Signal-dependent translocation of simian virus 40 large-T antigen into rat liver nuclei in a cell-free system.
An in vitro nuclear translocation system is described in which isolated rat liver nuclei were incubated in a defined buffered medium containing radiolabeled or fluorescently labeled exogenous proteins. The nuclei were rapidly recovered, extracted, and analyzed for the presence of associated radiolabeled or fluorescently labeled proteins. The isolated nuclei exhibited the same specificity for protein uptake as seen previously in vivo, accumulating simian virus 40 wild-type large-T antigen and p53 while excluding a cytoplasmic variant of large-T antigen (d10) and bovine serum albumin. The rapid nuclear accumulation of wild-type large-T antigen was shown to be selective and dependent upon the recognition of a wild-type nuclear location signal, ATP and temperature dependent, and unidirectional. Taken together, the data suggest that in our in vitro system the nuclear translocation of wild-type large-T antigen exhibits some of the characteristics of an active transport process.
Markland
W
W
Integrated Genetics, Framingham, Massachusetts 01701.
Smith
A E
AE
Roberts
B L
BL
D000818:Animals; D000952:Antigens, Polyomavirus Transforming / Q000302:isolation & purification; D001693:Biological Transport, Active; D002467:Cell Nucleus / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D005455:Fluorescent Antibody Technique; D005456:Fluorescent Dyes; D007163:Immunosorbent Techniques; D008099:Liver / Q000648:ultrastructure*; D008297:Male; D051379:Mice; D008970:Molecular Weight; D021382:Protein Sorting Signals / Q000502:physiology*; D011770:Pyruvate Kinase / Q000378:metabolism; D051381:Rats; D011919:Rats, Inbred Strains; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012235:Rhodamines; D013539:Simian virus 40 / Q000276:immunology*; D013462:Sulfur Radioisotopes
D000952:Antigens, Polyomavirus Transforming; D005456:Fluorescent Dyes; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D012235:Rhodamines; D013462:Sulfur Radioisotopes; C027755:rhodamine isothiocyanate; D011770:Pyruvate Kinase