Sequence elements in both subunits of the DNA fragmentation factor are essential for its nuclear transport.
DNA cleavage is a biochemical hallmark of apoptosis. In humans, apoptotic DNA cleavage is executed by DNA fragmentation factor (DFF) 40. In proliferating cells DFF40 is expressed in the presence of its chaperone and inhibitor DFF45, which results in the formation of the DFF complex. Here, we present a systematic analysis of the nuclear import of the DFF complex. Our in vitro experiments demonstrate that the importin alpha/beta-heterodimer mediates the translocation of the DFF complex from the cytoplasm to the nucleus. Both DFF subunits interact directly with the importin alpha/beta-heterodimer. However, importin alpha/beta binds more tightly to the DFF complex compared with the individual subunits. Additionally, the isolated C-terminal regions of both DFF subunits together bind importin alpha/beta more strongly than the individual C termini. Our results from in vivo studies reveal that the C-terminal regions of both DFF subunits harbor nuclear localization signals. Furthermore, nuclear import of the DFF complex requires the C-terminal regions of both subunits. In more detail, one basic cluster in the C-terminal region of each subunit, DFF40 (RLKRK) and DFF45 (KRAR), is essential for nuclear accumulation of the DFF complex. Based on these findings two alternative models for the interaction of importin alpha/beta with the DFF complex are presented.
Neimanis
Sonja
S
Institut für Biochemie und Molekulare Zellbiologie, Abteilung Molekularbiologie, Universität Göttingen, Humboldtallee 23, Göttingen 37073, Germany.
Albig
Werner
W
Doenecke
Detlef
D
Kahle
Joerg
J
D021581:Active Transport, Cell Nucleus / Q000502:physiology; D017209:Apoptosis / Q000502:physiology*; D051017:Apoptosis Regulatory Proteins / Q000378:metabolism*; D002467:Cell Nucleus / Q000378:metabolism*; D003593:Cytoplasm / Q000378:metabolism; D003851:Deoxyribonucleases / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D008954:Models, Biological; D046912:Multiprotein Complexes / Q000378:metabolism*; D019913:Nuclear Localization Signals / Q000378:metabolism; D000075223:Poly-ADP-Ribose Binding Proteins; D011485:Protein Binding / Q000502:physiology; D017434:Protein Structure, Tertiary / Q000502:physiology; D028901:alpha Karyopherins / Q000378:metabolism; D028961:beta Karyopherins / Q000378:metabolism
D051017:Apoptosis Regulatory Proteins; D046912:Multiprotein Complexes; D019913:Nuclear Localization Signals; D000075223:Poly-ADP-Ribose Binding Proteins; D028901:alpha Karyopherins; D028961:beta Karyopherins; C109664:caspase-activated DNase inhibitor; C113696:DFFB protein, human; D003851:Deoxyribonucleases