Import of nuclear deoxyribonucleic acid coded lysine-accepting transfer ribonucleic acid (anticodon C-U-U) into yeast mitochondria

Martin, Robert P.; Schneller, J.M.; Stahl, A; Dirheimer, G.

doi:10.1021/bi00588a021

Cited by 140 publications

(94 citation statements)

References 27 publications

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“…The import was carried out at 25-30°C for 20 min. Mitochondria were then treated with a mixture of RNases [3]. Mitoplasts were generated by hypotonic shock (10 mM HEPES-KOH, pH 6.8, l0 min at 0°C) and purified after addition of sorbitol to 0.44 M. Mitoplasts were lysed in 1% SDS, 0.1 M sodium acetate (pH 4.8) and 0.05% diethyl pyrocarbonate at 60°C and mitochondrial RNA was phenol-extracted at the same temperature.…”

Section: Methodsmentioning

confidence: 99%

“…This contrasts with the high efficiency of in vitro import of mitochondrial pre-proteins [13]. On the other hand, in vivo tRK1 is unequally distributed between the cytosol and mitochondria, only 2-3% being associated with the mitochondrial matrix [2,3]. Such an unequal compartmentalization is not unique for tRK1.…”

Section: Lysmentioning

confidence: 95%

“…This amount represents only 0.16% of the tRK1 molecules added to the import assay. The in vivo mitochondrial content of tRK1 can be estimated to be around 2.5 pmol per mg MP ( [2,3], I.A. Tarassov).…”

Section: Optimization Of the In Vitro Import Reaction Of Trk1mentioning

confidence: 99%

“…Various sets of cytosolic tRNAs are imported in different organisms; they range from a single tRNA in yeast [2,3] to the all of mitochondriaassociated tRNAs in trypanosomatids [4,5]. The recent development of in vitro and in vivo tRNA import test systems provided direct evidence for the existence of a mitochondrial tRNA import pathway [6][7][8][9][10].…”

Section: Introductionmentioning

confidence: 99%

“…To study these mechanisms, we have set up a specific in vitro import assay using isolated mitochondria of S. cerevisiae [7]. In this organism, a single Lys nuclear-coded tRNA, the tRNAcut: (tRK1) is in part associated with the mitochondrial matrix [2,3]. The mitochondrial concentration of tRK1 is comparable to that of minor native mitochondrial tRNAs.…”

Section: Introductionmentioning

confidence: 99%

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Mitochondrial import of a yeast cytoplasmic tRNA^Lys: possible roles of aminoacylation and modified nucleosides in subcellular partitioning

et al. 1996

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The yeast tRNAcu U is transcribed from a nuclear gene and then unequally redistributed between the cytosol (97-98%) and mitochondria (2-3%). We have optimized the conditions for its specific import into isolated mitochondria. However, only a minor fraction (about 0.5%) of the added tRNA was translocated into the organelles. An in vitro transcript, once aminoacylated, appeared to be a better import substrate than the natural tRNA which carries modified nucleosides. The tRNA is translocated across mitochondrial membranes in its aminoacylated form and remains relatively stable inside the organelle. Possible roles of aminoacylation, tRNA-protein interactions and nucleoside modification in subcellular partitioning of the tRNA are discussed. Lys

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Section: Methodsmentioning

confidence: 99%

Section: Lysmentioning

confidence: 95%

Section: Optimization Of the In Vitro Import Reaction Of Trk1mentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

Mitochondrial import of a yeast cytoplasmic tRNA^Lys: possible roles of aminoacylation and modified nucleosides in subcellular partitioning

et al. 1996

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In vivo import of a normal or mutagenized heterologous transfer RNA into the mitochondria of transgenic plants: towards novel ways of influencing mitochondrial gene expression?

et al. 1992

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Evidence that nuclear‐encoded RNAs are present inside mitochondria has been reported from a wide variety of organisms, and is presumed to be due to import of specific cytosolic RNAs. In plants, the first examples were the mitochondrial leucine transfer RNAs of bean. In all cases, the evidence is circumstantial, based on hybridization of the mitochondrial RNAs to nuclear and not mitochondrial DNA. Here we show that transgenic potato plants carrying a leucine tRNA gene from bean nuclear DNA contain RNA transcribed from the introduced gene both in the cytosol and inside mitochondria, providing proof that the mitochondrial leucine tRNA is derived from a nuclear gene and imported into the mitochondria. The same bean gene carrying a 4 bp insertion in the anticodon loop was also expressed in transgenic potato plants and the transcript found to be present inside mitochondria, suggesting that this natural RNA import system could eventually be used to introduce foreign RNA sequences into mitochondria.

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Mitochondrial import of a cytoplasmic lysine-tRNA in yeast is mediated by cooperation of cytoplasmic and mitochondrial lysyl-tRNA synthetases.

1995

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Cytoplasmic tRNA(Lys)CUU is the only nuclear‐encoded tRNA of Saccharomyces cerevisiae found to be associated with mitochondria. Selective import of this tRNA into isolated organelles requires cytoplasmic factors. Here we identify two of these factors as the cytoplasmic and mitochondrial lysyl‐tRNA synthetases. The cytoplasmic enzyme is obligatory for in vitro import of the deacylated, but not of the aminoacylated tRNA. We thus infer that it is needed for aminoacylation of the tRNA, which is a prerequisite for its import. The mitochondrial synthetase, which cannot aminoacylate tRN(Lys)CUU, is required for import of both aminoacylated and deacylated forms. Its depletion leads to a total arrest of tRNA import, in vitro and in vivo. The mitochondrial lysyl‐tRNA synthetase is able to form specific and stable RNP complexes with the amino‐acylated tRNA. Furthermore, an N‐terminal truncated form of the synthetase which cannot be targeted into mitochondria is unable to direct the import of the tRNA. We therefore hypothesize that the cytosolic precursor form of the mitochondrial synthetase has a carrier function for translocation of the tRNA across the mitochondrial membranes. However, cooperation of the two synthetases is not sufficient to direct tRNA import, suggesting the need of additional factor(s).

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Import of nuclear deoxyribonucleic acid coded lysine-accepting transfer ribonucleic acid (anticodon C-U-U) into yeast mitochondria

Cited by 140 publications

References 27 publications

Mitochondrial import of a yeast cytoplasmic tRNA^Lys: possible roles of aminoacylation and modified nucleosides in subcellular partitioning

Mitochondrial import of a yeast cytoplasmic tRNA^Lys: possible roles of aminoacylation and modified nucleosides in subcellular partitioning

In vivo import of a normal or mutagenized heterologous transfer RNA into the mitochondria of transgenic plants: towards novel ways of influencing mitochondrial gene expression?

Mitochondrial import of a cytoplasmic lysine-tRNA in yeast is mediated by cooperation of cytoplasmic and mitochondrial lysyl-tRNA synthetases.

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Import of nuclear deoxyribonucleic acid coded lysine-accepting transfer ribonucleic acid (anticodon C-U-U) into yeast mitochondria

Cited by 140 publications

References 27 publications

Mitochondrial import of a yeast cytoplasmic tRNALys: possible roles of aminoacylation and modified nucleosides in subcellular partitioning

Mitochondrial import of a yeast cytoplasmic tRNALys: possible roles of aminoacylation and modified nucleosides in subcellular partitioning

In vivo import of a normal or mutagenized heterologous transfer RNA into the mitochondria of transgenic plants: towards novel ways of influencing mitochondrial gene expression?

Mitochondrial import of a cytoplasmic lysine-tRNA in yeast is mediated by cooperation of cytoplasmic and mitochondrial lysyl-tRNA synthetases.

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Mitochondrial import of a yeast cytoplasmic tRNA^Lys: possible roles of aminoacylation and modified nucleosides in subcellular partitioning

Mitochondrial import of a yeast cytoplasmic tRNA^Lys: possible roles of aminoacylation and modified nucleosides in subcellular partitioning