Dynamic subcellular partitioning of the nucleolar transcription factor TIF-IA under ribotoxic stress.
TIF-IA is a basal transcription factor of RNA polymerase I (Pol I) that is a major target of the JNK2 signaling pathway in response to ribotoxic stress. Using advanced fluorescence microscopy and kinetic modeling we elucidated the subcellular localization of TIF-IA and its exchange dynamics between the nucleolus, nucleoplasm and cytoplasm upon ribotoxic stress. In steady state, the majority of (GFP-tagged) TIF-IA was in the cytoplasm and the nucleus, a minor portion (7%) localizing to the nucleoli. We observed a rapid shuttling of GFP-TIF-IA between the different cellular compartments with a mean residence time of approximately 130 s in the nucleus and only approximately 30 s in the nucleoli. The import rate from the cytoplasm to the nucleus was approximately 3-fold larger than the export rate, suggesting an importin/exportin-mediated transport rather than a passive diffusion. Upon ribotoxic stress, GFP-TIF-IA was released from the nucleoli with a half-time of approximately 24 min. Oxidative stress and inhibition of protein synthesis led to a relocation of GFP-TIF-IA with slower kinetics while osmotic stress had no effect. The observed relocation was much slower than the nucleo-cytoplasmic and nucleus-nucleolus exchange rates of GFP-TIF-IA, indicating a time-limiting step upstream of the JNK2 pathway. In support of this, time-course experiments on the activity of JNK2 revealed the activation of the JNK kinase as the rate-limiting step.
Szymański
Jedrzej
J
Cellular Biophysics Group, German Cancer Research Center, D-69120 Heidelberg, Germany.
Mayer
Christine
C
Hoffmann-Rohrer
Urs
U
Kalla
Claudia
C
Grummt
Ingrid
I
Weiss
Matthias
M
D002466:Cell Nucleolus / Q000502:physiology*; D002467:Cell Nucleus / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D015151:Immunoblotting; D047468:Immunoprecipitation; D048670:MAP Kinase Kinase 4 / Q000378:metabolism; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D018384:Oxidative Stress; D014176:Protein Biosynthesis / Q000187:drug effects; D012318:RNA Polymerase I / Q000235:genetics / Q000378:metabolism*; D012270:Ribosomes; D013347:Subcellular Fractions; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D014162:Transfection
D009687:Nuclear Proteins; D014157:Transcription Factors; C093517:transcriptional intermediary factor 1; D048670:MAP Kinase Kinase 4; D012318:RNA Polymerase I