Analysis of physiological meanings of mitochondrial localization of yeast tRNA splicing endonuclease

Research Project

Project/Area Number	12680609
Research Category	Grant-in-Aid for Scientific Research (C)
Allocation Type	Single-year Grants
Section	一般
Research Field	Structural biochemistry
Research Institution	Nagoya University
Principal Investigator	YOSHIHI Tbhn Nagova Univ. Res., Ctr. Of Material Science,Assoc, Prof, 物質科学国際研究センター, 助教授 (60212312)
Project Period (FY)	2000 – 2001
Project Status	Completed (Fiscal Year 2001)
Budget Amount *help	¥1,100,000 (Direct Cost: ¥1,100,000) Fiscal Year 2001: ¥1,100,000 (Direct Cost: ¥1,100,000)
Keywords	tRNA / splicing / nuclear-cytoplasmic transpo / endonuclease / mitochondria
Research Abstract	Primary transcripts of nuclear-encoded tRNA genes receive various processing to become mature tRNA functional for translation in cytoplasm. Among such processing, splicing is essential for maturation of intron-containing pre-RNAs.The splicing of pre-tRNAs has been believed to occur in the nuclei. In yeast, Saccharomyces cerevisiae, tRNA splicing endonuclease that cleaves exon-intron junctions of pre tRNAs consists offour subunits, Sen54p, Sen2p, Sen34p and SenlSp, and has been supposed as an integral membrane protein of inner uclear envelope. Through this research project, we show that the majority of Sen2p, Sen54p and the endonuclease activity are not localized in the nuclei but associated on the surface of mitochondria. Yeast mutant cells deficient in the endonuclease activity accumulate unspliced pre-tRNAs in cytosol. A Sen54p derivative artificially fixed on the mitochondrial surface by fusing a itochondrial targeting signal and a transmembrane domain ofTom70p (Tom70N), an integral membrane protein of outer mitochondrial membrane, can functionally replace authentic Sen54p. On the other hand, Sen54p derivatives with nuclear localization signals are not fully active. Partial deletion of the mitochondrial targeting signal in Sen54p compromises its function, but addition of Tom70N to this deletion mutant suppresses its defect. These results strongly suggest that an early step of tRNA splicing is not a nuclear event and that pre-tRNAs take a complex route via the mitochondrial surface during their maturation.