Mitochondrial Lysyl-tRNA Synthetase Independent Import of tRNA Lysine into Yeast Mitochondria

Aminoacyl tRNA synthetases play a central role in protein synthesis by charging tRNAs with amino acids. Yeast mitochondrial lysyl tRNA synthetase (Msk1), in addition to the aminoacylation of mitochondrial tRNA, also functions as a chaperone to facilitate the import of cytosolic lysyl tRNA. In this report, we show that human mitochondrial Kars (lysyl tRNA synthetase) can complement the growth defect associated with the loss of yeast Msk1 and can additionally facilitate the in vitro import of tRNA into mitochondria. Surprisingly, the import of lysyl tRNA can occur independent of Msk1 in vivo. This suggests that an alternative mechanism is present for the import of lysyl tRNA in yeast

Import of tRK1 into yeast (Fig. 2A), rat liver (Fig. 2B) and human mitochondria (Fig. 2C) in the presence of human mitochondrial lysyl-tRNA synthetase.

<p>Import of tRK1 into isolated yeast or mammalian mitochondria was performed in the presence or absence (lane 2) of either yeast or human mitochondrial lysyl-tRNA synthetase (lanes 3–7). The import efficiency was assessed by RNase protection assay followed by polyacrylamide gel electrophoresis and phosphorimaging. Lane 1 contains wild type yeast cytosol that presumably contains both cytosolic and mitochondrial lysyl- tRNA synthetase. Fig. 2D. Substitution of yeast cytosol with human cytosol for the import of tRK1 into either yeast or mammalian mitochondria. Import of tRK1 was carried out into yeast (lanes 1, 2, 4 & 9) and human mitochondria (lanes 3, 5, 6, 7 & 8) in the presence of wild type yeast cytosol (lanes 2 & 3), or <i>mskΔ</i> cytosol (lanes 4, 7 & 8) or human cytosol (lanes 5, 8 & 9). The import efficiency was assessed by RNase protection assay followed by electrophoresis. The bands were quantified and relative tRK1 import values were mentioned by taking maximum imported sample as a value of 1.</p

In vivo distribution of tRK1 in yeast cells.

<p>Isolated mitochondrial preparations were subjected to increasing concentrations of digitonin (Fig. 3A & B). Digitonin soluble (sup) and insoluble fractions (pellet) were separated by centrifugation. One set was used to analyze protein markers of outer membrane (porin), inter membrane space (CCPO), inner membrane (Tim 23) and the matrix (Put2) by SDS-PAGE followed by western blot (3A). In parallel, the other fraction was used to extract total RNAs and analyzed by northern blot for the presence of tRK1, tRK2 and tRK3 by using specific oligonucleotide probes. Isolated yeast mitochondrial preparations were treated with increasing concentration of digitonin from 0–0.2% (Fig. 3C) or 0.05% of digitonin (Fig. 3D) for 20 min on ice and reisolated the mitochondria by centrifugation. The pellet fraction was either treated with 250–750 units of MN (Fig. 3D) or 500 units of MN (Fig. 3C, lanes 5–8). Total RNA was extracted and analyzed by northern blot as above. Standard is the respective in vitro transcribed unlabeled tRNA that was used as a positive control except in the case of tCys and tPhe (Fig. 3C). Cytosolic fraction represents 2 µg of total RNA from the cytosol to show the levels of various tRNAs (Fig. 3C).</p

Confirmation of <i>MSK1</i> deletion in yeast strain by PCR analysis.

<p>Analytical PCR was performed with isolated genomic DNA from wild type and <i>msk1Δ</i> strain by using internal and upstream primers to detect the loss of <i>msk1</i> as mentioned in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035321#s4" target="_blank">Methods</a> Section. Lane 1 represents the wt <i>MSK1</i> gene, lane 2 represents the KAN marker at <i>MSK1</i> locus, lane 3 represents the internal fragment generated by using internal primer in wild type and lane 4 represents the lack of <i>MSK1</i> gene in <i>msk1Δ</i> strain.</p

Suppression of growth defect associated with yeast <i>MSK1</i> deletion by human mitochondrial lysyl-tRNA synthetase.

<p>Strains carrying various ectopic plasmids were grown on YEPD medium over night. 2 0D₆₀₀ unit cells were pelleted and suspended in 1 ml of water. The culture was serially diluted in 10-fold steps and 10 ul of each dilution was spotted onto YEPD and YEG plates. Two lanes of Δ/hmt<i>KARS</i> represent two spores originated from the single tetrad.</p

Presence of tRK1 in <i>msk1</i>Δ mitochondria.

<p>Mitochondria isolated from wild type or <i>msk1</i>Δ strains were treated with digitonin and MNase and total nucleic acids were separated on urea-acrylamide gel and analyzed by northern blot with respective probes. Total RNA represents the total cell RNA (2 µg) that was used to determine the levels of various tRNAs in the cytosol (Fig. 4A). <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035321#pone-0035321-g004" target="_blank">Figure 4B</a> represents the quantification of band intensities by densitometry.</p