Chemistry-based protein modification strategy for endocytic pathway analysis.
BACKGROUND
The integrated analysis of intracellular trafficking pathways is one of the current challenges in the field of cell biology, and functional proteomics has become a powerful technique for the large-scale identification of proteins or lipids and the elucidation of biological processes in their natural contexts. For this, new dynamic strategies must be devised to trace proteins that follow a specific pathway such that their initial and final destinations can be detected by automated means.


RESULTS
Here, we report a novel vectorial strategy for trafficking pathway analysis. This strategy is based on a chemical modification of plasma membrane proteins with a bSuPeR (biotinylated sulfation site peptide reagent) and metabolic labelling in the Golgi apparatus, such that plasma membrane proteins that traffic via the retrograde route become detectable in complex mixtures. Efficient synthesis schemes are presented for tailor-made chemical tools that are then applied to the step-by-step validation of the strategy, using a known retrograde cargo protein: the STxB (Shiga toxin B-subunit). bSuPeR modification at the plasma membrane does not affect STxB transport to the Golgi apparatus, where the protein is metabolically labelled, allowing its detection in cell lysates.


CONCLUSIONS
Our vectorial concept proposes a new chemical approach for traffic-based profiling of proteins that may prove to be applicable to the analysis of diverse endocytic pathways.
Christiano
Romain
R
Institut Curie - Centre de Recherche, Equipe Pharmacochimie, Chimie bioorganique, Vectorisation, 26 rue d'Ulm, 75248 Paris Cedex 05, France.
Amessou
Mohamed
M
Shi
Getao
G
Azoulay
Michel
M
Blanpain
Annick
A
Drobecq
Hervé
H
Melnyk
Oleg
O
Florent
Jean-Claude
JC
Johannes
Ludger
L
D002462:Cell Membrane / Q000378:metabolism; D004705:Endocytosis / Q000502:physiology*; D019084:Fluorescent Antibody Technique, Indirect; D006056:Golgi Apparatus / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D021381:Protein Transport / Q000502:physiology*; D040901:Proteomics / Q000379:methods*