Article

Cloning and functional characterization of mammalian homologues of the COPII component Sec23.

Department of Morphology, University of Geneva Medical Center, Switzerland.
Molecular Biology of the Cell (Impact Factor: 4.55). 11/1996; 7(10):1535-46. DOI: 10.1091/mbc.7.10.1535
Source: PubMed

ABSTRACT 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.

Download full-text

Full-text

Available from: Walter Reith, Jun 23, 2014
0 Followers
 · 
80 Views
  • Source
    [Show abstract] [Hide abstract]
    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 · 5.98 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    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 · 5.33 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Historically, Drosophila has been a model organism for studying molecular and developmental biology leading to many important discoveries in this field. More recently, the fruit fly has started to be used to address cell biology issues including studies of the secretory pathway, and more specifically on the functional integrity of the Golgi apparatus. A number of advances have been made that are reviewed below. Furthermore, with the development of RNAi technology, Drosophila tissue culture cells have been used to perform genome-wide screens addressing similar issues. Last, the Golgi function has been involved in specific developmental processes, thus shedding new light on the functions of a number of Golgi proteins.
    FEBS letters 10/2009; 583(23):3827-38. DOI:10.1016/j.febslet.2009.09.048 · 3.34 Impact Factor