Identification and characterization of mitochondrial targeting sequence of human apurinic/apyrimidinic endonuclease 1.
Dually targeted mitochondrial proteins usually possess an unconventional mitochondrial targeting sequence (MTS), which makes them difficult to predict by current bioinformatics approaches. Human apurinic/apyrimidinic endonuclease (APE1) plays a central role in the cellular response to oxidative stress. It is a dually targeted protein preferentially residing in the nucleus with conditional distribution in the mitochondria. However, the mitochondrial translocation mechanism of APE1 is not well characterized because it harbors an unconventional MTS that is difficult to predict by bioinformatics analysis. Two experimental approaches were combined in this study to identify the MTS of APE1. First, the interactions between the peptides from APE1 and the three purified translocase receptors of the outer mitochondrial membrane (Tom) were evaluated using a peptide array screen. Consequently, the intracellular distribution of green fluorescent protein-tagged, truncated, or mutated APE1 proteins was traced by tag detection. The results demonstrated that the only MTS of APE1 is harbored within residues 289-318 in the C terminus, which is normally masked by the intact N-terminal structure. As a dually targeted mitochondrial protein, APE1 possesses a special distribution pattern of different subcellular targeting signals, the identification of which sheds light on future prediction of MTSs.
Cancer Center, Cancer Center, Third Military Medical University, Chongqing 400042, China.
Mengxia
M
Li
Cancer Center, Cancer Center, Third Military Medical University, Chongqing 400042, China.
Zhaoyang
Z
Zhong
Cancer Center, Cancer Center, Third Military Medical University, Chongqing 400042, China.
Jianwu
J
Zhu
Department of Pathology of Research, Institute of Surgery, Daping Hospital, Third Military Medical University, Chongqing 400042, China.
Debing
D
Xiang
Cancer Center, Cancer Center, Third Military Medical University, Chongqing 400042, China.
Nan
N
Dai
Cancer Center, Cancer Center, Third Military Medical University, Chongqing 400042, China.
Xiaojing
X
Cao
Cancer Center, Cancer Center, Third Military Medical University, Chongqing 400042, China.
Yi
Y
Qing
Cancer Center, Cancer Center, Third Military Medical University, Chongqing 400042, China.
Zhenzhou
Z
Yang
Cancer Center, Cancer Center, Third Military Medical University, Chongqing 400042, China.
Jiayin
J
Xie
Department of Pathology of Research, Institute of Surgery, Daping Hospital, Third Military Medical University, Chongqing 400042, China.
Zengpeng
Z
Li
Department of Biology, University of Dallas, Irving, Texas 75062.
Laura
L
Baugh
Cancer Center, Cancer Center, Third Military Medical University, Chongqing 400042, China.
Ge
G
Wang
Cancer Center, Cancer Center, Third Military Medical University, Chongqing 400042, China. Electronic address: dongwang64@hotmail.com.
Dong
D
Wang
D000595:Amino Acid Sequence; D015153:Blotting, Western; D002846:Chromatography, Affinity; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D006367:HeLa Cells; D006801:Humans; D018613:Microscopy, Confocal; D008928:Mitochondria / Q000378:metabolism*; D013347:Subcellular Fractions / Q000201:enzymology; D013379:Substrate Specificity
D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase