scholarly journals Unconventional COPII-mediated secretory protein trafficking continues in the absence of the Sar1 GTPase

Author(s):  
William Kasberg ◽  
Peter Luong ◽  
Michael Hanna ◽  
Kayla Minushkin ◽  
Annabelle Tsao ◽  
...  

Abstract Coat protein complex II (COPII) plays an integral role in the packaging of secretory cargoes within membrane-enclosed transport carriers that leave the endoplasmic reticulum (ER) from discrete membrane subdomains. Lipid bilayer remodeling necessary for this process is driven initially by membrane penetration of the coat subunit Sar1 and further stabilized by assembly of a multi-layer complex of several COPII proteins. However, the relative contributions of these distinct factors to transport carrier formation and protein trafficking remain unclear. Here, we demonstrate that anterograde cargo transport from the ER continues in the absence of Sar1, although the unconventional carriers that form fail to efficiently deliver their contents to subsequent compartments in the secretory pathway. Instead, cargoes accumulate immediately adjacent to the perinuclear Golgi under these conditions, together with components of the COPII coat. Our findings highlight new mechanisms by which COPII promotes transport carrier biogenesis and strongly suggests that the Sar1 GTPase plays a critical role in transport carrier uncoating ahead of membrane fusion and secretory cargo delivery at acceptor compartments.

2017 ◽  
Vol 114 (37) ◽  
pp. E7707-E7716 ◽  
Author(s):  
Michael G. Hanna ◽  
Samuel Block ◽  
E. B. Frankel ◽  
Feng Hou ◽  
Adam Johnson ◽  
...  

The conserved coat protein complex II (COPII) mediates the initial steps of secretory protein trafficking by assembling onto subdomains of the endoplasmic reticulum (ER) in two layers to generate cargo-laden transport carriers that ultimately fuse with an adjacent ER–Golgi intermediate compartment (ERGIC). Here, we demonstrate that Trk-fused gene (TFG) binds directly to the inner layer of the COPII coat. Specifically, the TFG C terminus interacts with Sec23 through a shared interface with the outer COPII coat and the cargo receptor Tango1/cTAGE5. Our findings indicate that TFG binding to Sec23 outcompetes these other associations in a concentration-dependent manner and ultimately promotes outer coat dissociation. Additionally, we demonstrate that TFG tethers vesicles harboring the inner COPII coat, which contributes to their clustering between the ER and ERGIC in cells. Together, our studies define a mechanism by which COPII transport carriers are retained locally at the ER/ERGIC interface after outer coat disassembly, which is a prerequisite for fusion with ERGIC membranes.


Genetics ◽  
1996 ◽  
Vol 142 (2) ◽  
pp. 393-406 ◽  
Author(s):  
Linda J Wuestehube ◽  
Rainer Duden ◽  
Arlene Eun ◽  
Susan Hamamoto ◽  
Paul Korn ◽  
...  

Abstract We have isolated new temperature-sensitive mutations in five complementation groups, sec31-sec35, that are defective in the transport of proteins from the endoplasmic reticulum (ER) to the Golgi complex. The sec31-sec35 mutants and additional alleles of previously identified sec and vacuolar protein sorting (vps) genes were isolated in a screen based on the detection of α-factor precursor in yeast colonies replicated to and lysed on nitrocellulose filters. Secretory protein precursors accumulated in sec31-sec35 mutants at the nonpermissive temperature were core-glycosylated but lacked outer chain carbohydrate, indicating that transport was blocked after translocation into the ER but before arrival in the Golgi complex. Electron microscopy revealed that the newly identified sec mutants accumulated vesicles and membrane structures reminiscent of secretory pathway organelles. Complementation analysis revealed that sec32-1 is an allele of BOS1, a gene implicated in vesicle targeting to the Golgi complex, and sec33-1 is an allele of RET1, a gene that encodes the α subunit of coatomer.


2002 ◽  
Vol 13 (3) ◽  
pp. 880-891 ◽  
Author(s):  
Jacqueline Powers ◽  
Charles Barlowe

Erv14p is a conserved integral membrane protein that traffics in COPII-coated vesicles and localizes to the early secretory pathway in yeast. Deletion of ERV14 causes a defect in polarized growth because Axl2p, a transmembrane secretory protein, accumulates in the endoplasmic reticulum and is not delivered to its site of function on the cell surface. Herein, we show that Erv14p is required for selection of Axl2p into COPII vesicles and for efficient formation of these vesicles. Erv14p binds to subunits of the COPII coat and binding depends on conserved residues in a cytoplasmically exposed loop domain of Erv14p. When mutations are introduced into this loop, an Erv14p-Axl2p complex accumulates in the endoplasmic reticulum, suggesting that Erv14p links Axl2p to the COPII coat. Based on these results and further genetic experiments, we propose Erv14p coordinates COPII vesicle formation with incorporation of specific secretory cargo.


2007 ◽  
Vol 27 (10) ◽  
pp. 3716-3731 ◽  
Author(s):  
Zhao V. Wang ◽  
Todd D. Schraw ◽  
Ja-Young Kim ◽  
Tayeba Khan ◽  
Michael W. Rajala ◽  
...  

ABSTRACT Adiponectin is a secretory protein abundantly secreted from adipocytes. It assembles into a number of different higher-order complexes. Adipocytes maintain tight control over circulating plasma levels, suggesting the existence of a complex, highly regulated biosynthetic pathway. However, the critical mediators of adiponectin maturation within the secretory pathway have not been elucidated. Previously, we found that a significant portion of de novo-synthesized adiponectin is not secreted and retained in adipocytes. Here, we show that there is an abundant pool of properly folded adiponectin in the secretory pathway that is retained through thiol-mediated retention, as judged by the release of adiponectin in response to treatment of adipocytes with reducing agents. Adiponectin is covalently bound to the ER chaperone ERp44. An adiponectin mutant lacking cysteine 39 fails to stably interact with ERp44, demonstrating that this residue is the primary site mediating the covalent interaction. Another ER chaperone, Ero1-Lα, plays a critical role in the release of adiponectin from ERp44. Levels of both of these proteins are highly regulated in adipocytes and are influenced by the metabolic state of the cell. While less critical for the secretion of trimers, these chaperones play a major role in the assembly of higher-order adiponectin complexes. Our data highlight the importance of posttranslational events controlling adiponectin levels and the release of adiponectin from adipocytes. One mechanism for increasing circulating levels of specific adiponectin complexes by peroxisome proliferator-activated receptor gamma agonists may be selective upregulation of rate-limiting chaperones.


2004 ◽  
Vol 15 (2) ◽  
pp. 908-921 ◽  
Author(s):  
Gregory Huyer ◽  
Gaby L. Longsworth ◽  
Deborah L. Mason ◽  
Monica P. Mallampalli ◽  
J. Michael McCaffery ◽  
...  

The folding of nascent secretory and membrane proteins is monitored by the endoplasmic reticulum (ER) quality control system. Misfolded proteins are retained in the ER and can be removed by ER-associated degradation. As a model for the ER quality control of multispanning membrane proteins in yeast, we have been studying mutant forms of Ste6p. Here, we identify mislocalized mutant forms of Ste6p that induce the formation of, and localize to, prominent structures that are absent in normal cells. We have named these structures ER-associated compartments (ERACs), based on their juxtaposition to and connection with the ER, as observed by fluorescence and electron microscopy. ERACs comprise a network of tubulo-vesicular structures that seem to represent proliferated ER membranes. Resident ER lumenal and membrane proteins are present in ERACs in addition to their normal ER localization, suggesting there is no barrier for their entry into ERACs. However, the forms of Ste6p in ERACs are excluded from the ER and do not enter the secretory pathway; instead, they are ultimately targeted for ER-associated degradation. The presence of ERACs does not adversely affect secretory protein traffic through the ER and does not lead to induction of the unfolded protein response. We propose that ERACs may be holding sites to which misfolded membrane proteins are specifically diverted so as not to interfere with normal cellular functions. We discuss the likelihood that related ER membrane proliferations that form in response to certain other mutant or unassembled membrane proteins may be substantially similar to ERACs.


2002 ◽  
Vol 13 (8) ◽  
pp. 2639-2650 ◽  
Author(s):  
Christopher M. Cabral ◽  
Yan Liu ◽  
Kelley W. Moremen ◽  
Richard N. Sifers

Protein folding and quality control in the early secretory pathway function as posttranslational checkpoints in eukaryote gene expression. Herein, an aberrant form of the hepatic secretory protein α1-antitrypsin was stably expressed in a human embryonic kidney cell line to elucidate the mechanisms by which glycoprotein endoplasmic reticulum-associated degradation (GERAD) is administered in cells from higher eukaryotes. After biosynthesis, genetic variant PI Z underwent alternative phases of secretion and degradation, the latter of which was mediated by the proteasome. Degradation required release from calnexin- and asparagine-linked oligosaccharide modification by endoplasmic reticulum mannosidase I, the latter of which occurred as PI Z was bound to the molecular chaperone grp78/BiP. That a distinct GERAD program operates in human embryonic kidney cells was supported by the extent of PI Z secretion, apparent lack of polymerization, inability of calnexin to participate in the degradation process, and sequestration of the glycoprotein folding sensor UDP-glucose:glycoprotein glucosyltransferase in the Golgi complex. Because UDP-glucose:glycoprotein glucosyltransferase sustains calnexin binding, its altered distribution is consistent with a GERAD program that hinders the reentry of substrates into the calnexin cycle, allowing grp78/BiP to partner with a lectin, other than calnexin, in the recognition of a two-component GERAD signal to facilitate substrate recruitment. How the processing of a mutant protein, rather than the mutation itself, can contribute to disease pathogenesis, is discussed.


2012 ◽  
Vol 92 (2) ◽  
pp. 537-576 ◽  
Author(s):  
Christopher J. Guerriero ◽  
Jeffrey L. Brodsky

Protein folding is a complex, error-prone process that often results in an irreparable protein by-product. These by-products can be recognized by cellular quality control machineries and targeted for proteasome-dependent degradation. The folding of proteins in the secretory pathway adds another layer to the protein folding “problem,” as the endoplasmic reticulum maintains a unique chemical environment within the cell. In fact, a growing number of diseases are attributed to defects in secretory protein folding, and many of these by-products are targeted for a process known as endoplasmic reticulum-associated degradation (ERAD). Since its discovery, research on the mechanisms underlying the ERAD pathway has provided new insights into how ERAD contributes to human health during both normal and diseases states. Links between ERAD and disease are evidenced from the loss of protein function as a result of degradation, chronic cellular stress when ERAD fails to keep up with misfolded protein production, and the ability of some pathogens to coopt the ERAD pathway. The growing number of ERAD substrates has also illuminated the differences in the machineries used to recognize and degrade a vast array of potential clients for this pathway. Despite all that is known about ERAD, many questions remain, and new paradigms will likely emerge. Clearly, the key to successful disease treatment lies within defining the molecular details of the ERAD pathway and in understanding how this conserved pathway selects and degrades an innumerable cast of substrates.


2021 ◽  
Vol 26 (1) ◽  
Author(s):  
Alicja Koscielny ◽  
Ewa Liszewska ◽  
Katarzyna Machnicka ◽  
Michalina Wezyk ◽  
Katarzyna Kotulska ◽  
...  

Abstract Background Mammalian/mechanistic target of rapamycin (mTOR) complexes are essential for cell proliferation, growth, differentiation, and survival. mTORC1 hyperactivation occurs in the tuberous sclerosis complex (TSC). mTORC1 localizes to the surface of lysosomes, where Rheb activates it. However, mTOR was also found on the endoplasmic reticulum (ER) and Golgi apparatus (GA). Recent studies showed that the same inputs regulate ER-to-GA cargo transport and mTORC1 (e.g., the level of amino acids or energy status of the cell). Nonetheless, it remains unknown whether mTOR contributes to the regulation of cargo passage through the secretory pathway. Methods The retention using selective hooks (RUSH) approach was used to image movement of model cargo (VSVg) between the ER and GA in various cell lines in which mTOR complexes were inhibited. We also investigated VSVg trafficking in TSC patient fibroblasts. Results We found that mTOR inhibition led to the overall enhancement of VSVg transport through the secretory pathway in PC12 cells and primary human fibroblasts. Also, in TSC1-deficient cells, VSVg transport was enhanced. Conclusions Altogether, these data indicate the involvement of mTOR in the regulation of ER-to-GA cargo transport and suggest that impairments in exocytosis may be an additional cellular process that is disturbed in TSC.


Blood ◽  
2006 ◽  
Vol 107 (1) ◽  
pp. 222-231 ◽  
Author(s):  
Jennifer S. Carew ◽  
Steffan T. Nawrocki ◽  
Yelena V. Krupnik ◽  
Kenneth Dunner ◽  
David J. McConkey ◽  
...  

Abstract Previous studies showed that chronic lymphocytic leukemia (CLL) cells exhibit certain mitochondrial abnormalities including mtDNA mutations, increased superoxide generation, and aberrant mitochondrial biogenesis, which are associated with impaired apoptosis and reduced sensitivity to fludarabine. Here we report that CLL cells and multiple myeloma cells are highly sensitive to brefeldin A, an inhibitor of endoplasmic reticulum (ER) to Golgi protein transport currently being developed as a novel anticancer agent in a prodrug formulation. Of importance, brefeldin A effectively induced apoptosis in fludarabine-refractory CLL cells. Disruption of protein trafficking by brefeldin A caused the sequestration of the prosurvival factors APRIL and VEGF in the ER, leading to abnormal ER swelling and a decrease in VEGF secretion. Such ER stress and blockage of secretory protein traffic eventually resulted in Golgi collapse, activation of caspases, and cell death. Notably, the cellular sensitivity to this compound appeared to be independent of p53 status. Taken together, these findings suggest that malignant B cells may be highly dependent on ER-Golgi protein transport and that targeting this process may be a promising therapeutic strategy for B-cell malignancies, especially for those that respond poorly to conventional treatments.


2013 ◽  
Vol 126 (12) ◽  
pp. 2641-2655 ◽  
Author(s):  
E. Gutierrez-Martinez ◽  
I. Fernandez-Ulibarri ◽  
F. Lazaro-Dieguez ◽  
L. Johannes ◽  
S. Pyne ◽  
...  

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