scholarly journals Sec16 influences transitional ER sites by regulating rather than organizing COPII

2013 ◽  
Vol 24 (21) ◽  
pp. 3406-3419 ◽  
Author(s):  
Nike Bharucha ◽  
Yang Liu ◽  
Effrosyni Papanikou ◽  
Conor McMahon ◽  
Masatoshi Esaki ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Coat Protein ◽  
Pichia Pastoris ◽  
Protein Complex ◽  
The Other ◽  
Yeast Pichia Pastoris ◽  
Functional Regions ◽  
Conserved Domain ◽  
Copii Vesicles ◽  
Negative Regulatory Role

During the budding of coat protein complex II (COPII) vesicles from transitional endoplasmic reticulum (tER) sites, Sec16 has been proposed to play two distinct roles: negatively regulating COPII turnover and organizing COPII assembly at tER sites. We tested these ideas using the yeast Pichia pastoris. Redistribution of Sec16 to the cytosol accelerates tER dynamics, supporting a negative regulatory role for Sec16. To evaluate a possible COPII organization role, we dissected the functional regions of Sec16. The central conserved domain, which had been implicated in coordinating COPII assembly, is actually dispensable for normal tER structure. An upstream conserved region (UCR) localizes Sec16 to tER sites. The UCR binds COPII components, and removal of COPII from tER sites also removes Sec16, indicating that COPII recruits Sec16 rather than the other way around. We propose that Sec16 does not in fact organize COPII. Instead, regulation of COPII turnover can account for the influence of Sec16 on tER sites.

scholarly journals Erv26p Directs Pro-Alkaline Phosphatase into Endoplasmic Reticulum–derived Coat Protein Complex II Transport Vesicles

2006 ◽  
Vol 17 (11) ◽  
pp. 4780-4789 ◽  
Author(s):  
Catherine A. Bue ◽  
Christine M. Bentivoglio ◽  
Charles Barlowe
Keyword(s):  
Alkaline Phosphatase ◽  
Endoplasmic Reticulum ◽  
Coat Protein ◽  
Membrane Protein ◽  
Protein Complex ◽  
Integral Membrane Protein ◽  
Secretory Proteins ◽  
Complex Ii ◽  
Transport Vesicles ◽  
Copii Vesicles

Secretory proteins are exported from the endoplasmic reticulum (ER) in transport vesicles formed by the coat protein complex II (COPII). We detected Erv26p as an integral membrane protein that was efficiently packaged into COPII vesicles and cycled between the ER and Golgi compartments. The erv26Δ mutant displayed a selective secretory defect in which the pro-form of vacuolar alkaline phosphatase (pro-ALP) accumulated in the ER, whereas other secretory proteins were transported at wild-type rates. In vitro budding experiments demonstrated that Erv26p was directly required for packaging of pro-ALP into COPII vesicles. Moreover, Erv26p was detected in a specific complex with pro-ALP when immunoprecipitated from detergent-solublized ER membranes. Based on these observations, we propose that Erv26p serves as a transmembrane adaptor to link specific secretory cargo to the COPII coat. Because ALP is a type II integral membrane protein in yeast, these findings imply that an additional class of secretory cargo relies on adaptor proteins for efficient export from the ER.

scholarly journals Small sequence variations between two mammalian paralogs of the small GTPase SAR1 underlie functional differences in coat protein complex II assembly

2020 ◽  
Vol 295 (25) ◽  
pp. 8401-8412 ◽  
Author(s):  
David B. Melville ◽  
Sean Studer ◽  
Randy Schekman
Keyword(s):  
Endoplasmic Reticulum ◽  
Coat Protein ◽  
Binding Site ◽  
Protein Complex ◽  
Small Gtpase ◽  
The Other ◽  
Complex Ii ◽  
Gtp Binding ◽  
Lipoprotein Secretion ◽  
Coat Protein Complex

Vesicles that are coated by coat protein complex II (COPII) are the primary mediators of vesicular traffic from the endoplasmic reticulum to the Golgi apparatus. Secretion-associated Ras-related GTPase 1 (SAR1) is a small GTPase that is part of COPII and, upon GTP binding, recruits the other COPII proteins to the endoplasmic reticulum membrane. Mammals have two SAR1 paralogs that genetic data suggest may have distinct physiological roles, e.g. in lipoprotein secretion in the case of SAR1B. Here we identified two amino acid clusters that have conserved SAR1 paralog–specific sequences. We observed that one cluster is adjacent to the SAR1 GTP-binding pocket and alters the kinetics of GTP exchange. The other cluster is adjacent to the binding site for two COPII components, SEC31 homolog A COPII coat complex component (SEC31) and SEC23. We found that the latter cluster confers to SAR1B a binding preference for SEC23A that is stronger than that of SAR1A for SEC23A. Unlike SAR1B, SAR1A was prone to oligomerize on a membrane surface. SAR1B knockdown caused loss of lipoprotein secretion, overexpression of SAR1B but not of SAR1A could restore secretion, and a divergent cluster adjacent to the SEC31/SEC23-binding site was critical for this SAR1B function. These results highlight that small primary sequence differences between the two mammalian SAR1 paralogs lead to pronounced biochemical differences that significantly affect COPII assembly and identify a specific function for SAR1B in lipoprotein secretion, providing insights into the mechanisms of large cargo secretion that may be relevant for COPII-related diseases.

scholarly journals Sorting of GPI-anchored proteins into ER exit sites by p24 proteins is dependent on remodeled GPI

2011 ◽  
Vol 194 (1) ◽  
pp. 61-75 ◽  
Author(s):  
Morihisa Fujita ◽  
Reika Watanabe ◽  
Nina Jaensch ◽  
Maria Romanova-Michaelides ◽  
Tadashi Satoh ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Coat Protein ◽  
Posttranslational Modification ◽  
Protein Complex ◽  
Er Exit Sites ◽  
Ph Dependent ◽  
Copii Vesicles ◽  
Attachment Proteins ◽  
Acidic Compartments ◽  
P24 Proteins

Glycosylphosphatidylinositol (GPI) anchoring of proteins is a posttranslational modification occurring in the endoplasmic reticulum (ER). After GPI attachment, proteins are transported by coat protein complex II (COPII)-coated vesicles from the ER. Because GPI-anchored proteins (GPI-APs) are localized in the lumen, they cannot interact with cytosolic COPII components directly. Receptors that link GPI-APs to COPII are thought to be involved in efficient packaging of GPI-APs into vesicles; however, mechanisms of GPI-AP sorting are not well understood. Here we describe two remodeling reactions for GPI anchors, mediated by PGAP1 and PGAP5, which were required for sorting of GPI-APs to ER exit sites. The p24 family of proteins recognized the remodeled GPI-APs and sorted them into COPII vesicles. Association of p24 proteins with GPI-APs was pH dependent, which suggests that they bind in the ER and dissociate in post-ER acidic compartments. Our results indicate that p24 complexes act as cargo receptors for correctly remodeled GPI-APs to be sorted into COPII vesicles.

scholarly journals TANGO1 and SEC12 are copackaged with procollagen I to facilitate the generation of large COPII carriers

2018 ◽  
Vol 115 (52) ◽  
pp. E12255-E12264 ◽  
Author(s):  
Lin Yuan ◽  
Samuel J. Kenny ◽  
Juliet Hemmati ◽  
Ke Xu ◽  
Randy Schekman
Keyword(s):  
Endoplasmic Reticulum ◽  
Coat Protein ◽  
Protein Complex ◽  
Coated Vesicles ◽  
Initiation Factor ◽  
Interacting Protein ◽  
Complex Ii ◽  
Transport Vesicles ◽  
Er Exit Sites ◽  
Copii Vesicles

Large coat protein complex II (COPII)-coated vesicles serve to convey the large cargo procollagen I (PC1) from the endoplasmic reticulum (ER). The link between large cargo in the lumen of the ER and modulation of the COPII machinery remains unresolved. TANGO1 is required for PC secretion and interacts with PC and COPII on opposite sides of the ER membrane, but evidence suggests that TANGO1 is retained in the ER, and not included in normal size (<100 nm) COPII vesicles. Here we show that TANGO1 is exported out of the ER in large COPII-coated PC1 carriers, and retrieved back to the ER by the retrograde coat, COPI, mediated by the C-terminal RDEL retrieval sequence of HSP47. TANGO1 is known to target the COPII initiation factor SEC12 to ER exit sites through an interacting protein, cTAGE5. SEC12 is important for the growth of COPII vesicles, but it is not sorted into small budded vesicles. We found both cTAGE5 and SEC12 were exported with TANGO1 in large COPII carriers. In contrast to its exclusion from small transport vesicles, SEC12 was particularly enriched around ER membranes and large COPII carriers that contained PC1. We constructed a split GFP system to recapitulate the targeting of SEC12 to PC1 via the luminal domain of TANGO1. The minimal targeting system enriched SEC12 around PC1 and generated large PC1 carriers. We conclude that TANGO1, cTAGE5, and SEC12 are copacked with PC1 into COPII carriers to increase the size of COPII, thus ensuring the capture of large cargo.

scholarly journals Expression of GA Coat Protein-Derived Mosaic Virus-Like Particles in Saccharomyces cerevisiae and Packaging in vivo of mRNAs into Particles

2012 ◽  
Vol 66 (6) ◽  
pp. 234-241
Author(s):  
Arnis Strods ◽  
Dagnija Argule ◽  
Indulis Cielens ◽  
Ludmila Jackeviča ◽  
Regīna Renhofa
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Coat Protein ◽  
Pichia Pastoris ◽  
Mosaic Virus ◽  
Expression Vector ◽  
Expression Systems ◽  
Coding Sequences ◽  
Yeast Pichia Pastoris ◽  
Virus Like Particles

Our previous research showed that the best yield of virus-like particles (VLPs) formed by RNA bacteriophage GA coat protein was obtained by expression in yeast Pichia pastoris, while other used expression systems in Saccharomyces cerevisiae gave much lower amounts of capsids. The main reasons to attempt further studies in Saccharomyces cerevisiae were to improve the yield of GA-based VLPs using constructs with optimised nucleotide triplets in coding sequences, and to exploit the possibilities of the two-promoter Gal1/Gal10 system of expression vector pESC-URA for production of the desired mosaic VLPs and for packaging of mRNAs into VLPs in vivo

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 Sec24C mediates a Golgi-independent trafficking pathway that is required for tonoplast localization of ABCC1 and ABCC2

2020 ◽  
Author(s):  
Qiao-Yan Lv ◽  
Yi-Qun Gao ◽  
Ya-Ling Wang ◽  
Chu-Ying Zhang ◽  
Zhen-Fei Chao ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Coat Protein ◽  
Golgi Apparatus ◽  
Genetic Screening ◽  
Biological Process ◽  
Loss Of Function ◽  
Independent Manner ◽  
Trafficking Pathway ◽  
Copii Vesicles ◽  
Target Locations

Abstract Protein sorting is an essential biological process in all organisms. Trafficking membrane proteins generally relies on the sorting machinery of the Golgi apparatus. However, many proteins have been found to be delivered to target locations via Golgi-independent pathways, but the mechanisms underlying this delivery system remain unknown. Here, we report that Sec24C, a component of coat protein complex II (COPII) vesicles, mediates the direct secretory trafficking of the phytochelatin transporters ABCC1 and ABCC2 from the endoplasmic reticulum (ER) to prevacuolar compartments (PVCs). After performing a genetic screening, we found that Sec24C loss-of-function mutants are hypersensitive to cadmium (Cd) and arsenic (As) treatments due to mislocalization of ABCC1 and ABCC2, which results in defects in the vacuole compartmentalization of the toxic metals. Further studies showed that Sec24C recognizes ABCC1 and ABCC2 through direct interactions to mediate their exit from the ER to PVCs in a Golgi-independent manner. These findings expand our understanding of Golgi-independent trafficking as well as COPII vesicles.

scholarly journals UNC93B1 mediates differential trafficking of endosomal TLRs

eLife ◽  
2013 ◽  
Vol 2 ◽  
Author(s):  
Bettina L Lee ◽  
Joanne E Moon ◽  
Jeffrey H Shu ◽  
Lin Yuan ◽  
Zachary R Newman ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Autoimmune Diseases ◽  
Protein Complex ◽  
Secretory Pathway ◽  
Transmembrane Protein ◽  
Adaptor Protein ◽  
Regulatory Effects ◽  
Copii Vesicles

UNC93B1, a multipass transmembrane protein required for TLR3, TLR7, TLR9, TLR11, TLR12, and TLR13 function, controls trafficking of TLRs from the endoplasmic reticulum (ER) to endolysosomes. The mechanisms by which UNC93B1 mediates these regulatory effects remain unclear. Here, we demonstrate that UNC93B1 enters the secretory pathway and directly controls the packaging of TLRs into COPII vesicles that bud from the ER. Unlike other COPII loading factors, UNC93B1 remains associated with the TLRs through post-Golgi sorting steps. Unexpectedly, these steps are different among endosomal TLRs. TLR9 requires UNC93B1-mediated recruitment of adaptor protein complex 2 (AP-2) for delivery to endolysosomes while TLR7, TLR11, TLR12, and TLR13 utilize alternative trafficking pathways. Thus, our study describes a mechanism for differential sorting of endosomal TLRs by UNC93B1, which may explain the distinct roles played by these receptors in certain autoimmune diseases.

scholarly journals Phosphatidic acid phospholipase A1 mediates ER–Golgi transit of a family of G protein–coupled receptors

2014 ◽  
Vol 206 (1) ◽  
pp. 79-95 ◽  
Author(s):  
Govind Kunduri ◽  
Changqing Yuan ◽  
Velayoudame Parthibane ◽  
Katherine M. Nyswaner ◽  
Ritu Kanwar ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Coat Protein ◽  
Membrane Proteins ◽  
Phosphatidic Acid ◽  
G Protein ◽  
Golgi Complex ◽  
G Protein Coupled Receptors ◽  
Phospholipase A1 ◽  
Copii Vesicles ◽  
G Protein Coupled

The coat protein II (COPII)–coated vesicular system transports newly synthesized secretory and membrane proteins from the endoplasmic reticulum (ER) to the Golgi complex. Recruitment of cargo into COPII vesicles requires an interaction of COPII proteins either with the cargo molecules directly or with cargo receptors for anterograde trafficking. We show that cytosolic phosphatidic acid phospholipase A1 (PAPLA1) interacts with COPII protein family members and is required for the transport of Rh1 (rhodopsin 1), an N-glycosylated G protein–coupled receptor (GPCR), from the ER to the Golgi complex. In papla1 mutants, in the absence of transport to the Golgi, Rh1 is aberrantly glycosylated and is mislocalized. These defects lead to decreased levels of the protein and decreased sensitivity of the photoreceptors to light. Several GPCRs, including other rhodopsins and Bride of sevenless, are similarly affected. Our findings show that a cytosolic protein is necessary for transit of selective transmembrane receptor cargo by the COPII coat for anterograde trafficking.

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