Regulation of Sar1 NH2 terminus by GTP binding and hydrolysis promotes membrane deformation to control COPII vesicle fission

The mechanisms by which the coat complex II (COPII) coat mediates membrane deformation and vesicle fission are unknown. Sar1 is a structural component of the membrane-binding inner layer of COPII (Bi, X., R.A. Corpina, and J. Goldberg. 2002. Nature. 419:271–277). Using model liposomes we found that Sar1 uses GTP-regulated exposure of its NH2-terminal tail, an amphipathic peptide domain, to bind, deform, constrict, and destabilize membranes. Although Sar1 activation leads to constriction of endoplasmic reticulum (ER) membranes, progression to effective vesicle fission requires a functional Sar1 NH2 terminus and guanosine triphosphate (GTP) hydrolysis. Inhibition of Sar1 GTP hydrolysis, which stabilizes Sar1 membrane binding, resulted in the formation of coated COPII vesicles that fail to detach from the ER. Thus Sar1-mediated GTP binding and hydrolysis regulates the NH2-terminal tail to perturb membrane packing, promote membrane deformation, and control vesicle fission.

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

Molecular Biology of the Cell ◽

10.1091/mbc.01-10-0499 ◽

2002 ◽

Vol 13 (3) ◽

pp. 880-891 ◽

Cited By ~ 81

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.

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COPII Vesicle Transport Is Required for Rotavirus NSP4 Interaction with the Autophagy Protein LC3 II and Trafficking to Viroplasms

Journal of Virology ◽

10.1128/jvi.01341-19 ◽

2019 ◽

Vol 94 (1) ◽

Cited By ~ 5

Author(s):

Sue E. Crawford ◽

Jeanette M. Criglar ◽

Zheng Liu ◽

James R. Broughman ◽

Mary K. Estes

Keyword(s):

Endoplasmic Reticulum ◽

Virus Replication ◽

Nonstructural Protein ◽

Vesicle Transport ◽

Host Protein ◽

Cellular Membranes ◽

Particle Assembly ◽

Cellular Autophagy ◽

Copii Vesicle ◽

Copii Vesicles

ABSTRACT Many viruses that replicate in the cytoplasm dramatically remodel and stimulate the accumulation of host cell membranes for efficient replication by poorly understood mechanisms. For rotavirus, a critical step in virion assembly requires the accumulation of membranes adjacent to virus replication centers called viroplasms. Early electron microscopy studies describe viroplasm-associated membranes as “swollen” endoplasmic reticulum (ER). We previously demonstrated that rotavirus infection initiates cellular autophagy and that membranes containing the autophagy marker protein LC3 and the rotavirus ER-synthesized transmembrane glycoprotein NSP4 traffic to viroplasms, suggesting that NSP4 must exit the ER. This study aimed to address the mechanism of NSP4 exit from the ER and determine whether the viroplasm-associated membranes are ER derived. We report that (i) NSP4 exits the ER in COPII vesicles, resulting in disrupted COPII vesicle transport and ER exit sites; (ii) COPII vesicles are hijacked by LC3 II, which interacts with NSP4; and (iii) NSP4/LC3 II-containing membranes accumulate adjacent to viroplasms. In addition, the ER transmembrane proteins SERCA and calnexin were not detected in viroplasm-associated membranes, providing evidence that the rotavirus maturation process of “budding” occurs through autophagy-hijacked COPII vesicle membranes. These findings reveal a new mechanism for rotavirus maturation dependent on intracellular host protein transport and autophagy for the accumulation of membranes required for virus replication. IMPORTANCE In a morphogenic step that is exceedingly rare for nonenveloped viruses, immature rotavirus particles assemble in replication centers called viroplasms, and bud through cytoplasmic cellular membranes to acquire the outer capsid proteins for infectious particle assembly. Historically, the intracellular membranes used for particle budding were thought to be endoplasmic reticulum (ER) because the rotavirus nonstructural protein NSP4, which interacts with the immature particles to trigger budding, is synthesized as an ER transmembrane protein. This present study shows that NSP4 exits the ER in COPII vesicles and that the NSP4-containing COPII vesicles are hijacked by the cellular autophagy machinery, which mediates the trafficking of NSP4 to viroplasms. Changing the paradigm for rotavirus maturation, we propose that the cellular membranes required for immature rotavirus particle budding are not an extension of the ER but are COPII-derived autophagy isolation membranes.

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Cis and trans interactions between atlastin molecules during membrane fusion

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1504368112 ◽

2015 ◽

Vol 112 (15) ◽

pp. E1851-E1860 ◽

Cited By ~ 43

Author(s):

Tina Y. Liu ◽

Xin Bian ◽

Fabian B. Romano ◽

Tom Shemesh ◽

Tom A. Rapoport ◽

...

Keyword(s):

Endoplasmic Reticulum ◽

Transition State ◽

Membrane Fusion ◽

Lipid Bilayer ◽

Comprehensive Model ◽

Gtp Hydrolysis ◽

Supported Lipid Bilayer ◽

Gtp Binding ◽

Cis And Trans

Atlastin (ATL), a membrane-anchored GTPase that mediates homotypic fusion of endoplasmic reticulum (ER) membranes, is required for formation of the tubular network of the peripheral ER. How exactly ATL mediates membrane fusion is only poorly understood. Here we show that fusion is preceded by the transient tethering of ATL-containing vesicles caused by the dimerization of ATL molecules in opposing membranes. Tethering requires GTP hydrolysis, not just GTP binding, because the two ATL molecules are pulled together most strongly in the transition state of GTP hydrolysis. Most tethering events are futile, so that multiple rounds of GTP hydrolysis are required for successful fusion. Supported lipid bilayer experiments show that ATL molecules sitting on the same (cis) membrane can also undergo nucleotide-dependent dimerization. These results suggest that GTP hydrolysis is required to dissociate cis dimers, generating a pool of ATL monomers that can dimerize with molecules on a different (trans) membrane. In addition, tethering and fusion require the cooperation of multiple ATL molecules in each membrane. We propose a comprehensive model for ATL-mediated fusion that takes into account futile tethering and competition between cis and trans interactions.

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

The Journal of Cell Biology ◽

10.1083/jcb.200408135 ◽

2004 ◽

Vol 167 (6) ◽

pp. 997-1003 ◽

Cited By ~ 67

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.

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Sar1 assembly regulates membrane constriction and ER export

The Journal of Cell Biology ◽

10.1083/jcb.201004132 ◽

2010 ◽

Vol 190 (1) ◽

pp. 115-128 ◽

Cited By ~ 50

Author(s):

Kimberly R. Long ◽

Yasunori Yamamoto ◽

Adam L. Baker ◽

Simon C. Watkins ◽

Carolyn B. Coyne ◽

...

Keyword(s):

Endoplasmic Reticulum ◽

Membrane Tension ◽

Conserved Residues ◽

Terminal Loop ◽

Membrane Attachment ◽

Copii Vesicles ◽

Guanosine Triphosphatase ◽

Er Export ◽

Lipid Tubules ◽

And Control

The guanosine triphosphatase Sar1 controls the assembly and fission of COPII vesicles. Sar1 utilizes an amphipathic N-terminal helix as a wedge that inserts into outer membrane leaflets to induce vesicle neck constriction and control fission. We hypothesize that Sar1 organizes on membranes to control constriction as observed with fission proteins like dynamin. Sar1 activation led to membrane-dependent oligomerization that transformed giant unilamellar vesicles into small vesicles connected through highly constricted necks. In contrast, membrane tension provided through membrane attachment led to organization of Sar1 in ordered scaffolds that formed rigid, uniformly nonconstricted lipid tubules to suggest that Sar1 organization regulates membrane constriction. Sar1 organization required conserved residues located on a unique C-terminal loop. Mutations in this loop did not affect Sar1 activation or COPII recruitment and enhanced membrane constriction, yet inhibited Sar1 organization and procollagen transport from the endoplasmic reticulum (ER). Sar1 activity was directed to liquid-disordered lipid phases. Thus, lipid-directed and tether-assisted Sar1 organization controls membrane constriction to regulate ER export.

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GTP hydrolysis by complexes of the signal recognition particle and the signal recognition particle receptor.

The Journal of Cell Biology ◽

10.1083/jcb.123.4.799 ◽

1993 ◽

Vol 123 (4) ◽

pp. 799-807 ◽

Cited By ~ 42

Author(s):

T Connolly ◽

R Gilmore

Keyword(s):

Endoplasmic Reticulum ◽

Protein Translocation ◽

Signal Recognition Particle ◽

Hydrolysis Rate ◽

Endoplasmic Reticulum Membrane ◽

Signal Recognition ◽

Gtp Hydrolysis ◽

Receptor Complexes ◽

Gtp Binding ◽

Hydrolysis Activity

Translocation of proteins across the endoplasmic reticulum membrane is a GTP-dependent process. The signal recognition particle (SRP) and the SRP receptor both contain subunits with GTP binding domains. One GTP-dependent reaction during protein translocation is the SRP receptor-mediated dissociation of SRP from the signal sequence of a nascent polypeptide. Here, we have assayed the SRP and the SRP receptor for GTP binding and hydrolysis activities. GTP hydrolysis by SRP was not detected, so the maximal GTP hydrolysis rate for SRP was estimated to be < 0.002 mol GTP hydrolyzed x mol of SRP-1 x min-1. The intrinsic GTP hydrolysis activity of the SRP receptor ranged between 0.02 and 0.04 mol GTP hydrolyzed x mol of SRP receptor-1 x min-1. A 40-fold enhancement of GTP hydrolysis activity relative to that observed for the SRP receptor alone was obtained when complexes were formed between SRP and the SRP receptor. GTP hydrolysis activity was inhibited by GDP, but not by ATP. Extended incubation of the SRP or the SRP receptor with GTP resulted in substoichiometric quantities of protein-bound ribonucleotide. SRP-SRP receptor complexes engaged in GTP hydrolysis were found to contain a minimum of one bound guanine ribonucleotide per SRP-SRP receptor complex. We conclude that the GTP hydrolysis activity described here is indicative of one of the GTPase cycles that occur during protein translocation across the endoplasmic reticulum.

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mBet3p is required for homotypic COPII vesicle tethering in mammalian cells

The Journal of Cell Biology ◽

10.1083/jcb.200603044 ◽

2006 ◽

Vol 174 (3) ◽

pp. 359-368 ◽

Cited By ~ 60

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.

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