Modulation of nucleocytoplasmic trafficking by retention in cytoplasm or nucleus.
Nuclear protein transport processes have largely been studied using in vitro semi-intact cell systems where high concentrations of nuclear localizing substrates are used, and cytoplasmic components such as the microtubule (MT) network, are either absent or damaged. Here we use the fluorescence recovery after photobleaching (FRAP) technique to analyze the nucleocytoplasmic flux of distinct fluorescently tagged proteins over time in living cultured cells. FRAP was performed in different parts of the cell to analyze the kinetics of nucleocytoplasmic trafficking and intranuclear/cytoplasmic mobility of the tumor suppressor Rb protein and a SV40 large tumor antigen (T-ag) derivative containing the nuclear localization sequence (NLS), both fused to green fluorescent protein (GFP). The results indicate that proteins carrying the T-ag NLS are highly mobile in the nucleus and cytoplasm. Rb, in contrast, is largely immobile in both cellular compartments, with similar nuclear import and export kinetics. Rb nuclear export was CRM-1-mediated, with its reduced mobility in the cytoplasm in part due to association with MTs. Overall our results show that nuclear and cytoplasm retention modulates the rates of nuclear protein import and export in intact cells.
Roth
Daniela M
DM
Nuclear Signalling Laboratory, Department of Biochemistry and Molecular Biology, Monash University, Monash, Victoria 3800, Australia.
Harper
Ian
I
Pouton
Colin W
CW
Jans
David A
DA
D021581:Active Transport, Cell Nucleus; D000818:Animals; D000952:Antigens, Polyomavirus Transforming / Q000378:metabolism; D019556:COS Cells; D002467:Cell Nucleus / Q000378:metabolism*; D002522:Chlorocebus aethiops; D003593:Cytoplasm / Q000378:metabolism*; D028884:Karyopherins / Q000378:metabolism; D007700:Kinetics; D019913:Nuclear Localization Signals; D018160:Receptors, Cytoplasmic and Nuclear / Q000378:metabolism; D016160:Retinoblastoma Protein / Q000378:metabolism; D014162:Transfection; D000097606:Exportin 1 Protein
D000952:Antigens, Polyomavirus Transforming; D028884:Karyopherins; D019913:Nuclear Localization Signals; D018160:Receptors, Cytoplasmic and Nuclear; D016160:Retinoblastoma Protein; D000097606:Exportin 1 Protein