Identification and characterization of the mitochondrial targeting sequence and mechanism in human citrate synthase

Tsung Lin Cheng, Ching Chun Liao, Wen Hui Tsai, Chin Chih Lin, Chin Wei Yeh, Chiao Fang Teng, Wen Tsan Chang

研究成果: Article同行評審

34 引文 斯高帕斯(Scopus)

摘要

Citrate synthase (CS), the first and rate-limiting enzyme of the tricarboxylic acid (TCA) cycle, plays a decisive role in regulating energy generation of mitochondrial respiration. Most mitochondrial proteins are synthesized in the cytoplasm as preproteins with an amino (N)-terminal mitochondrial targeting sequence (MTS) that directs mitochondria-specific sorting of the preprotein. However, the MTS and targeting mechanism of the human CS protein are not fully characterized. The human CS gene is a single nuclear gene which transcribes into two mRNA variants, isoform a (CSa) and b (CSb), by alternative splicing of exon 2. CSa encodes 466 amino acids, including a putative N-terminal MTS, while CSb expresses 400 residues with a shorter N terminus, lacking the MTS. Our results indicated that CSa is localized in the mitochondria and the N-terminal 27 amino acids, including a well-conserved RXY ↓ (S/A) motif (the RHAS sequence), can efficiently target the enhanced green fluorescent protein (EGFP) into the mitochondria. Furthermore, site-directed mutagenesis analysis of the conserved basic amino acids and serine/threonine residues revealed that the R9 residue is essential but all serine/threonine residues are dispensable in the mitochondrial targeting function. Moreover, RNA interference (RNAi)-mediated gene silencing of the preprotein import receptors, including TOM20, TOM22, and TOM70, showed that all three preprotein import receptors are required for transporting CSa into the mitochondria. In conclusion, we have experimentally identified the mitochondrial targeting sequence of human CSa and elucidated its targeting mechanism. These results provide an important basis for the study of mitochondrial dysfunction due to aberrant CSa trafficking.

原文English
頁(從 - 到)1002-1015
頁數14
期刊Journal of Cellular Biochemistry
107
發行號5
DOIs
出版狀態Published - 2009 8月 1

All Science Journal Classification (ASJC) codes

  • 生物化學
  • 分子生物學
  • 細胞生物學

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