Different dynamics in nuclear entry of subunits of the repair/transcription factor TFIIH.
We report here the different ways in which four subunits of the basal transcription/repair factor TFIIH (XPB, XPD, p62 and p44) and the damage recognition XPC repair protein can enter the nucleus. We examined their nuclear localization by transiently expressing the gene products tagged with the enhanced green fluorescent protein (EGFP) in transfected 3T3 cells. In agreement with the identification of more than one putative nuclear localization signal (NLS) in their protein sequences, XPB, XPC, p62 and p44 chimeras were rapidly sorted to the nucleus. In contrast, the XPD-EGFP chimeras appeared mainly localized in the cytoplasm, with a minor fraction of transfectants showing the EGFP-based fluorescence also in the nucleus. The ability of the XPD chimeras to enter the nucleus was confirmed by western blotting on fractionated cell extracts and by functional complementation of the repair defect in the UV5 rodent cells, mutated in the XPD homologous gene. By deletion mutagenesis, we were unable to identify any sequence specific for nuclear localization. In particular, deletion of the putative NLS failed to affect subcellular localization and, conversely, the C-terminal part of XPD containing the putative NLS showed no specific nuclear accumulation. These findings suggest that the nuclear entry of XPD depends on its complexation with other proteins in the cytoplasm, possibly other components of the TFIIH complex.
Santagati
F
F
Istituto di Genetica Biochimica ed Evoluzionistica del CNR, Via Abbiategrasso 207, 27100 Pavia, Italy.
Botta
E
E
Stefanini
M
M
Pedrini
A M
AM
D016475:3T3 Cells; D000818:Animals; D001692:Biological Transport; D002467:Cell Nucleus / Q000378:metabolism*; D002470:Cell Survival / Q000235:genetics / Q000528:radiation effects; D004265:DNA Helicases; D004260:DNA Repair; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D049452:Green Fluorescent Proteins; D006367:HeLa Cells; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008856:Microscopy, Fluorescence; D021122:Protein Subunits; D011506:Proteins / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D051758:Transcription Factor TFIIH; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D019473:Transcription Factors, TFII; D014162:Transfection; D051759:Xeroderma Pigmentosum Group D Protein
D004268:DNA-Binding Proteins; D008164:Luminescent Proteins; D021122:Protein Subunits; D011506:Proteins; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D019473:Transcription Factors, TFII; C501002:Xpc protein, mouse; D049452:Green Fluorescent Proteins; D051758:Transcription Factor TFIIH; C086984:XPC protein, human; D004265:DNA Helicases; D051759:Xeroderma Pigmentosum Group D Protein; C497292:ERCC2 protein, human; C497293:Ercc2 protein, mouse