Volume 6 issue 7 July 2005
Translocation Biosensors To Study Signal Specific Nucleo-Cytoplasmic Transport, Protease Activity And Protein-Protein Interactions
Shirley K. Knauer, Sabrina Moodt, Thorsten Berg, Urban Liebel, Rainer Pepperkok and Roland H. Stauber

SUPLEMENTARY MATERIAL:

Supplementary Methods
Plasmids
Plasmid p3-Exportin 1-HA, encoding HA-tagged Exportin 1 (7), pc3-E1B55K, encoding the Adenovirus type 5 E1B-55K protein (45), pc3-Vpr, encoding the HIV-1 Vpr protein (48), pc3-GR, encoding the human glucocorticoid receptor alpha (48), pc3-PKI-BFP, encoding a PKI-BFP fusion (7), pc3 eIF-5A, encoding the eukaryotic initiation factor 5A (eIF-5A) (45), pc3p53, encoding human p53 (45), pc3mdm2, encoding the human mdm2 protein (45), pc3-E1BAP5, encoding the human E1B-AP5 protein (45), p3CANc/VSV-G, encoding the carboxy terminus of CAN/Nup214 (7) have been described. Plasmid pBrev14-GFP produces a cytoplasmic Rev-GFP mutant (Rev14-GFP) (23). pSTAT1-GFP encodes a human STAT1-GFP fusion (4). pDS-RED-N1 encodes the red-fluorescent protein (RFP) (BD Biosciences). pBABE confers puromycin resistance. pCaspase3-Sensor (BD Biosciences) encodes a fusion protein composed of the mitogen-activated kinase kinase (MAPKK) NES (26), followed by CS3, GFP and three copies of the SV40 NLS. pHA-CMV-bFosGC (FosGC) and pFLAG-CMV2-bJunYN (JunGN), encoding the IDs of human Fos and Jun fused to a C- (aa 155-238) or N-terminal (aa 1-154) fragment of GFP, respectively, have been described (12).

Supplementary Online Tables

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Supplementary Figure S1
Application of transport biosensors to identify compounds or proteins that interfere with nucleo-cytoplasmic transport. Expression of the RevNES sensor was induced by Dox and (A) cells were incubated with taxol or nocodazol or (B) transfected with the PKI-BFP or the CanC expression plasmids (3 µg each) together with 0.3 µg of the RFP expression plasmid and analyzed by fluorescence microscopy. (A) Inhibition of microtubule metabolism resulted in cell rounding but did not block nuclear export (upper panel) nor interfered with nuclear import (lower panel). (B) Overexpression of PKI-BFP or CanC inhibited nuclear export and resulted in the nuclear accumulation of the RevNES-sensor. Scale bars, 10 µm.

View Fig. S1 (.tif)

Supplementary Figure S2
Nuclear translocation of the biosensors as the principle for cell based screening applications. The cellular biosensors are composed of GST, GFP and rational combinations of nuclear import and export signals. Addition of regulatory sequences resulted in three classes of biosensors applicable for the identification of signal specific nuclear transport inhibitors, small molecules that interfere with protease activity and compounds that modulate protein-protein interactions in living cells. Nuclear accumulation of the cytoplasmic biosensors serves as the indicator, which can be induced by interference with nuclear export, induction of protease activity or formation of highly specific protein complexes.

View Fig. S2 (.tif)