Fluorogenic Probing of Membrane Protein Trafficking

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Fluorogenic Probing of Membrane Protein Trafficking, Bioconjugate Chem. 2018

 

Plasma membrane resident proteins are in charge of vital functions for cells and organisms. Membrane receptors relay external signals inside cells. Transporters are involved in the movement of ions, small molecules, and macromolecules across the cell membrane. Cell adhesion molecules dictate how cells interact with other cells or with the extracellular matrix. Defects within the traffi cking of membrane proteins are associated with many important diseases. Tools that enable simple and efficient study of the secretion, transportation, and recycling of membrane proteins are thus fundamental for both understanding membrane protein functions and developing innovative cell assays for the discovery of small molecules able to restore genetic mutation-induced trafficking defects.

 

 

In order to broaden the toolbox available for the study of membrane proteins, in particular, in terms of spectral properties and tag size, we developed an approach based on the chemicalgenetic fluorescent marker FAST (fluorescence-activating and absorption-shifting tag), which has been recently developed for the selective labeling of fusion proteins in living cells. FAST is a small kDa protein tag evolved to form noncovalent and reversible fluorescent complexes with cell-permeant and nontoxic 4-hydroxybenzylidene rhodanine (HBR) analogs. 

 

Because they fluoresce only when immobilized within FAST (because of conformational locking) and undergo a >80 nm red-shift in absorption upon binding (due to selective binding of their anionic form), HBR analogs do not exhibit nonspecific fluorescence in cells, enabling one to image without the need for washing. FAST was shown to be functional in various subcellular localizations and expression systems (e.g., bacteria, yeast, mammalian cells) including multicellular organisms (e.g., zebrafish embryo).  An important feature of FAST is the ability to tune its properties by changing the chemical structure of the applied fluorogen.

 

 

Résumé: 

Bioconjugate Chem. 2018

 

Methods to differentially label cell-surface and intracellular membrane proteins are indispensable for understanding their function and the regulation of their trafficking. We present an effi cient strategy for the rapid and selective fluorescent labeling of membrane proteins based on the chemical-genetic fl uorescent marker FAST (fluorescence activating and absorption-shifting tag). Cell-surface FASTtagged proteins could be selectively and rapidly labeled using fluorogenic membrane-impermeant 4-hydroxybenzylidene rhodanine (HBR) analogs. This approach allows the study of protein trafficking at the plasma membrane with various fluorometric techniques, and opens exciting prospects for the high-throughput screening of small molecules able to restore disease-related trafficking defects.

Group: 
Chimie Biophysique
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Références: 

Fluorogenic Probing of Membrane Protein Trafficking

 

Chenge Li,Aurélien Mourton,Marie-Aude Plamont,Vanessa Rodrigues,Isabelle Aujard,Michel Volovitch,Thomas Le Saux,Franck Perez,Sophie Vriz,Ludovic Jullien,Alain Joliot and Arnaud Gautier

 

Bioconjugate Chem. 2018

 

DOI : 10.1021/acs.bioconjchem.8b00180