PIN-G Reporter for Imaging and Defining Trafficking Signals in Membrane Proteins
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.
Key wordsGreen fluorescent protein (GFP) live imaging trafficking epitope tags reporter constructs photoactivation lentivirus
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.
- 6.Patterson, G. H. (2008) Photoactivation and imaging of photoactivatable fluorescent proteins. Curr Protoc Cell Biol Chapter 21: Unit 21.6.Google Scholar