The use of green fluorescent protein (GFP) chimeras to illuminate the secretory pathway in living cells has provided a wealth of information on the mechanisms of protein retention, sorting, and recycling. A wide variety of microscopic techniques, including time-lapse imaging, double-labeling, quantitation, photobleaching, and energy transfer approaches, have been utilized to explore the organization of the early secretory pathway. In this chapter we focus on the application of GFP technology to gain insight into the dynamics of ERGIC-53, a putative cargo receptor localized to the early secretory pathway, and the way in which photobleaching approaches have provided insight into its transport.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Altan-Bonnet, N., Sougrat, R., and Lippincott-Schwartz, J. (2004) Molecular basis for Golgi maintenance and biogenesis. Curr. Opin. Cell Biol. 16, 364–372.
Lee, M. C. S., Miller, E. A., Goldberg, J., Orci, L., and Schekman, R. (2004) Bi-directional protein transport between the ER and Golgi. Annu. Rev. Cell Dev. Biol. 20, 87–123.
Ward, T. H. and Lippincott-Schwartz, J. (2006) The uses of green fluorescent protein in mammalian cells, in Green Fluorescent Protein: Properties, Applications, and Protocols, 2nd ed. ( Chalfie, M., and Kain, S. R., eds.), John Wiley, Hoboken, NJ, pp. 305–337.
Schweizer, A., Fransen, J. A., Bächi, T., Ginsel, L., and Hauri, H. P. (1988) Identification, by a monoclonal antibody, of a 53-kDa protein associated with a tubulo-vesicular compartment at the cis-side of the Golgi apparatus. J. Cell Biol. 107, 1643–1653.
Lahtinen, U., Dahllöf, B., and Saraste, J. (1992) Characterization of a 58 kDa cis-Golgi protein in pancreatic exocrine cells. J. Cell Sci. 103, 321–333.
Hauri, H. P., Kappeler, F., Andersson, H., and Appenzeller, C. (2000) ERGIC-53 and traffic in the secretory pathway. J. Cell Sci. 113, 587–596.
Klumperman, J., Schweizer, A., Clausen, H., et al. (1998) The recycling pathway of protein ERGIC-53 and dynamics of the ER-Golgi intermediate compartment. J. Cell Sci. 111, 3411–3425.
Kappeler, F., Klopfenstein, D. R. C., Foguet, M., Paccaud, J. P., and Hauri, H.-P. (1997) The recycling of ERGIC-53 in the early secretory pathway: ERGIC-53 carries a cytosolic endoplasmic reticulum-exit determinant interacting with COPII. J. Biol. Chem. 272, 31801–31808.
Itin, C., Schindler, R., and Hauri, H.-P. (1995) Targeting of protein ERGIC-53 to the ER/ERGIC/cis-Golgi recycling pathway. J. Cell Biol. 131, 57–67.
Ward, T. H., Polishchuk, R. S., Caplan, S., Hirschberg, K., and Lippincott-Schwartz, J. (2001) Maintenance of Golgi structure and function depends on the integrity of ER export. J. Cell Biol. 155, 557–570.
Lippincott-Schwartz, J., Altan-Bonnet, N., and Patterson, G. H. (2003) Photobleaching and photoactivation: following protein dynamics in living cells. Nat. Cell Biol. 5, S7–S14.
Nufer, O., Kappeler, F., Guldbrandsen, S., and Hauri, H.-P. (2003) ER export of ERGIC-53 is controlled by cooperation of targeting determinants in all three of its domains. J. Cell Sci. 116, 4429–4440.
Lippincott-Schwartz, J., Donaldson, J. G., Schweizer, A., et al. (1990) Microtubule-dependent retrograde transport of proteins into the ER in the presence of brefeldin A suggests an ER recycling pathway. Cell 60, 821–836.
Saraste, J. and Svensson, K. (1991) Distribution of the intermediate elements operating in ER to Golgi transport. J. Cell Sci. 100, 415–430.
Phair, R. D. and Misteli, T. (2001) Kinetic modelling approaches to in vivo imaging. Nat. Rev. Mol. Cell Biol. 2, 898–907.
Klausner, R. D., Donaldson, J. G., and Lippincott-Schwartz, J. (1992) Brefeldin A: insights into the control of membrane traffic and organelle structure. J. Cell Biol. 116, 1071–1080.
Cole, N. B., Sciaky, N., Marotta, A., Song, J., and Lippincott-Schwartz, J. (1996) Golgi dispersal during microtubule disruption: regeneration of Golgi stacks at peripheral endoplasmic reticulum exit sites. Mol. Biol. Cell 7, 631–650.
Sannerud, R., Marie, M., Nizak, C., et al. (2006) Rab1 defines a novel pathway connecting the pre-Golgi intermediate compartment with the cell periphery. Mol. Biol. Cell 17, 1514–1526.
Nehls, S., Snapp, E. L., Cole, N. B., et al. (2000) Dynamics and retention of misfolded proteins in native ER membranes. Nat. Cell Biol. 2, 288–295.
Ben-Tekaya, H., Miura, K., Pepperkok, R., and Hauri, H.-P. (2005) Live imaging of bidirectional traffic from the ERGIC. J. Cell Sci. 118, 357–367.
Zacharias, D. A., Violin, J. D., Newton, A. C., and Tsien, R. Y. (2002) Partitioning of lipid-modified monomeric GFPs into membrane microdomains of live cells. Science 296, 913–916.
Campbell, R. E., Tour, O., Palmer, A. E., et al. (2002) A monomeric red fluorescent protein. Proc. Natl. Acad. Sci. USA 99, 7877–7882.
Shaner, N. C., Campbell, R. E., Steinbach, P. A., Giepmans, B. N. G, Palmer, A. E., and Tsien, R. Y. (2004) Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein. Nat. Biotechnol. 22, 1567–1572.
Zaal, K. J. M., Smith, C. L., Polishchuk, R. S., et al. (1999) Golgi membranes are absorbed into and reemerge from the ER during mitosis. Cell 99, 589–601.
Gerlich, D., Beaudouin, J., Kalbfuss, B., Daigle, N., Eils, R., and Ellenberg, J. (2003) Global chromosome positions are transmitted through mitosis in mammalian cells. Cell 112, 751–764.
Cole, N. B., Smith, C. L., Sciaky, N., Terasaki, M., Edidin, M., and Lippincott- Schwartz, J. (1996) Diffusional mobility of Golgi proteins in membranes of living cells. Science 273, 797–801.
Sciaky, N., Presley, J., Smith, C., et al. (1997) Golgi tubule traffic and the effects of brefeldin A visualized in living cells. J. Cell Biol. 139, 1137–1155.
Acknowledgments
This work was supported by a Royal Society Dorothy Hodgkin Fellowship.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2007 Humana Press Inc.
About this protocol
Cite this protocol
Ward, T.H. (2007). Trafficking Through the Early Secretory Pathway of Mammalian Cells. In: van der Giezen, M. (eds) Protein Targeting Protocols. Methods in Molecular Biology™, vol 390. Humana Press. https://doi.org/10.1007/978-1-59745-466-7_19
Download citation
DOI: https://doi.org/10.1007/978-1-59745-466-7_19
Publisher Name: Humana Press
Print ISBN: 978-1-58829-702-0
Online ISBN: 978-1-59745-466-7
eBook Packages: Springer Protocols