Faculty Opinions recommendation of Inhibiting endoplasmic reticulum (ER)-associated degradation of misfolded Yor1p does not permit ER export despite the presence of a diacidic sorting signal.

2007 ◽  
Author(s):  
Davis Ng
Keyword(s):  
Endoplasmic Reticulum ◽  
Sorting Signal ◽  
Er Export

scholarly journals Limited ER quality control for GPI-anchored proteins

2016 ◽  
Vol 213 (6) ◽  
pp. 693-704 ◽  
Author(s):  
Natalia Sikorska ◽  
Leticia Lemus ◽  
Auxiliadora Aguilera-Romero ◽  
Javier Manzano-Lopez ◽  
Howard Riezman ◽  
...  
Keyword(s):  
Quality Control ◽  
Endoplasmic Reticulum ◽  
Protein Folding ◽  
Misfolded Proteins ◽  
Control Mechanisms ◽  
Gpi Anchor ◽  
Er Quality Control ◽  
Entire Class ◽  
Er Export ◽  
Export Signal

Endoplasmic reticulum (ER) quality control mechanisms target terminally misfolded proteins for ER-associated degradation (ERAD). Misfolded glycophosphatidylinositol-anchored proteins (GPI-APs) are, however, generally poor ERAD substrates and are targeted mainly to the vacuole/lysosome for degradation, leading to predictions that a GPI anchor sterically obstructs ERAD. Here we analyzed the degradation of the misfolded GPI-AP Gas1* in yeast. We could efficiently route Gas1* to Hrd1-dependent ERAD and provide evidence that it contains a GPI anchor, ruling out that a GPI anchor obstructs ERAD. Instead, we show that the normally decreased susceptibility of Gas1* to ERAD is caused by canonical remodeling of its GPI anchor, which occurs in all GPI-APs and provides a protein-independent ER export signal. Thus, GPI anchor remodeling is independent of protein folding and leads to efficient ER export of even misfolded species. Our data imply that ER quality control is limited for the entire class of GPI-APs, many of them being clinically relevant.

scholarly journals Capacity of the Golgi Apparatus for Biogenesis from the Endoplasmic Reticulum

2003 ◽  
Vol 14 (12) ◽  
pp. 5011-5018 ◽  
Author(s):  
Sapna Puri ◽  
Adam D. Linstedt
Keyword(s):  
Endoplasmic Reticulum ◽  
Golgi Apparatus ◽  
Self Assembly ◽  
De Novo ◽  
Brefeldin A ◽  
The Other ◽  
Matrix Proteins ◽  
Er Export ◽  
Golgi Matrix

It is unclear whether the mammalian Golgi apparatus can form de novo from the ER or whether it requires a preassembled Golgi matrix. As a test, we assayed Golgi reassembly after forced redistribution of Golgi matrix proteins into the ER. Two conditions were used. In one, ER redistribution was achieved using a combination of brefeldin A (BFA) to cause Golgi collapse and H89 to block ER export. Unlike brefeldin A alone, which leaves matrix proteins in relatively large remnant structures outside the ER, the addition of H89 to BFA-treated cells caused ER accumulation of all Golgi markers tested. In the other, clofibrate treatment induced ER redistribution of matrix and nonmatrix proteins. Significantly, Golgi reassembly after either treatment was robust, implying that the Golgi has the capacity to form de novo from the ER. Furthermore, matrix proteins reemerged from the ER with faster ER exit rates. This, together with the sensitivity of BFA remnants to ER export blockade, suggests that presence of matrix proteins in BFA remnants is due to cycling via the ER and preferential ER export rather than their stable assembly in a matrix outside the ER. In summary, the Golgi apparatus appears capable of efficient self-assembly.

scholarly journals Nm23H2 Facilitates Coat Protein Complex II Assembly and Endoplasmic Reticulum Export in Mammalian Cells

2005 ◽  
Vol 16 (2) ◽  
pp. 835-848 ◽  
Author(s):  
Lori Kapetanovich ◽  
Cassandra Baughman ◽  
Tina H. Lee
Keyword(s):  
Endoplasmic Reticulum ◽  
Coat Protein ◽  
Protein Complex ◽  
Mammalian Cells ◽  
Complex Ii ◽  
Permeabilized Cells ◽  
Er Export ◽  
Coat Protein Complex

The cytosolic coat protein complex II (COPII) mediates vesicle formation from the endoplasmic reticulum (ER) and is essential for ER-to-Golgi trafficking. The minimal machinery for COPII assembly is well established. However, additional factors may regulate the process in mammalian cells. Here, a morphological COPII assembly assay using purified COPII proteins and digitonin-permeabilized cells has been applied to demonstrate a role for a novel component of the COPII assembly pathway. The factor was purified and identified by mass spectrometry as Nm23H2, one of eight isoforms of nucleoside diphosphate kinase in mammalian cells. Importantly, recombinant Nm23H2, as well as a catalytically inactive version, promoted COPII assembly in vitro, suggesting a noncatalytic role for Nm23H2. Consistent with a function for Nm23H2 in ER export, Nm23H2 localized to a reticular network that also stained for the ER marker calnexin. Finally, an in vivo role for Nm23H2 in COPII assembly was confirmed by isoform-specific knockdown of Nm23H2 by using short interfering RNA. Knockdown of Nm23H2, but not its most closely related isoform Nm23H1, resulted in diminished COPII assembly at steady state and reduced kinetics of ER export. These results strongly suggest a previously unappreciated role for Nm23H2 in mammalian ER export.

scholarly journals Potential Role for Protein Kinases in Regulation of Bidirectional Endoplasmic Reticulum-to-Golgi Transport Revealed by Protein Kinase Inhibitor H89

2000 ◽  
Vol 11 (8) ◽  
pp. 2577-2590 ◽  
Author(s):  
Tina H. Lee ◽  
Adam D. Linstedt
Keyword(s):  
Endoplasmic Reticulum ◽  
Protein Kinase ◽  
Protein Kinase A ◽  
Protein Kinases ◽  
Potential Role ◽  
Kinase Inhibitor ◽  
Transport Processes ◽  
Protein Kinase Inhibitors ◽  
Er Export ◽  
Kinase A

Recent evidence suggests a regulatory connection between cell volume, endoplasmic reticulum (ER) export, and stimulated Golgi-to-ER transport. To investigate the potential role of protein kinases we tested a panel of protein kinase inhibitors for their effect on these steps. One inhibitor, H89, an isoquinolinesulfonamide that is commonly used as a selective protein kinase A inhibitor, blocked both ER export and hypo-osmotic-, brefeldin A-, or nocodazole-induced Golgi-to-ER transport. In contrast, H89 did not block the constitutive ER Golgi-intermediate compartment (ERGIC)-to-ER and Golgi-to-ER traffic that underlies redistribution of ERGIC and Golgi proteins into the ER after ER export arrest. Surprisingly, other protein kinase A inhibitors, KT5720 and H8, as well as a set of protein kinase C inhibitors, had no effect on these transport processes. To test whether H89 might act at the level of either the coatomer protein (COP)I or the COPII coat protein complex we examined the localization of βCOP and Sec13 in H89-treated cells. H89 treatment led to a rapid loss of Sec13-labeled ER export sites but βCOP localization to the Golgi was unaffected. To further investigate the effect of H89 on COPII we developed a COPII recruitment assay with permeabilized cells and found that H89 potently inhibited binding of exogenous Sec13 to ER export sites. This block occurred in the presence of guanosine-5′-O-(3-thio)triphosphate, suggesting that Sec13 recruitment is inhibited at a step independent of the activation of the GTPase Sar1. These results identify a requirement for an H89-sensitive factor(s), potentially a novel protein kinase, in recruitment of COPII to ER export sites, as well as in stimulated but not constitutive Golgi-to-ER transport.

scholarly journals Collagen has a unique SEC24 preference for efficient export from the endoplasmic reticulum

2021 ◽  
Author(s):  
Chung-Ling Lu ◽  
Jacob Cain ◽  
Jon Brudvig ◽  
Steven Ortmeier ◽  
Simeon A. Boyadjiev ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Osteogenesis Imperfecta ◽  
Tissue Specificity ◽  
Bone Development ◽  
Coat Proteins ◽  
Tissue Specific ◽  
Secretion Process ◽  
Collagen Secretion ◽  
Copii Vesicle ◽  
Er Export

ABSTRACTProcollagen requires COPII coat proteins for export from the endoplasmic reticulum (ER). SEC24 is the major component of the COPII proteins that selects cargo during COPII vesicle assembly. There are four paralogs (A to D) of SEC24 in mammals, which are classified into two subgroups. Pathological mutations in SEC24D cause osteogenesis imperfecta with craniofacial dysplasia in humans and sec24d mutant fish also recapitulate this phenotypes. Consistent with the skeletal phenotypes, the secretion of collagen was severely defective in mutant fish, emphasizing the importance of SEC24D in collagen secretion. However, SEC24D patient-derived fibroblasts show only a mild secretion phenotype, suggesting tissue-specificity in the secretion process. To explore this possibility, we generated Sec24d knockout (KO) mice. Homozygous KO mice died prior to bone development. When we analyzed embryonic and extraembryonic tissues of mutant animals, we observed tissue-dependent defects of procollagen processing and ER export. The spacial patterns of these defects mirrored with SEC24B deficiency. By systematically knocking down the expression of Sec24 paralogs, we determined that, in addition to SEC24C and SEC24D, SEC24A and SEC24B also contribute to collagen secretion. In contrast, fibronectin 1 preferred either SEC24C or SEC24D. On the basis of our results, we propose that procollagen interacts with multiple SEC24 paralogs for efficient export from the ER, and that this is the basis for tissue-specific phenotypes resulting from SEC24 paralog deficiency.

scholarly journals Endoplasmic Reticulum Export of Glycosyltransferases Depends on Interaction of a Cytoplasmic Dibasic Motif with Sar1

2003 ◽  
Vol 14 (9) ◽  
pp. 3753-3766 ◽  
Author(s):  
Claudio G. Giraudo ◽  
Hugo J.F. Maccioni
Keyword(s):  
Endoplasmic Reticulum ◽  
Membrane Proteins ◽  
Type I ◽  
Type Ii ◽  
Transport Vesicles ◽  
Microsomal Membranes ◽  
Copii Vesicles ◽  
Er Export ◽  
Selective Process ◽  
Selection Of

Membrane proteins exit the endoplasmic reticulum (ER) in COPII-transport vesicles. ER export is a selective process in which transport signals present in the cytoplasmic tail (CT) of cargo membrane proteins must be recognized by coatomer proteins for incorporation in COPII vesicles. Two classes of ER export signals have been described for type I membrane proteins, the diacidic and the dihydrophobic motifs. Both motifs participate in the Sar1-dependent binding of Sec23p–Sec24p complex to the CTs during early steps of cargo selection. However, information concerning the amino acids in the CTs that interact with Sar1 is lacking. Herein, we describe a third class of ER export motif, [RK](X)[RK], at the CT of Golgi resident glycosyltransferases that is required for these type II membrane proteins to exit the ER. The dibasic motif is located proximal to the transmembrane border, and experiments of cross-linking in microsomal membranes and of binding to immobilized peptides showed that it directly interacts with the COPII component Sar1. Sar1GTP-bound to immobilized peptides binds Sec23p. Collectively, the present data suggest that interaction of the dibasic motif with Sar1 participates in early steps of selection of Golgi resident glycosyltransferases for transport in COPII vesicles.

scholarly journals Cholera Toxin Is Exported from Microsomes by the Sec61p Complex

2000 ◽  
Vol 148 (6) ◽  
pp. 1203-1212 ◽  
Author(s):  
Anton Schmitz ◽  
Helga Herrgen ◽  
Alexandra Winkeler ◽  
Volker Herzog
Keyword(s):  
Endoplasmic Reticulum ◽  
Cholera Toxin ◽  
De Novo ◽  
Bacterial Toxins ◽  
Protein Export ◽  
In Vitro Translation ◽  
Unknown Mechanism ◽  
A Cell ◽  
Er Export

After endocytosis cholera toxin is transported to the endoplasmic reticulum (ER), from where its A1 subunit (CTA1) is assumed to be transferred to the cytosol by an as-yet unknown mechanism. Here, export of CTA1 from the ER to the cytosol was investigated in a cell-free assay using either microsomes loaded with CTA1 by in vitro translation or reconstituted microsomes containing CTA1 purified from V. cholerae. Export of CTA1 from the microsomes was time- and adenosine triphosphate–dependent and required lumenal ER proteins. By coimmunoprecipitation CTA1 was shown to be associated during export with the Sec61p complex, which mediates import of proteins into the ER. Export of CTA1 was inhibited when the Sec61p complexes were blocked by nascent polypeptides arrested during import, demonstrating that the export of CTA1 depended on translocation-competent Sec61p complexes. Export of CTA1 from the reconstituted microsomes indicated the de novo insertion of the toxin into the Sec61p complex from the lumenal side. Our results suggest that Sec61p complex–mediated protein export from the ER is not restricted to ER-associated protein degradation but is also used by bacterial toxins, enabling their entry into the cytosol of the target cell.

scholarly journals The Sar1 Gtpase Coordinates Biosynthetic Cargo Selection with Endoplasmic Reticulum Export Site Assembly

2001 ◽  
Vol 152 (1) ◽  
pp. 213-230 ◽  
Author(s):  
Meir Aridor ◽  
Kenneth N. Fish ◽  
Sergei Bannykh ◽  
Jacques Weissman ◽  
Theresa H. Roberts ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Small Gtpase ◽  
Live Cells ◽  
Tubular Structures ◽  
Golgi Transport ◽  
Er Export

Cargo selection and export from the endoplasmic reticulum is mediated by the COPII coat machinery that includes the small GTPase Sar1 and the Sec23/24 and Sec13/31 complexes. We have analyzed the sequential events regulated by purified Sar1 and COPII coat complexes during synchronized export of cargo from the ER in vitro. We find that activation of Sar1 alone, in the absence of other cytosolic components, leads to the formation of ER-derived tubular domains that resemble ER transitional elements that initiate cargo selection. These Sar1-generated tubular domains were shown to be transient, functional intermediates in ER to Golgi transport in vitro. By following cargo export in live cells, we show that ER export in vivo is also characterized by the formation of dynamic tubular structures. Our results demonstrate an unanticipated and novel role for Sar1 in linking cargo selection with ER morphogenesis through the generation of transitional tubular ER export sites.

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