and 100 control participants. SEC23B transcript level were studied by quantitative PCR (qPCR) in peripheral erythroid precursors and lymphocytes from the patients and healthy control participants. Sec23B protein content was analyzed by immunoblotting in samples of erythroblast cells from CDAII patients and healthy controls. All of the investigated cases carried SEC23B mutations on both alleles, with the exception of two patients in which a single heterozygous mutation was found. We identified 15 different SEC23B mutations, of which four represent novel mutations: p.Gln214Stop, p.Thr485Ala, p.Val637Gly, and p.Ser727Phe. The CDAII patients exhibited a 40-60% decrease of SEC23B mRNA levels in erythroid precursors when compared with the corresponding cell type from healthy participants. The largest decrease was observed in compound heterozygote patients with missense/nonsense mutations. In three patients, Sec23B protein levels were evaluated in erythroid precursors and found to be strictly correlated with the reduction observed at the transcript level. We also demonstrate that Sec23B mRNA expression levels in lymphocytes and erythroblasts are similar. In this study, we identified four novel SEC23B mutations associated with CDAII disease. We also demonstrate that the genetic alteration results in a significant decrease of SEC23B transcript in erythroid precursors. Similar down-regulation was observed in peripheral lymphocytes, suggesting that the use of these cells might be sufficient in the identification of Sec23B gene alterations. Finally, we demonstrate that decreased Sec23B protein levels in erythroid precursors correlate with down-regulation of the SEC23B mRNA transcript.
    Orphanet Journal of Rare Diseases 12/2011; 6(1):89. DOI:10.1186/1750-1172-6-89 · 3.36 Impact Factor
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