Identification and characterization of the mitochondrial targeting sequence and mechanism in human citrate synthase.
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 downward arrow (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.
Cheng
Tsung-Lin
TL
Institute of Basic Medical Sciences, National Cheng Kung University Medical College, Tainan, Taiwan 701, ROC.
Liao
Ching-Chun
CC
Tsai
Wen-Hui
WH
Lin
Chin-Chih
CC
Yeh
Chin-Wei
CW
Teng
Chiao-Fang
CF
Chang
Wen-Tsan
WT
D000595:Amino Acid Sequence; D001483:Base Sequence; D002460:Cell Line; D002950:Citrate (si)-Synthase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017124:Conserved Sequence; D015971:Gene Expression Regulation, Enzymologic; D055785:Gene Knockdown Techniques; D006801:Humans; D042541:Intracellular Space / Q000201:enzymology; D008928:Mitochondria / Q000378:metabolism*; D024101:Mitochondrial Proteins / Q000378:metabolism; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011498:Protein Precursors / Q000378:metabolism; D021382:Protein Sorting Signals; D021381:Protein Transport; D034741:RNA, Small Interfering / Q000378:metabolism
D024101:Mitochondrial Proteins; D050505:Mutant Proteins; D011498:Protein Precursors; D021382:Protein Sorting Signals; D034741:RNA, Small Interfering; D002950:Citrate (si)-Synthase