Sequence Elements in Both Subunits of the DNA Fragmentation Factor Are Essential for Its Nuclear Transport

Abstract

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 ␣/␤-heterodimer mediates the translocation of the DFF complex from the cytoplasm to the nucleus. Both DFF subunits interact directly with the importin ␣/␤-heterodimer. However, importin ␣/␤ 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 ␣/␤ 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 ␣/␤ with the DFF complex are presented.Apoptosis is an evolutionarily conserved process that plays an important role in development and tissue homeostasis (1). Apoptotic signaling is ultimately transduced by a family of cysteine proteases called caspases, which are the central executioners of apoptosis (2). One of the biochemical hallmarks of apoptosis is nucleosomal DNA fragmentation (3) mainly caused by the DNA fragmentation factor 40 (DFF40, 2 also known as caspase-activated DNase, CAD) (reviewed in Ref. 4). In proliferating cells DFF40 is expressed in the presence of DFF45 (inhibitor of CAD), which has a dual role as chaperone and as inhibitor of DFF40 (5, 6). During apoptosis active DFF40 is released from the DFF complex due to cleavage of DFF45 executed by caspase-3 and caspase-7 (7, 8). A structural feature of both DFF40 and DFF45 is the N-terminal cell death-inducing DFF45-like effector domain that mediates the interaction between DFF40 and DFF45 (9 -11). Recently, it was shown that the DFF complex is composed of two DFF40 and two DFF45 subunits (12). The tetrameric DFF complex is located in the nucleus (13,14), and sequence analysis revealed two putative nuclear localization signals (NLSs). According to this prediction, the C-terminal portion of murine DFF40 contains a monopartite NLS, and the C-terminal sequences of murine and human DFF45 exhibit a bipartite NLS (5, 14). Although previous studies showed that both C-terminal regions are involved in nuclear translocation of the DFF complex (13, 14), the nuclear transport mechanism has not been characterized so far.The nuclear transport of proteins harboring a NLS in their amino acid sequence is mediated by soluble nuclear import receptors also known as importins or karyopherins. NLSs ca...

Roles of the Major Apoptotic Nuclease-DNA Fragmentation Factor-in Biology and Disease

“…It has been shown recently that nuclear import of DFF depends on the importin a/b heterodimer. Efficient binding to the importin heterodimer and nuclear import of DFF requires the presence of the NLS localized in the Ctermini of both DFF40 and DFF45 [45]. Three major chromatin proteins were the first factors identified that stimulate DNA cleavage by DFF: histone H1, HMGB1/2 and topoisomerase II [14,15,33,38].…”

“…Although DFF was initially purified from cytoplasmic extracts [13,16], the nuclease actually resides in the nucleus of normal healthy cells [14,[42][43][44]; the presence of DFF in cytoplasmic extracts results from artifactual "leakage" of nuclear proteins during cell fractionation [39,42]. Both DFF40 and DFF45 each possess their own nuclear localization sequence (NLS) located in their C-termini [42][43][44] and these two separate NLSs synergize for targeting DFF to the nucleus [45]. DFF35 resides in the cytoplasm because of a splicedout NLS in its C-terminus [16,43].…”

Oligosaccharide‐Mediated Nuclear Transport of Nanoparticles

“…Digitonin permeabilization has been used often in the study of biochemical processes that are related to the import and export of nuclear proteins. [14][15][16] Although live cells become semi-intact upon digitonin treatment due to the leakage of cytoplasmic proteins through the plasma membrane, the nuclear membrane is left intact. Import buffer (pH 7.3, 20 mm HEPES, 110 mm KOAc, 5 mm NaOAc, 2 mm MgA C H T U N G T R E N N U N G (OAc) 2 , 0.5 mm EGTA) that contained the sugar-displaying CdTe QDs (0.6 mg mL À1 ,…”

Histone H1 subtype preferences of DFF40 and possible nuclear localization of DFF40/45 in normal and trichostatin A-treated NB4 leukemic cells

“…Each subunit possesses its own nuclear localization sequence (NLS) but DFF40 cannot enter the nucleus alone. It needs DFF45 for proper folding so that the NLS of DFF40 may be exposed [12].…”