Human APE2 protein is mostly localized in the nuclei and to some extent in the mitochondria, while nuclear APE2 is partly associated with proliferating cell nuclear antigen.
In human cells APE1 is the major AP endonuclease and it has been reported to have no functional mitochondrial targeting sequence (MTS). We found that APE2 protein possesses a putative MTS. When its N-terminal 15 amino acid residues were fused to the N-terminus of green fluorescent protein and transiently expressed in HeLa cells the fusion protein was localized in the mitochondria. By electron microscopic immunocytochemistry we detected authentic APE2 protein in mitochondria from HeLa cells. Western blotting of the subcellular fraction of HeLa cells revealed most of the APE2 protein to be localized in the nuclei. We found a putative proliferating cell nuclear antigen (PCNA)-binding motif in the C-terminal region of APE2 and showed this motif to be functional by immunoprecipitation and in vitro pull-down binding assays. Laser scanning immunofluorescence microscopy of HeLa cells demonstrated both APE2 and PCNA to form foci in the nucleus and also to be co-localized in some of the foci. The incubation of HeLa cells in HAT medium containing deoxyuridine significantly increased the number of foci in which both molecules were co-localized. Our results suggest that APE2 participates in both nuclear and mitochondrial BER and also that nuclear APE2 functions in the PCNA-dependent BER pathway.
Tsuchimoto
D
D
Department of Biochemistry, Medical Institute of Bioregulation, Kyushu University, Fukuoka 812-8582, Japan.
Sakai
Y
Y
Sakumi
K
K
Nishioka
K
K
Sasaki
M
M
Fujiwara
T
T
Nakabeppu
Y
Y
D000595:Amino Acid Sequence; D001665:Binding Sites; D001692:Biological Transport / Q000235:genetics; D019757:Carbon-Oxygen Lyases / Q000235:genetics / Q000378:metabolism*; D002467:Cell Nucleus / Q000201:enzymology*; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase; D043223:Deoxyribonuclease IV (Phage T4-Induced); D015971:Gene Expression Regulation, Enzymologic; D049452:Green Fluorescent Proteins; D006367:HeLa Cells; D006801:Humans; D019169:Jurkat Cells; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D018613:Microscopy, Confocal; D008928:Mitochondria / Q000201:enzymology*; D008969:Molecular Sequence Data; D018809:Proliferating Cell Nuclear Antigen / Q000378:metabolism*; D011485:Protein Binding; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D014018:Tissue Distribution
D018076:DNA, Complementary; D008164:Luminescent Proteins; D018809:Proliferating Cell Nuclear Antigen; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D043223:Deoxyribonuclease IV (Phage T4-Induced); D019757:Carbon-Oxygen Lyases; C085108:APEX1 protein, human; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase