scholarly journals Reconstitution of COPII vesicle fusion to generate a pre-Golgi intermediate compartment

2004 ◽  
Vol 167 (6) ◽  
pp. 997-1003 ◽  
Author(s):  
Dalu Xu ◽  
Jesse C. Hay
Keyword(s):  
Endoplasmic Reticulum ◽  
Membrane Fusion ◽  
Secretory Pathway ◽  
Golgi Stack ◽  
Golgi Membranes ◽  
Transport Vesicles ◽  
Copii Vesicle ◽  
Intermediate Compartment ◽  
Copii Vesicles

What is the first membrane fusion step in the secretory pathway? In mammals, transport vesicles coated with coat complex (COP) II deliver secretory cargo to vesicular tubular clusters (VTCs) that ferry cargo from endoplasmic reticulum exit sites to the Golgi stack. However, the precise origin of VTCs and the membrane fusion step(s) involved have remained experimentally intractable. Here, we document in vitro direct tethering and SNARE-dependent fusion of endoplasmic reticulum–derived COPII transport vesicles to form larger cargo containers. The assembly did not require detectable Golgi membranes, preexisting VTCs, or COPI function. Therefore, COPII vesicles appear to contain all of the machinery to initiate VTC biogenesis via homotypic fusion. However, COPI function enhanced VTC assembly, and early VTCs acquired specific Golgi components by heterotypic fusion with Golgi-derived COPI vesicles.

scholarly journals Erv14p Directs a Transmembrane Secretory Protein into COPII-coated Transport Vesicles

2002 ◽  
Vol 13 (3) ◽  
pp. 880-891 ◽  
Author(s):  
Jacqueline Powers ◽  
Charles Barlowe
Keyword(s):  
Endoplasmic Reticulum ◽  
Secretory Pathway ◽  
Integral Membrane Protein ◽  
Secretory Protein ◽  
Conserved Residues ◽  
Early Secretory Pathway ◽  
Transport Vesicles ◽  
Copii Vesicle ◽  
Copii Vesicles ◽  
Selection Of

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.

scholarly journals MIA3/TANGO1 enables efficient secretion by constraining COPII vesicle budding

2021 ◽  
Author(s):  
Janine McCaughey ◽  
Judith M. Mantell ◽  
Chris R. Neal ◽  
Kate Heesom ◽  
David J. Stephens
Keyword(s):  
Endoplasmic Reticulum ◽  
Light Microscopy ◽  
Secretory Pathway ◽  
Genome Engineering ◽  
High Volume ◽  
Vesicle Budding ◽  
Early Secretory Pathway ◽  
Copii Vesicle ◽  
Intermediate Compartment ◽  
Golgi Organization

AbstractComplex machinery is required to drive secretory cargo export from the endoplasmic reticulum. In vertebrates, this includes transport and Golgi organization protein 1 (TANGO1), encoded by the Mia3 gene. Here, using genome engineering of human cells light microscopy, secretion assays, and proteomics, we show loss of Mia3/TANGO1 results in formation of numerous vesicles and a loss of early secretory pathway integrity. This restricts secretion not only of large proteins like procollagens but of all types of secretory cargo. Our data shows that Mia3/TANGO1 constrains the propensity of COPII to form vesicles promoting instead the formation of the ER-Golgi intermediate compartment. Thus, Mia3/TANGO1 facilities the secretion of complex and high volume cargoes from vertebrate cells.

scholarly journals ER/Golgi Intermediates Acquire Golgi Enzymes by Brefeldin a–Sensitive Retrograde Transport in Vitro

1999 ◽  
Vol 147 (7) ◽  
pp. 1457-1472 ◽  
Author(s):  
Chung-Chih Lin ◽  
Harold D. Love ◽  
Jennifer N. Gushue ◽  
John J.M. Bergeron ◽  
Joachim Ostermann
Keyword(s):  
Secretory Pathway ◽  
Mutant Cell ◽  
Brefeldin A ◽  
Retrograde Transport ◽  
Secretory Proteins ◽  
Intermediate Step ◽  
Golgi Stack ◽  
Transport Vesicles ◽  
Direct Fusion

Secretory proteins exit the ER in transport vesicles that fuse to form vesicular tubular clusters (VTCs) which move along microtubule tracks to the Golgi apparatus. Using the well-characterized in vitro approach to study the properties of Golgi membranes, we determined whether the Golgi enzyme NAGT I is transported to ER/Golgi intermediates. Secretory cargo was arrested at distinct steps of the secretory pathway of a glycosylation mutant cell line, and in vitro complementation of the glycosylation defect was determined. Complementation yield increased after ER exit of secretory cargo and was optimal when transport was blocked at an ER/Golgi intermediate step. The rapid drop of the complementation yield as secretory cargo progresses into the stack suggests that Golgi enzymes are preferentially targeted to ER/Golgi intermediates and not to membranes of the Golgi stack. Two mechanisms for in vitro complementation could be distinguished due to their different sensitivities to brefeldin A (BFA). Transport occurred either by direct fusion of preexisting transport intermediates with ER/Golgi intermediates, or it occurred as a BFA-sensitive and most likely COP I–mediated step. Direct fusion of ER/Golgi intermediates with cisternal membranes of the Golgi stack was not observed under these conditions.

scholarly journals KDEL and KKXX Retrieval Signals Appended to the Same Reporter Protein Determine Different Trafficking between Endoplasmic Reticulum, Intermediate Compartment, and Golgi Complex

2003 ◽  
Vol 14 (3) ◽  
pp. 889-902 ◽  
Author(s):  
Mariano Stornaiuolo ◽  
Lavinia V. Lotti ◽  
Nica Borgese ◽  
Maria-Rosaria Torrisi ◽  
Giovanna Mottola ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Steady State ◽  
Golgi Complex ◽  
Secretory Pathway ◽  
Molecular Mechanisms ◽  
Reporter Protein ◽  
Transport Vesicles ◽  
Efficient Retrieval ◽  
Intermediate Compartment ◽  
Almost All

Many endoplasmic reticulum (ER) proteins maintain their residence by dynamic retrieval from downstream compartments of the secretory pathway. In previous work we compared the retrieval process mediated by the two signals, KKMP and KDEL, by appending them to the same neutral reporter protein, CD8, and found that the two signals determine a different steady-state localization of the reporter. CD8-K (the KDEL-bearing form) was restricted mainly to the ER, whereas CD8-E19 (the KKMP-bearing form) was distributed also to the intermediate compartment and Golgi complex. To investigate whether this different steady-state distribution reflects a difference in exit rates from the ER and/or in retrieval, we have now followed the first steps of export of the two constructs from the ER and their trafficking between ER and Golgi complex. Contrary to expectation, we find that CD8-K is efficiently recruited into transport vesicles, whereas CD8-E19 is not. Thus, the more restricted ER localization of CD8-K must be explained by a more efficient retrieval to the ER. Moreover, because most of ER resident CD8-K is not O-glycosylated but almost all CD8-E19 is, the results suggest that CD8-K is retrieved from the intermediate compartment, before reaching the Golgi, whereO-glycosylation begins. These results illustrate how different retrieval signals determine different trafficking patterns and pose novel questions on the underlying molecular mechanisms.

scholarly journals Poliovirus Infection Transiently Increases COPII Vesicle Budding

2012 ◽  
Vol 86 (18) ◽  
pp. 9675-9682 ◽  
Author(s):  
Meg Trahey ◽  
Hyung Suk Oh ◽  
Craig E. Cameron ◽  
Jesse C. Hay
Keyword(s):  
Golgi Apparatus ◽  
Secretory Pathway ◽  
Vesicle Formation ◽  
Vesicle Budding ◽  
Precursor Pool ◽  
Copii Vesicle ◽  
Early Increase ◽  
Vesicle Biogenesis ◽  
Intermediate Compartment ◽  
Copii Vesicles

Poliovirus (PV) requires membranes of the host cell's secretory pathway to generate replication complexes (RCs) for viral RNA synthesis. Recent work identified the intermediate compartment and the Golgi apparatus as the precursors of the replication “organelles” of PV (N. Y. Hsu et al., Cell 141:799–811, 2010). In this study, we examined the effect of PV on COPII vesicles, the secretory cargo carriers that bud from the endoplasmic reticulum and homotypically fuse to form the intermediate compartment that matures into the Golgi apparatus. We found that infection by PV results in a biphasic change in functional COPII vesicle biogenesis in cells, with an early enhancement and subsequent inhibition. Concomitant with the early increase in COPII vesicle formation, we found an increase in the membrane fraction of Sec16A, a key regulator of COPII vesicle formation. We suggest that the early burst in COPII vesicle formation detected benefits PV by increasing the precursor pool required for the formation of its RCs.

scholarly journals Distinct stages in the recognition, sorting, and packaging of proTGFα into COPII-coated transport vesicles

2016 ◽  
Vol 27 (12) ◽  
pp. 1938-1947 ◽  
Author(s):  
Pengcheng Zhang ◽  
Randy Schekman
Keyword(s):  
Secretory Pathway ◽  
Transforming Growth Factor ◽  
Transmembrane Protein ◽  
Transforming Growth Factor Α ◽  
Transport Vesicles ◽  
Cargo Protein ◽  
Copii Vesicles ◽  
Factor Α ◽  
Cytosolic Factor

In addition to its role in forming vesicles from the endoplasmic reticulum (ER), the coat protein complex II (COPII) is also responsible for selecting specific cargo proteins to be packaged into COPII transport vesicles. Comparison of COPII vesicle formation in mammalian systems and in yeast suggested that the former uses more elaborate mechanisms for cargo recognition, presumably to cope with a significantly expanded repertoire of cargo that transits the secretory pathway. Using proTGFα, the transmembrane precursor of transforming growth factor α (TGFα), as a model cargo protein, we demonstrate in cell-free assays that at least one auxiliary cytosolic factor is specifically required for the efficient packaging of proTGFα into COPII vesicles. Using a knockout HeLa cell line generated by CRISPR/Cas9, we provide functional evidence showing that a transmembrane protein, Cornichon-1 (CNIH), acts as a cargo receptor of proTGFα. We show that both CNIH and the auxiliary cytosolic factor(s) are required for efficient recruitment of proTGFα to the COPII coat in vitro. Moreover, we provide evidence that the recruitment of cargo protein by the COPII coat precedes and may be distinct from subsequent cargo packaging into COPII vesicles.

scholarly journals Overexpression of Sly41 suppresses COPII vesicle–tethering deficiencies by elevating intracellular calcium levels

2016 ◽  
Vol 27 (10) ◽  
pp. 1635-1649 ◽  
Author(s):  
Indrani Mukherjee ◽  
Charles Barlowe
Keyword(s):  
Secretory Pathway ◽  
Cytosolic Calcium ◽  
Live Cells ◽  
Rab Gtpase ◽  
Multicopy Suppressor ◽  
Vesicle Tethering ◽  
Copii Vesicle ◽  
Copii Vesicles ◽  
Polytopic Membrane Protein

SLY41 was identified as a multicopy suppressor of loss of Ypt1, a Rab GTPase essential for COPII vesicle tethering at the Golgi complex. SLY41 encodes a polytopic membrane protein with homology to a class of solute transporter proteins, but how overexpression suppresses vesicle-tethering deficiencies is not known. Here we show that Sly41 is efficiently packaged into COPII vesicles and actively cycles between the ER and Golgi compartments. SLY41 displays synthetic negative genetic interactions with PMR1, which encodes the major Golgi-localized Ca2+/Mn2+transporter and suggests that Sly41 influences cellular Ca2+and Mn2+homeostasis. Experiments using the calcium probe aequorin to measure intracellular Ca2+concentrations in live cells reveal that Sly41 overexpression significantly increases cytosolic calcium levels. Although specific substrates of the Sly41 transporter were not identified, our findings indicate that localized overexpression of Sly41 to the early secretory pathway elevates cytosolic calcium levels to suppress vesicle-tethering mutants. In vitro SNARE cross-linking assays were used to directly monitor the influence of Ca2+on tethering and fusion of COPII vesicles with Golgi membranes. Strikingly, calcium at suppressive concentrations stimulated SNARE-dependent membrane fusion when vesicle-tethering activity was reduced. These results show that calcium positively regulates the SNARE-dependent fusion stage of ER–Golgi transport.

scholarly journals Distinct stages in the recognition, sorting and packaging of proTGFα into COPII coated transport vesicles

2016 ◽  
Author(s):  
Pengcheng Zhang ◽  
Randy Schekman
Keyword(s):  
Secretory Pathway ◽  
Transforming Growth Factor ◽  
Transmembrane Protein ◽  
Transport Vesicles ◽  
Cargo Protein ◽  
Cargo Proteins ◽  
Factor Alpha ◽  
Copii Vesicles ◽  
Cytosolic Factor

AbstractIn addition to its role in forming vesicles from the endoplasmic reticulum (ER), the coat protein complex II (COPII) is also responsible for selecting specific cargo proteins to be packaged into COPII transport vesicles. Comparison of COPII vesicle formation in mammalian systems and in yeast suggested that the former employs more elaborate mechanisms for cargo recognition, presumably to cope with a significantly expanded repertoire of cargo that transits the secretory pathway. Using proTGFα, the transmembrane precursor of transforming growth factor alpha (TGFα), as a model cargo protein, we demonstrate in cell-free assays that at least one auxiliary cytosolic factor is specifically required for the efficient packaging of proTGFα into COPII vesicles. Using a knockout HeLa cell line generated by CRISPR/Cas9, we provide functional evidence showing that a transmembrane protein, Cornichon-1 (CNIH), acts as a cargo receptor of proTGFα. We show that both CNIH and the auxiliary cytosolic factor(s) are required for efficient recruitment of proTGFα to the COPII coat in vitro. Moreover, we provide evidence that the recruitment of cargo protein by the COPII coat precedes and may be distinct from subsequent cargo packaging into COPII vesicles.AbbreviationsCNIHCornichon

scholarly journals mBet3p is required for homotypic COPII vesicle tethering in mammalian cells

2006 ◽  
Vol 174 (3) ◽  
pp. 359-368 ◽  
Author(s):  
Sidney Yu ◽  
Ayano Satoh ◽  
Marc Pypaert ◽  
Karl Mullen ◽  
Jesse C. Hay ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Endoplasmic Reticulum ◽  
Mammalian Cells ◽  
Yeast Saccharomyces Cerevisiae ◽  
Vesicle Tethering ◽  
Transitional Endoplasmic Reticulum ◽  
Large Complex ◽  
Copii Vesicle ◽  
Copii Vesicles

TRAPPI is a large complex that mediates the tethering of COPII vesicles to the Golgi (heterotypic tethering) in the yeast Saccharomyces cerevisiae. In mammalian cells, COPII vesicles derived from the transitional endoplasmic reticulum (tER) do not tether directly to the Golgi, instead, they appear to tether to each other (homotypic tethering) to form vesicular tubular clusters (VTCs). We show that mammalian Bet3p (mBet3p), which is the most highly conserved TRAPP subunit, resides on the tER and adjacent VTCs. The inactivation of mBet3p results in the accumulation of cargo in membranes that colocalize with the COPII coat. Furthermore, using an assay that reconstitutes VTC biogenesis in vitro, we demonstrate that mBet3p is required for the tethering and fusion of COPII vesicles to each other. Consistent with the proposal that mBet3p is required for VTC biogenesis, we find that ERGIC-53 (VTC marker) and Golgi architecture are disrupted in siRNA-treated mBet3p-depleted cells. These findings imply that the TRAPPI complex is essential for VTC biogenesis.

scholarly journals Proteomic Profiling of Mammalian COPII Vesicles

2018 ◽  
Author(s):  
Frank Adolf ◽  
Manuel Rhiel ◽  
Bernd Hessling ◽  
Andrea Hellwig ◽  
Felix T. Wieland
Keyword(s):  
Intracellular Transport ◽  
Spectrometric Analysis ◽  
Secretory Proteins ◽  
Proteomic Profiling ◽  
Vesicular Carriers ◽  
Copii Vesicle ◽  
Cargo Proteins ◽  
Core Proteome ◽  
Copii Vesicles

AbstractIntracellular transport and homeostasis of the endomembrane system in eukaryotic cells depend on formation and fusion of vesicular carriers. COPII vesicles export newly synthesized secretory proteins from the endoplasmic reticulum (ER). They are formed by sequential recruitment of the small GTP binding protein Sar1, the inner coat complex Sec23/24, and the outer coat complex Sec13/31. In order to investigate the roles of mammalian Sec24 isoforms in cargo sorting, we have combined in vitro COPII vesicle reconstitutions with SILAC-based mass spectrometric analysis. This approach enabled us to identify the core proteome of mammalian COPII vesicles. Comparison of the proteomes generated from vesicles with different Sec24 isoforms confirms several established isoform-dependent cargo proteins, and identifies ERGIC1 and CNIH1 as novel Sec24C‐ and Sec24A-specific cargo proteins, respectively. Proteomic analysis of vesicles reconstituted with a Sec24C mutant, bearing a compromised binding site for the ER-to-Golgi QSNARE Syntaxin5, revealed that the SM/Munc18 protein SCFD1 binds to Syntaxin5 prior to its sorting into COPII vesicles. Furthermore, analysis of Sec24D mutants implicated in the development of a syndromic form of osteogenesis imperfecta showed sorting defects for the three ER-to-Golgi QSNAREs Syntaxin5, GS27, and Bet1.

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