Analysis of nuclear transport signals in the human apurinic/apyrimidinic endonuclease (APE1/Ref1)

Abstract

The mammalian abasic-endonuclease1/redox-factor1 (APE1/Ref1) is an essential protein whose subcellular distribution depends on the cellular physiological status. However, its nuclear localization signals have not been studied in detail. We examined nuclear translocation of APE1, by monitoring enhanced green fluorescent protein (EGFP) fused to APE1. APE1's nuclear localization was significantly decreased by deleting 20 amino acid residues from its N-terminus. Fusion of APE1's N-terminal 20 residues directed nuclear localization of EGFP. An APE1 mutant lacking the seven N-terminal residues (ND7 APE1) showed nearly normal nuclear localization, which was drastically reduced when the deletion was combined with the E12A/D13A double mutation. On the other hand, nearly normal nuclear localization of the full-length E12A/D13A mutant suggests that the first 7 residues and residues 8–13 can independently promote nuclear import. Both far-western analyses and immuno-pull-down assays indicate interaction of APE1 with karyopherin alpha 1 and 2, which requires the 20 N-terminal residues and implicates nuclear importins in APE1's nuclear translocation. Nuclear accumulation of the ND7 APE1(E12A/D13A) mutant after treatment with the nuclear export inhibitor leptomycin B suggests the presence of a previously unidentified nuclear export signal, and the subcellular distribution of APE1 may be regulated by both nuclear import and export.

Cytoplasmic Localization and Redox Cysteine Residue of APE1/Ref-1 Are Associated with Its Anti-Inflammatory Activity in Cultured Endothelial Cells

“…Some evidence suggests that APE1/Ref-1 expression and subcellular localization are finely tuned. The subcellular distribution of APE1/Ref-1 may be regulated by both nuclear import and export systems (Jackson et al, 2005). APE1/Ref-1 undergoes active shuttling between the cytoplasm and nucleus in response to oxidative (Angkeow et al, 2002;Bhakat et al, 2003;Tell et al, 2000a) and nitrosative stress (Qu et al, 2007).…”

“…Specifically, the N-terminal region contains the redox regulatory domain characterized by 2 critical cysteine residues, C65 and C93 (Xanthoudakis et al, 1992;1994). Nuclear localization of APE1/Ref-1 is controlled by the first 20-35 amino acids at the N-terminal sequence (Jackson et al, 2005;Jeon et al, 2004). It also has been reported that nitrosation of APE1/Ref-1 leads to cytoplasmic localization in a chromosome region maintenance 1 (CRM1)-independent process (Qu et al, 2007).…”

Early steps in the DNA base excision/single-strand interruption repair pathway in mammalian cells

“…However, we have also shown that the extract of mitochondria (containing processing peptidases) but not of nuclei or cytosol, cleaves recombinant hAPE1 to generate an mtAPE-sized product, implying that the removal of NLS is necessary for mitochondrial import of APE. At the same time, deletion mapping of hAPE1 helped us identify a bipartite NLS as well as a nuclear export signal in the N-terminal segment of the APE1 polypeptide [110].…”

“…This segment contains the nuclear localization signal (NLS) [110] and is also required for the transcriptional regulation function unique to APE1 and unrelated to BER [111,112]. We recently discovered that human APE1 (hAPE1) is acetylated in vivo at Lys 6 / Lys 7 which modulates its regulatory but not endonuclease activity [113].…”

Emerging Roles of the Nucleolus in Regulating the DNA Damage Response: The Noncanonical DNA Repair Enzyme APE1/Ref-1 as a Paradigmatical Example

“…Three functionally independent domains can be distinguished within the APE1 protein ( Fig. 5): (i) the first 33-35 amino acid region consists of a structurally disordered segment (133) essential for the interaction with other proteins (148,149) and harboring sites for PTMs (14,24,70,89,159,165,166) and RNA interaction (89); (ii) the redox domain is located in a region between amino acids 35 and 127; and (iii) the DNA repair domain, which spans the C-terminal domain of the protein from about residue 61 onwards (42,68). Whereas the APE1 C-terminal domain involved in AP endonuclease activity is conserved from bacteria to humans, the N-terminal region is unique to mammals suggesting a recent acquisition during evolution.…”