PIN-G--a novel reporter for imaging and defining the effects of trafficking signals in membrane proteins.
BACKGROUND
The identification of protein trafficking signals, and their interacting mechanisms, is a fundamental objective of modern biology. Unfortunately, the analysis of trafficking signals is complicated by their topography, hierarchical nature and regulation. Powerful strategies to test candidate motifs include their ability to direct simpler reporter proteins, to which they are fused, to the appropriate cellular compartment. However, present reporters are limited by their endogenous expression, paucity of cloning sites, and difficult detection in live cells.


RESULTS
Consequently, we have engineered a mammalian expression vector encoding a novel trafficking reporter--pIN-G--consisting of a simple, type I integral protein bearing permissive intra/extracellular cloning sites, green fluorescent protein (GFP), cMyc and HA epitope tags. Fluorescence imaging, flow cytometry and biochemical assays of transfected HEK293 cells, confirm the size, topology and surface expression of PIN-G. Moreover, a pIN-G fusion construct, containing a Trans-Golgi Network (TGN) targeting determinant, internalises rapidly from the cell surface and localises to the TGN. Additionally, another PIN-G fusion protein and its mutants reveal trafficking determinants in the cytoplasmic carboxy terminus of Kv1.4 voltage-gated potassium channels.


CONCLUSIONS
Together, these data indicate that pIN-G is a versatile, powerful, new reporter for analysing signals controlling membrane protein trafficking, surface expression and dynamics.
McKeown
Lynn
L
Faculty of Life Sciences, University of Manchester, Manchester, M13 9PT, UK. lynn.mckeown@manchester.ac.uk
Robinson
Philip
P
Greenwood
Sam M
SM
Hu
Weiwen
W
Jones
Owen T
OT
D002460:Cell Line; D006801:Humans; D007668:Kidney / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism* / Q000648:ultrastructure*; D008856:Microscopy, Fluorescence / Q000379:methods*; D025941:Protein Interaction Mapping / Q000379:methods*; D021382:Protein Sorting Signals / Q000502:physiology*; D021381:Protein Transport / Q000502:physiology*
D008565:Membrane Proteins; D021382:Protein Sorting Signals