Identification and characterization of the mitochondrial targeting sequence and mechanism in human citrate synthase
- 28 May 2009
- journal article
- research article
- Published by Wiley in Journal of Cellular Biochemistry
- Vol. 107 (5), 1002-1015
- https://doi.org/10.1002/jcb.22200
Abstract
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. J. Cell. Biochem. 107: 1002–1015, 2009.Keywords
This publication has 44 references indexed in Scilit:
- Living with death: the evolution of the mitochondrial pathway of apoptosis in animalsCell Death & Differentiation, 2008
- A novel siRNA validation system for functional screening and identification of effective RNAi probes in mammalian cellsBiochemical and Biophysical Research Communications, 2006
- Integrative Analysis of the Mitochondrial Proteome in YeastPLoS Biology, 2004
- Molecular Chaperones Hsp90 and Hsp70 Deliver Preproteins to the Mitochondrial Import Receptor Tom70Cell, 2003
- A System for Stable Expression of Short Interfering RNAs in Mammalian CellsScience, 2002
- Identification of a Mammalian Homologue of the Fungal Tom70 Mitochondrial Precursor Protein Import Receptor as a Thyroid Mitochondrial Precursor Protein Import Receptor as a Thyroid Hormone‐Regulated Gene in Specific Brain RegionsJournal of Neurochemistry, 1999
- A Receptor for the Import of Proteins into Human MitochondriaJBIC Journal of Biological Inorganic Chemistry, 1996
- A human homolog of the mitochondrial protein import receptor Mom19 can assemble with the yeast mitochondrial receptor complexFEBS Letters, 1995
- Identification of the human mitochondrial protein import receptor, huMas20p. Complementation of Δmas20 in yeastFEBS Letters, 1995
- The cleavable prepiece of an imported mitochondrial protein is sufficient to direct cytosolic dihydrofolate reductase into the mitochondrial matrixFEBS Letters, 1984