Abstract
The identification of motifs that control the intracellular trafficking of proteins is a fundamental objective of cell biology. Once identified, such regions should, in principle, be both necessary and sufficient to direct any randomly distributed protein, acting as a reporter, to the subcellular compartment in question. However, most reporter proteins have limited versatility owing to their endogenous expression and limited modes of detection – especially in live cells. To surmount such limitations, we engineered a plasmid – pIN-G – encoding an entirely artificial, type I transmembrane reporter protein (PIN-G), containing HA, cMyc and GFP epitope, and fluorescence tags. Although originally designed for trafficking studies, pIN technology is a powerful tool applicable to almost every area of biology. Here we describe the methodologies used routinely in analyzing pIN constructs and some of their derivatives.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
McKeown, L., Robinson, P., Greenwood, S. M., Hu, W., Jones, O.T, (2006) PIN-G - a novel reporter for imaging and defining the effects of trafficking signals in membrane proteins. BMC Biotechnol 6 , 15.
Bonifacino, J. S., Cosson, P., and Klausner, R. D. (1990) Colocalized transmembrane determinants for ER degradation and subunit assembly explain the intracellular fate of TCR chains. Cell 63 , 503–513.
Gu, C., Jan, Y. N., and Jan, L. Y. (2003) A conserved domain in axonal targeting of Kv1 (Shaker) voltage-gated potassium channels. Science 301 , 646–649.
Jones, V. C., McKeown, L., Verkhratsky, A., Jones, O. T. (2008) LV-pIN-KDEL: a novel lentiviral vector demonstrates the morphology, dynamics and continuity of the endoplasmic reticulum in live neurones. BMC Neurosci 9 , 10.
Prescott, M., Battad, J. M., Wilmann, P. G., Rossjohn, J., and Devenish, R. J. (2006) Recent advances in all-protein chromophore technology. Biotechnol Annu Rev 12 , 31–66.
Patterson, G. H. (2008) Photoactivation and imaging of photoactivatable fluorescent proteins. Curr Protoc Cell Biol Chapter 21: Unit 21.6.
Patterson, G. H., and Lippincott-Schwartz, J. A. (2002) Photoactivatable GFP for selective photolabeling of proteins and cells. Science 297 , 1873–1877.
Follenzi, A., and Naldini, L. (2002) HIV-based vectors. Preparation and use. Methods Mol Med 69 , 259–274.
Acknowledgments
This work was supported by funds from the Biotechnology and Biological Sciences Research Council UK: BBSRC, BB/D008891/1. We are indebted to Professor P.-L. Nicotera and Dr. D. Bano (University of Leicester, UK) for their gift of the lentiviral packaging system and advice on its use. We also thank Jane Kott and the University of Manchester Bioimaging Facility for imaging support.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2009 Humana Press, a part of Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Mckeown, L., Jones, V.C., Jones, O.T. (2009). PIN-G Reporter for Imaging and Defining Trafficking Signals in Membrane Proteins. In: Rich, P., Douillet, C. (eds) Bioluminescence. Methods in Molecular Biology™, vol 574. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-60327-321-3_19
Download citation
DOI: https://doi.org/10.1007/978-1-60327-321-3_19
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-60327-320-6
Online ISBN: 978-1-60327-321-3
eBook Packages: Springer Protocols