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Non-invasive topology analysis of membrane proteins in the secretory pathway

We present a novel method to experimentally visualize in vivo the topology of transmembrane proteins residing in the endoplasmic reticulum (ER) membrane or passing through the secretory pathway on their way to their final destination. This approach, so-called redox-based topology analysis (ReTA), is...

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Bibliographic Details
Main Authors: Brach, Thorsten (Author) , Soyk, Sebastian (Author) , Hinz, Giselbert (Author) , Hell, Rüdiger (Author) , Meyer, Andreas (Author)
Format: Article (Journal)
Language:English
Published: 7 November 2008
In: The plant journal
Year: 2009, Volume: 57, Issue: 3, Pages: 534-541
ISSN:1365-313X
DOI:10.1111/j.1365-313X.2008.03704.x
Online Access:Verlag, kostenfrei, Volltext: http://dx.doi.org/10.1111/j.1365-313X.2008.03704.x
Verlag, kostenfrei, Volltext: http://onlinelibrary.wiley.com/doi/10.1111/j.1365-313X.2008.03704.x/abstract
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Author Notes:Thorsten Brach, Sebastian Soyk, Christopher Müller, Giselbert Hinz, Rüdiger Hell, Federica Brandizzi and Andreas J. Meyer
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Summary:We present a novel method to experimentally visualize in vivo the topology of transmembrane proteins residing in the endoplasmic reticulum (ER) membrane or passing through the secretory pathway on their way to their final destination. This approach, so-called redox-based topology analysis (ReTA), is based on fusion of transmembrane proteins with redox-sensitive GFP (roGFP) and ratiometric imaging. The ratio images provide direct information on the orientation of roGFP relative to the membrane as the roGFP fluorescence alters with changes in the glutathione redox potential across the ER membrane. As proof of concept, we produced binary read-outs using oxidized roGFP inside the ER lumen and reduced roGFP on the cytosolic side of the membrane for both N- and C-terminal fusions of single and multi-spanning membrane proteins. Further, successive deletion of hydrophobic domains from the C-terminus of the K/HDEL receptor ERD2 resulted in alternating localization of roGFP and a topology model for AtERD2 with six transmembrane domains.
Item Description:Gesehen am 09.05.2017
Physical Description:Online Resource
ISSN:1365-313X
DOI:10.1111/j.1365-313X.2008.03704.x

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