Insights from reconstitution reactions of COPII vesicle formation using pure components and low mechanical perturbation
-
Sebastian Daum
Abstract
As shape transformations of membranes are vital for intracellular trafficking, it is crucial to understand both the mechanics and the biochemistry of these processes. The interplay of these two factors constitutes an experimental challenge, however, because biochemical experiments are not tailored to the investigation of mechanical processes, and biophysical studies using model membranes are not capable of emulating native biological complexity. Reconstituted liposome-based model systems have been widely used for investigating the formation of transport vesicles by the COPII complex that naturally occurs at the endoplasmic reticulum. Here we have revisited these model systems, to address the influence of lipid composition, GTP hydrolyzing conditions and mechanical perturbation on the experimental outcome. We observed that the lipid-dependence of COPII-induced membrane remodeling differs from that predicted based on the lipid-dependence of COPII membrane binding. Under GTP non-hydrolyzing conditions, a structured coat was seen while GTP-hydrolyzing conditions yielded uncoated membranes as well as membranes coated by a thick protein coat of rather unstructured appearance. Detailed up-to-date protocols for purifications of Saccharomyces cerevisiae COPII proteins and for reconstituted reactions using these proteins with giant liposomes are also provided.
Acknowledgments
We thank Gerd Hause for his support with electron microscopy at the University of Halle, Karin Breunig and Markus Pietzsch for advice concerning yeast cell culture, Randy Schekman and Bob Lesch for protocols, expression strains and discussions and Claudia Müller for technical assistance. Financial support from the Deutsche Forschungsgemeinschaft within Graduiertenkolleg 1026, from the Bundesministerium für Bildung und Forschung (FKZ 03Z2HN22) and from the state of Saxony-Anhalt (Landesgraduiertenfoerderung and European Regional Development Grants 124112001 and 1241090001) is gratefully acknowledged.
Conflict of interest statement
Funding: Bundesministerium für Bildung und Forschung, (Grant/Award Number: ‘03Z2HN22’) Deutsche Forschungsgemeinschaft, (Grant/Award Number: ‘GRK 1026’). State of Saxony-Anhalt (Landesgraduiertenförderung and <softenter;European Regional Developments (Grant/Award Numbers 124112001 and 1241090001).
References
Adolf, F., Herrmann, A., Hellwig, A., Beck, R., Brugger, B., and Wieland, F.T. (2013). Scission of COPI and COPII vesicles is independent of GTP hydrolysis. Traffic. 14, 922–932.10.1111/tra.12084Suche in Google Scholar
Akaaboune, M., Grady, R.M., Turney, S., Sanes, J.R., and Lichtman, J.W. (2002). Neurotransmitter receptor dynamics studied in vivo by reversible photo-unbinding of fluorescent ligands. Neuron. 34, 865–876.10.1016/S0896-6273(02)00739-0Suche in Google Scholar
Antonny, B., Madden, D., Hamamoto, S., Orci, L., and Schekman, R. (2001). Dynamics of the COPII coat with GTP and stable analogues. Nat. Cell Biol. 3, 531–537.10.1038/35078500Suche in Google Scholar
Bacia, K., Futai, E., Prinz, S., Meister, A., Daum, S., Glatte, D., Briggs, J.A.G., and Schekman, R. (2011). Multibudded tubules formed by COPII on artificial liposomes. Sci. Rep. 1, 17.10.1038/srep00017Suche in Google Scholar
Barlowe, C. (1995). COPII: a membrane coat that forms endoplasmic reticulum-derived vesicles. FEBS Lett. 369, 93–96.10.1016/0014-5793(95)00618-JSuche in Google Scholar
Barlowe, C. (1997). Coupled ER to Golgi transport reconstituted with purified cytosolic proteins. J. Cell Biol. 139, 1097–1108.10.1083/jcb.139.5.1097Suche in Google Scholar
Barlowe, C., d’Enfert, C., and Schekman, R. (1993). Purification and characterization of SAR1p, a small GTP-binding protein required for transport vesicle formation from the endoplasmic reticulum. J. Biol. Chem. 268, 873–879.10.1016/S0021-9258(18)54015-1Suche in Google Scholar
Barlowe, C., Orci, L., Yeung, T., Hosobuchi, M., Hamamoto, S., Salama, N., Rexach, M.F., Ravazzola, M., Amherdt, M., and Schekman, R. (1994). COPII: a membrane coat formed by Sec proteins that drive vesicle budding from the endoplasmic reticulum. Cell 77, 895–907.10.1016/0092-8674(94)90138-4Suche in Google Scholar
Bashkirov, P.V., Akimov, S.A., Evseev, A.I., Schmid, S.L., Zimmerberg, J., and Frolov, V.A. (2008). GTPase cycle of dynamin is coupled to membrane squeeze and release, leading to spontaneous fission. Cell 135, 1276–1286.10.1016/j.cell.2008.11.028Suche in Google Scholar PubMed PubMed Central
Bi, X., Corpina, R.A., and Goldberg, J. (2002). Structure of the Sec23/24-Sar1 pre-budding complex of the COPII vesicle coat. Nature 419, 271–277.10.1038/nature01040Suche in Google Scholar PubMed
Bi, X., Mancias, J.D., and Goldberg, J. (2007). Insights into COPII coat nucleation from the structure of Sec23.Sar1 complexed with the active fragment of Sec31. Dev. Cell. 13, 635–645.10.1016/j.devcel.2007.10.006Suche in Google Scholar
Boucrot, E., Pick, A., Camdere, G., Liska, N., Evergren, E., McMahon, H.T., and Kozlov, M.M. (2012). Membrane fission is promoted by insertion of amphipathic helices and is restricted by crescent BAR domains. Cell 149, 124–136.10.1016/j.cell.2012.01.047Suche in Google Scholar
Brandizzi, F. and Barlowe, C. (2013). Organization of the ER-Golgi interface for membrane traffic control. Nature reviews. Mol. Cell Biol. 14, 382–392.10.1038/nrm3588Suche in Google Scholar
Bromley, E.H., Kuwada, N.J., Zuckermann, M.J., Donadini, R., Samii, L., Blab, G.A., Gemmen, G.J., Lopez, B.J., Curmi, P.M., Forde, N.R., et al. (2009). The Tumbleweed: towards a synthetic proteinmotor. HFSP J. 3, 204–212.10.2976/1.3111282Suche in Google Scholar
Cai, H., Yu, S., Menon, S., Cai, Y., Lazarova, D., Fu, C., Reinisch, K., Hay, J.C., and Ferro-Novick, S. (2007). TRAPPI tethers COPII vesicles by binding the coat subunit Sec23. Nature 445, 941–944.10.1038/nature05527Suche in Google Scholar
Futai, E., Hamamoto, S., Orci, L., and Schekman, R. (2004). GTP/GDP exchange by Sec12p enables COPII vesicle bud formation on synthetic liposomes. EMBO J. 23, 4146–4155.10.1038/sj.emboj.7600428Suche in Google Scholar
Heinze, K.G., Costantino, S., De Koninck, P., and Wiseman, P.W. (2009). Beyond photobleaching, laser illumination unbinds fluorescent proteins. J. Phys. Chem. B 113, 5225–5233.10.1021/jp8060152Suche in Google Scholar
Huang, M., Weissman, J.T., Beraud-Dufour, S., Luan, P., Wang, C., Chen, W., Aridor, M., Wilson, I.A., and Balch, W.E. (2001). Crystal structure of Sar1-GDP at 1.7 A resolution and the role of the NH2 terminus in ER export. J. Cell Biol. 155, 937–948.10.1083/jcb.200106039Suche in Google Scholar
Karatekin, E., Sandre, O., Guitouni, H., Borghi, N., Puech, P.H., and Brochard-Wyart, F. (2003). Cascades of transient pores in giant vesicles: line tension and transport. Biophys. J. 84, 1734–1749.10.1016/S0006-3495(03)74981-9Suche in Google Scholar
Kim, J., Hamamoto, S., Ravazzola, M., Orci, L., and Schekman, R. (2005). Uncoupled packaging of amyloid precursor protein and presenilin 1 into coat protein complex II vesicles. J. Biol. Chem. 28, 7758–7768.10.1074/jbc.M411091200Suche in Google Scholar PubMed
Kuehn, M.J. and Schekman, R. (1997). COPII and secretory cargo capture into transport vesicles. Curr. Opin. Cell Biol. 9, 477–483.10.1016/S0955-0674(97)80022-1Suche in Google Scholar
Lee, M.C., Orci, L., Hamamoto, S., Futai, E., Ravazzola, M., and Schekman, R. (2005). Sar1p N-terminal helix initiates membrane curvature and completes the fission of a COPII vesicle. Cell 122, 605–617.10.1016/j.cell.2005.07.025Suche in Google Scholar
Liu, A.P. and Fletcher, D.A. (2009). Biology under construction: in vitro reconstitution of cellular function. Nature reviews. Mol. Cell Biol. 10, 644–650.10.1038/nrm2746Suche in Google Scholar
Long, K.R., Yamamoto, Y., Baker, A.L., Watkins, S.C., Coyne, C.B., Conway, J.F., and Aridor, M. (2010). Sar1 assembly regulates membrane constriction and ER export. J. Cell Biol. 190, 115–128.10.1083/jcb.201004132Suche in Google Scholar
Lord, C., Bhandari, D., Menon, S., Ghassemian, M., Nycz, D., Hay, J., Ghosh, P., and Ferro-Novick, S. (2011). Sequential interactions with Sec23 control the direction of vesicle traffic. Nature 473, 181–186.10.1038/nature09969Suche in Google Scholar
Lord, C., Ferro-Novick, S., and Miller, E.A. (2013). The highly conserved COPII coat complex sorts cargo from the endoplasmic reticulum and targets it to the Golgi. Cold Spring Harb. Perspect. Biol. 5. pii: a013367.Suche in Google Scholar
Matsuoka, K. and Schekman, R. (2000). The use of liposomes to study COPII- and COPI-coated vesicle formation and membrane protein sorting. Methods 20, 417–428.10.1006/meth.2000.0955Suche in Google Scholar
Matsuoka, K., Orci, L., Amherdt, M., Bednarek, S.Y., Hamamoto, S., Schekman, R., and Yeung, T. (1998). COPII-coated vesicle formation reconstituted with purified coat proteins and chemically defined liposomes. Cell 9, 263–275.10.1016/S0092-8674(00)81577-9Suche in Google Scholar
Miller, E.A., Beilharz, T.H., Malkus, P.N., Lee, M.C., Hamamoto, S., Orci, L., and Schekman, R. (2003). Multiple cargo binding sites on the COPII subunit Sec24p ensure capture of diverse membrane proteins into transport vesicles. Cell 114, 497–509.10.1016/S0092-8674(03)00609-3Suche in Google Scholar
Neumuller, K.G., Elsayad, K., Reisecker, J.M., Waxham, M.N., and Heinze, K.G. (2010). Photounbinding of calmodulin from a family of CaM binding peptides. PloS one. 5, e14050.10.1371/journal.pone.0014050Suche in Google Scholar PubMed PubMed Central
Oka, T. and Nakano, A. (1994). Inhibition of GTP hydrolysis by Sar1p causes accumulation of vesicles that are a functional intermediate of the ER-to-Golgi transport in yeast. J. Cell Biol. 124, 425–434.10.1083/jcb.124.4.425Suche in Google Scholar PubMed PubMed Central
Saito, Y., Kimura, K., Oka, T., and Nakano, A. (1998). Activities of mutant Sar1 proteins in guanine nucleotide binding, GTP hydrolysis, and cell-free transport from the endoplasmic reticulum to the Golgi apparatus. J. Biochem. 124, 816–823.10.1093/oxfordjournals.jbchem.a022185Suche in Google Scholar PubMed
Salama, N.R., Yeung, T., and Schekman, R.W. (1993). The Sec13p complex and reconstitution of vesicle budding from the ER with purified cytosolic proteins. EMBO J. 12, 4073–4082.10.1002/j.1460-2075.1993.tb06091.xSuche in Google Scholar PubMed PubMed Central
Salama, N.R., Chuang, J.S., and Schekman, R.W. (1997). Sec31 encodes an essential component of the COPII coat required for transport vesicle budding from the endoplasmic reticulum. Mol. Biol. Cell 8, 205–217.10.1091/mbc.8.2.205Suche in Google Scholar PubMed PubMed Central
Sanborn, J., Oglecka, K., Kraut, R.S., and Parikh, A.N. (2013). Transient pearling and vesiculation of membrane tubes under osmotic gradients. Faraday Discuss. 161, 167–176; discussion 273–303.10.1039/C2FD20116JSuche in Google Scholar PubMed
Schwille, P. and Diez, S. (2009). Synthetic biology of minimal systems. Crit. Rev. Chem. Mol Biol. 44, 223–242.10.1080/10409230903074549Suche in Google Scholar PubMed
Settles, E.I., Loftus, A.F., McKeown, A.N., and Parthasarathy, R. (2010). The vesicle trafficking protein Sar1 lowers lipid membrane rigidity. Biophys. J. 99, 1539–1545.10.1016/j.bpj.2010.06.059Suche in Google Scholar PubMed PubMed Central
Supek, F., Madden, D.T., Hamamoto, S., Orci, L., and Schekman, R. (2002). Sec16p potentiates the action of COPII proteins to bud transport vesicles. J. Cell Biol. 158, 1029–1038.10.1083/jcb.200207053Suche in Google Scholar PubMed PubMed Central
Venditti, R., Wilson, C., and De Matteis, M.A. (2014). Exiting the ER: what we know and what we don’t. Trends Cell Biol. 24, 9–18.10.1016/j.tcb.2013.08.005Suche in Google Scholar PubMed
Vetter, I.R. and Wittinghofer, A. (2001). The guanine nucleotide-binding switch in three dimensions. Science 294, 1299–1304.10.1126/science.1062023Suche in Google Scholar PubMed
Yamanushi, T., Hirata, A., Oka, T., and Nakano, A. (1996). Characterization of yeast sar1 temperature-sensitive mutants, which are defective in protein transport from the endoplasmic reticulum. J Biochem. 120, 452–458.10.1093/oxfordjournals.jbchem.a021432Suche in Google Scholar PubMed
Zanetti, G., Pahuja, K.B., Studer, S., Shim, S., and Schekman, R. (2012). COPII and the regulation of protein sorting in mammals. Nat. Cell Biol. 14, 20–28.10.1038/ncb2390Suche in Google Scholar PubMed
Zanetti, G., Prinz, S., Daum, S., Meister, A., Schekman, R., Bacia, K., and Briggs, J.A. (2013). The structure of the COPII transport-vesicle coat assembled on membranes. eLife 2, e00951.10.7554/eLife.00951.018Suche in Google Scholar
Zhu, T.F., Adamala, K., Zhang, N., and Szostak, J.W. (2012). Photochemically driven redox chemistry induces protocell membrane pearling and division. Proc. Nat. Acad. Sci. USA 109, 9828–9832.10.1073/pnas.1203212109Suche in Google Scholar PubMed PubMed Central
©2014 by Walter de Gruyter Berlin/Boston
Artikel in diesem Heft
- Frontmatter
- Guest Editorial
- Highlight: conformational transitions in macromolecular interactions
- Single-molecule spectroscopy of unfolded proteins and chaperonin action
- Influence of the polypeptide environment next to amyloidogenic peptides on fibril formation
- Structure of large dsDNA viruses
- Functional aspects of extracellular cyclophilins
- Generic tools for conditionally altering protein abundance and phenotypes on demand
- Structural insights into calmodulin/Munc13 interaction
- Interaction of linear polyamines with negatively charged phospholipids: the effect of polyamine charge distance
- Interaction of the human N-Ras protein with lipid raft model membranes of varying degrees of complexity
- Lanthanides as substitutes for calcium ions in the activation of plant α-type phospholipase D
- Insights from reconstitution reactions of COPII vesicle formation using pure components and low mechanical perturbation
- Identification of key residues in the formate channel FocA that control import and export of formate
- Twin-arginine translocation-arresting protein regions contact TatA and TatB
- Biophysical and biochemical analysis of hnRNP K: arginine methylation, reversible aggregation and combinatorial binding to nucleic acids
- An ancient oxidoreductase making differential use of its cofactors
- Biophysical characterization of polyomavirus minor capsid proteins
- Structural basis for PTPA interaction with the invariant C-terminal tail of PP2A
- Correlating structure and ligand affinity in drug discovery: a cautionary tale involving second shell residues
- Thermodynamic signatures in macromolecular interactions involving conformational flexibility
Artikel in diesem Heft
- Frontmatter
- Guest Editorial
- Highlight: conformational transitions in macromolecular interactions
- Single-molecule spectroscopy of unfolded proteins and chaperonin action
- Influence of the polypeptide environment next to amyloidogenic peptides on fibril formation
- Structure of large dsDNA viruses
- Functional aspects of extracellular cyclophilins
- Generic tools for conditionally altering protein abundance and phenotypes on demand
- Structural insights into calmodulin/Munc13 interaction
- Interaction of linear polyamines with negatively charged phospholipids: the effect of polyamine charge distance
- Interaction of the human N-Ras protein with lipid raft model membranes of varying degrees of complexity
- Lanthanides as substitutes for calcium ions in the activation of plant α-type phospholipase D
- Insights from reconstitution reactions of COPII vesicle formation using pure components and low mechanical perturbation
- Identification of key residues in the formate channel FocA that control import and export of formate
- Twin-arginine translocation-arresting protein regions contact TatA and TatB
- Biophysical and biochemical analysis of hnRNP K: arginine methylation, reversible aggregation and combinatorial binding to nucleic acids
- An ancient oxidoreductase making differential use of its cofactors
- Biophysical characterization of polyomavirus minor capsid proteins
- Structural basis for PTPA interaction with the invariant C-terminal tail of PP2A
- Correlating structure and ligand affinity in drug discovery: a cautionary tale involving second shell residues
- Thermodynamic signatures in macromolecular interactions involving conformational flexibility