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

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

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

Ndfip Proteins Target Robo Receptors for Degradation and Allow Commissural Axons to Cross the Midline in the Developing Spinal Cord

Summary: Commissural axons initially respond to attractive signals at the midline, but once they cross, they become sensitive to repulsive cues. This switch prevents axons from re-entering the midline. In insects and mammals, negative regulation of Roundabout (Robo) receptors prevents premature response to the midline repellant Slit. In Drosophila, the endosomal protein Commissureless (Comm) prevents Robo1 surface expression before midline crossing by diverting Robo1 into late endosomes. Notably, Comm is not conserved in vertebrates. We identified two Nedd-4-interacting proteins, Ndfip1 and Ndfip2, that act analogously to Comm to localize Robo1 to endosomes. Ndfip proteins recruit Nedd4 E3 ubiquitin ligases to promote Robo1 ubiquitylation and degradation. Ndfip proteins are expressed in commissural axons in the developing spinal cord and removal of Ndfip proteins results in increased Robo1 expression and reduced midline crossing. Our results define a conserved Robo1 intracellular sorting mechanism between flies and mammals to avoid premature responsiveness to Slit. : In order to cross the midline in the developing spinal cord, commissural axons must prevent premature responses to the midline repellant Slit. Here Gorla et al. define a key role for the Ndfip adaptor proteins in preventing the surface expression of the Robo1 receptor prior to midline crossing. Keywords: axon guidance, spinal cord, midline, repulsion, Robo, Slit, Ndfip, E3 ubiquitin ligase, Nedd4, Commissureles

Video_3_Decreased anterograde transport coupled with sustained retrograde transport contributes to reduced axonal mitochondrial density in tauopathy neurons.AVI

Mitochondria are essential organelle required for neuronal homeostasis. Mitochondria supply ATP and buffer calcium at synaptic terminals. However, the complex structural geometry of neurons poses a unique challenge in transporting mitochondria to synaptic terminals. Kinesin motors supply mitochondria to the axonal compartments, while cytoplasmic dynein is required for retrograde transport. Despite the importance of presynaptic mitochondria, how and whether axonal mitochondrial transport and distribution are altered in tauopathy neurons remain poorly studied. In the current study, we have shown that anterograde transport of mitochondria is reduced in P301L neurons, while there is no change in the retrograde transport. Consistently, axonal mitochondrial abundance is reduced in P301L neurons. We further studied the possible role of two opposing motor proteins on mitochondrial transport and found that mitochondrial association of kinesin is decreased significantly in P301L cells. Interestingly, fitting our experimental data into mathematical equations suggested a possible rise in dynein activity to maintain retrograde flux in P301L cells. Our data indicate that decreased kinesin-mediated transport coupled with sustained retrograde transport might reduce axonal mitochondria in tauopathy neurons, thus contributing to the synaptic deficits in Alzheimer’s disease (AD) and other tauopathies.</p