A ubiquitin-based assay for the cytosolic uptake of protein transduction domains.
Protein transduction domains (PTDs) are promising tools for transducing presynthesized polypeptides across the plasma membrane. However, the development and optimization of PTDs are hampered by many technical problems and artifacts resulting notably from the tight binding of PTDs to the cell surface and the difficulty in discriminating, through imagery analyses, truly cytosolic from cytoplasmic vesicular compartments. To circumvent these problems, we have developed an unambiguous enzymatic assay of the cytosolic uptake of PTD-driven proteins, based on the processing by ubiquitin-specific C-terminal proteases (DUBs). This method, coupled with fluorometry and fluorescence microscopy, shows that the TAT PTD derived from human immunodeficiency virus type 1 is rapidly taken up by cells but fails to reach their cytosol, except when dendritic cells, which are known to take up circulating antigens for cross-presentation, are used. In addition to its usefulness in assessing cytosolic uptake, DUB processing of PTD-linked proteins can ensure the intracellular release of cargo proteins, which might prove helpful for MHC-I-based vaccination or intracellular delivery of biologically active polypeptides.
Loison
Fabien
F
UMR6026, Intracellular Protein Homeostasis, Universite de Rennes1-CNRS, Campus de Beaulieu, Bâtiment 13, 35042 Rennes Cedex, France.
Nizard
Philippe
P
Sourisseau
Tony
T
Le Goff
Pascale
P
Debure
Laure
L
Le Drean
Yves
Y
Michel
Denis
D
D000818:Animals; D015153:Blotting, Western; D002478:Cells, Cultured; D002522:Chlorocebus aethiops; D003600:Cytosol / Q000378:metabolism*; D003713:Dendritic Cells / Q000378:metabolism; D005434:Flow Cytometry; D015696:Gene Products, tat / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008856:Microscopy, Fluorescence; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D025801:Ubiquitin / Q000235:genetics / Q000378:metabolism*
D015696:Gene Products, tat; D011993:Recombinant Fusion Proteins; D025801:Ubiquitin