Identification and Characterization of Mitochondrial Targeting Sequence of Human Apurinic/Apyrimidinic Endonuclease 1

Cancer Center, Institute of Surgery, Daping Hospital, Third Military Medical University, Chongqing 400042, China.
Journal of Biological Chemistry (Impact Factor: 4.57). 03/2010; 285(20):14871-81. DOI: 10.1074/jbc.M109.069591
Source: PubMed


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.

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    • "Bax GTPTWQTVTIFVAGVLTASLTIWKKMG Bax OMM [113] TOM MVGRNSAIAAGVCGALFIGYCIYFDRKRRSDPN TOM20 OMM [112] XL RKGQERFNRWFLTGMTVAGVVLLGSLFSRK Bcl-XL OMM [112] CCO MSVLTPLLLRGLTGSARRLPVPRAKIHSL Cyt. C Oxidase Subunit VIII IMM [112] Cox8 MSVLTPLLLRSLTGSARRLMVPRAQV Cox8 IMM [136] [137] APE1 HSLLPALCDSKIRSKALGSDHCPITLYLAL APE1 Matrix [66] cMTS APVLSDFPLLQAFKTRISNIPTIKKFL QPGSQRKPPPDGPYVEVVRTVLKF Glutathione S-Transferase 4A-4 Matrix [134] [135] MnSOD MLSRAVCGTSRQLAPALGYLGSRQ Mn-Superoxide Dismutase Matrix [66] OTC MLFNLRILLNNAAFRNGHN FMVRNFRCGQPLQNKVQ Ornithine Transcarbamylase Matrix [112] oxidative stress, they may emerge as immortalized cells and initiate tumor progression [4]. The activity of some tyrosine kinases and phosphatases depends on the levels of ROS, specifically H 2 O 2 . "
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    • "Subsequently, the molecular chaperones allow proteins destined for the mitochondria to interact with translocase in the outer membrane of the TOM complex. Three subunits of this complex (Tom20, Tom22 and Tom70) function as import receptors for proteins containing an MTS [2] [5] [8] [11] [44]. To elucidate the interaction between rpS3 and TOM receptors in the cytosol, we performed a "
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    • "Recent studies have suggested the hypothesis of the involvement of a redox-mediating folding process of APE1 that would affect its subcellular distribution (Li et al. 2010; Vascotto et al. 2011). Increased cellular redox status would result in differential interaction of APE1 with mitochondrial transport proteins increasing the mitochondrial import into mitochondria after increased oxidative stress (Li et al. 2010; Vascotto et al. 2011). "
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