Carrier-independent nuclear import of the transcription factor PU.1 via RanGTP-stimulated binding to Nup153.
PU.1 is a transcription factor of the Ets family with important functions in hematopoietic cell differentiation. Using green fluorescent protein-PU.1 fusions, we show that the Ets DNA binding domain of PU.1 is necessary and sufficient for its nuclear localization. Fluorescence and ultrastructural nuclear import assays showed that PU.1 nuclear import requires energy but not soluble carriers. PU.1 interacted directly with two nucleoporins, Nup62 and Nup153. The binding of PU.1 to Nup153, but not to Nup62, increased dramatically in the presence of RanGMPPNP, indicating the formation of a PU.1.RanGTP.Nup153 complex. The Ets domain accounted for the bulk of the interaction of PU.1 with Nup153 and RanGMPPNP. Because Nup62 is located close to the midplane of the nuclear pore complex whereas Nup153 is at its nuclear side, these findings suggest a model whereby RanGTP propels PU.1 toward the nuclear side of the nuclear pore complex by increasing its affinity for Nup153. This notion was confirmed by ultrastructural studies using gold-labeled PU.1 in permeabilized cells.
Zhong
Hualin
H
Laboratory of Cell Biology, Howard Hughes Medical Institute, New York, New York 10031, USA.
Takeda
Akiko
A
Nazari
Reza
R
Shio
Helen
H
Blobel
Günter
G
Yaseen
Nabeel R
NR
D021581:Active Transport, Cell Nucleus; D001692:Biological Transport; D002467:Cell Nucleus / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D007150:Immunohistochemistry; D008854:Microscopy, Electron; D008856:Microscopy, Fluorescence; D028861:Nuclear Pore Complex Proteins / Q000737:chemistry*; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D014176:Protein Biosynthesis; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D011518:Proto-Oncogene Proteins / Q000737:chemistry*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D015534:Trans-Activators / Q000737:chemistry*; D014157:Transcription Factors / Q000378:metabolism; D014162:Transfection; D020931:ran GTP-Binding Protein / Q000737:chemistry*; D000099232:Proto-Oncogene Protein Spi-1
D049452:Green Fluorescent Proteins; D028861:Nuclear Pore Complex Proteins; D011518:Proto-Oncogene Proteins; D011994:Recombinant Proteins; D015534:Trans-Activators; D014157:Transcription Factors; D020931:ran GTP-Binding Protein; D000099232:Proto-Oncogene Protein Spi-1; C079377:NUP153 protein, human