Leucine-induced localization of Leucyl-tRNA synthetase in lysosome membrane

doi:10.1016/j.bbrc.2017.09.008

Biochemical and Biophysical Research Communications

Volume 493, Issue 2, 18 November 2017, Pages 1129-1135

https://doi.org/10.1016/j.bbrc.2017.09.008 Get rights and content

Highlights

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LRS activates mTORC1 via intracellular leucine sensing.
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We used STED microscopy and TEM to observe effect of leucine on LRS localization in lysosomes.
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LRS translocated to lysosomes and autophagy decreased after adding leucine.
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BC-LI-0186 inhibited translocation of LRS.
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LRS inhibition or LRS-mediated mTORC1 suppression can be a new therapeutic target.

Abstract

Leucyl-tRNA synthetase (LRS) plays major roles in providing leucine-tRNA and activating mechanistic target of rapamycin complex 1 (mTORC1) through intracellular leucine sensing. mTORC1 activated by amino acids affects the influence on physiology functions including cell proliferation, protein synthesis and autophagy in various organisms. Biochemical results demonstrating leucine sensing have been published, but visual results are lacking. Therefore, we observed the location of LRS with and without leucine using stimulated emission depletion (STED) microscopy one of the super-resolution microscopy and transmission electron microscopy (TEM). This revealed that LRS was translocated to the lysosome on addition of leucine. The translocation was inhibited by treatment with compound BC-LI-0186, disrupting the interaction between RagD and LRS. Immuno-TEM revealed a clear decrease in LRS translocation to the lysosome on addition of the inhibitor. This direct visualization of leucine sensing and LRS translocation to the lysosome was related to mTORC1 activation. To study the relationship between mTORC1 activation and LRS translocation, we monitored the change in autophagy for each condition using TEM and CLSM. The results showed a decrease in autophagy on addition of leucine, demonstrating crosstalk between leucine sensing, LRS translocation, RagD interaction, and mTORC1 activation.

Introduction

Lysosomes are key cellular organelles that play a role in catabolism by degrading extracellular and intracellular materials. Specifically, mechanistic target of rapamycin complex 1 (mTORC1) localizes in its active form to the surface of lysosomes and regulates energy levels, growth signals, and nutrients along with other functions [1]. The mechanisms for tight crosstalk between mTORC1 activity and lysosomal function have been reported. Multiprotein complexes on the lysosomal surface, including Rag GTPases and a v-ATPase for an amino acid-sensing device, have been shown to influence catabolism at different levels through mTORC1 regulation in lysosomes [2], [3]. Leucyl-tRNA synthetase (LRS) is well-known as one of the aminoacyl-tRNA synthetases (ARSs), which charge amino acids to their cognate tRNAs. In our previous paper, LRS was reported as an mTORC1-associated protein by leucine sensing, and it also functions as an activator of mTORC1 by converting RagD^GTP to RagD^GDP [4], [5]. In the results, when the leucine-binding ability of LRS was ablated, the mTORC1 pathway to this amino acid was desensitized. In addition, co-immunoprecipitation results showed that the C-terminal of LRS directly interacts with RagD GTPase in an amino acid-dependent manner, and it functions as the GTPase-activating protein for Rag GTPase to activate mTORC1. LRS was found in the endomembrane fraction with mTORC1 after adding leucine. This was also confirmed by the decrease of the double-labeled signal of eGFP-LRS and LysoTracker when leucine was absent. Therefore, we suggested that LRS translocation to the lysosome through leucine is involved in mTORC1 activation. To directly visualize whether LRS translocates to the lysosome, to determine its location in the lysosome if it does, and to determine if it is involved in mTORC1 activation, we used stimulated emission depletion (STED) microscopy and transmission electron microscopy (TEM). STED microscopy allows the ability to overcome the diffraction limit of light (4–600 nm) for investigating the detailed location of a molecule with high resolution [6]. Recently, STED microscopy with high resolution (∼50 nm) observed the substructure of bio-molecule in organelle and exact length of DNA which can't be detected by CLSM [7], [8], [9]. In addition, two-color STED has been developed to simultaneously visualize two different proteins in a cell with high resolution [10]. We used new two-color STED microscopy using two STED beams (800 nm, 592 nm) to directly observe whether lysosome-associated membrane glycoprotein 2 (LAMP2) and LRS co-localized in lysosomes after adding leucine. In addition, anti-LRS immunogold-labeled lysosomes were observed using TEM to visualize whether LRS was translocated to the lysosome in the presence of leucine. We also investigated the location of LRS after treatment with compound BC-LI-0186 [11], an inhibitor of the GTPase-activating function that binds to the RagD-interacting site of LRS, to confirm LRS translocation to the lysosome. To investigate mTORC1 activation caused by the translocation, we monitored the amount of autophagy using TEM and CLSM. This approach would demonstrate the relationship between leucine sensing, LRS translocation, and mTORC1 activation. A decrease in autophagy would indicate mTORC1 activation, whereas an increase in autophagy would indicate its inactivation. In this study, we used STED microscopy and immuno-TEM to demonstrate whether LRS translocates to the lysosome in presence of leucine and used TEM and CLSM to demonstrate whether the translocation results in a decrease of autophagy suggesting mTORC1 activation.

Section snippets

Cell preparation

HEK293T and HeLa cells were cultured in RPMI and DMEM medium supplemented with 10% heat-inactivated fetal bovine serum and 1% penicillin-streptomycin at 37 °C in a 5% CO₂ atmosphere. Leucine-free media was used to culture cells for 1 h for leucine deficient condition. For leucine sufficient condition, leucine-free media was used for 50 min, which was later replaced with complete media including leucine for 10 min. To investigate the role of BC-LI-0186, 10 μM of the compound was added to

Co-localization of LRS and LAMP2 through CLSM showed leucine-dependent LRS translocation to the lysosome

To observe LRS translocation to the lysosome in the presence of leucine, LRS and lysosomes were labeled and imaged using CLSM. LRS (green) and LAMP2 (white) in HeLa cells incubated in the absence of leucine (−Leu) and the presence of leucine (+Leu) were observed using CLSM (Fig. 1). Co-localization (blue) demonstrated that direct LRS translocation to the lysosome is leucine-dependent. To quantify the co-localization of LRS and LAMP2, the effective co-local factor (ECF; supplementary material)

Discussion

Lysosomes are membrane-bound organelles essential for the formation of endosomes, phagosomes, and autophagosomes, and the low pH in lysosomes results in the degradation of macromolecules [16]. It is known that mutations in some lysosomal membrane proteins that play multiple roles in acidification of the lysosomal matrix, sequestration of lysosomal enzymes, fusion with other organelles, and transport of degradation products to the cytoplasm lead to disorders in humans [17]. In the lysosomal

Acknowledgements

These works were supported by Global Frontier Research Grant (NRF-2014M3A6A4075063, NRF-2015M3A6A4065729) and the Basic Science Research Program (NRF-2015R1C1A1A02037153).

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Skeletal muscle is a highly dynamic organ that is essential for locomotion as well as endocrine regulation in all populations of horses. However, despite the importance of adequate muscle development and maintenance, the mechanisms underlying protein anabolism in horses on different diets, exercise programs, and at different life stages remain obscure. Mechanistic target of rapamycin (mTOR) is a key component of the protein synthesis pathway and is regulated by biological factors such as insulin and amino acid availability. Providing a diet ample in vital amino acids, such as leucine and glutamine, is essential in activating sensory pathways that recruit mTOR to the lysosome and assist in the translation of important downstream targets. When the diet is well balanced, mitochondrial biogenesis and protein synthesis are activated in response to increased exercise bouts in the performing athlete. It is important to note that the mTOR kinase pathways are multifaceted and very complex, with several binding partners and targets that lead to specific functions in protein turnover of the cell, and ultimately, the capacity to maintain or grow muscle mass. Further, these pathways are likely altered across the lifespan, with an emphasis of growth in young horses while decreases in musculature with aged horses appears to be attributable to degradation or other regulators of protein synthesis rather than alterations in the mTOR pathway. Previous work has begun to pinpoint ways in which the mTOR pathway is influenced by diet, exercise, and age; however, future research is warranted to quantify the functional outcomes related to changes in mTOR. Promisingly, this could provide direction on appropriate management techniques to support skeletal muscle growth and maximize athletic potential in differing equine populations.
Aminoacyl-tRNA synthetases in cell signaling
2020, Enzymes
Citation Excerpt :
Thus, the noncanonical function of LeuRS in regulating mTORC1 in animal cells is unusual in that the mechanism takes advantage of both canonical and noncanonical domains. Leucine-stimulated translocation of LeuRS to the lysosome, and enhanced autophagy, was supported visually by stimulated emission depletion microscopy, as well as by transmission electron microscopy [86]. Translocation was blocked by BC-LI-0186, which disrupts LeuRS-RagD interaction [87].
Aminoacyl-tRNA synthetases (ARSs) are a family of essential “housekeeping” enzymes ubiquitous in the three major domains of life. ARSs uniquely connect the essential minimal units of both major oligomer classes—the 3-nucleotide codons of oligonucleotides and the amino acids of proteins. They catalyze the esterification of amino acids to the 3′-end of cognate transfer RNAs (tRNAs) bearing the correct anticodon triplet to ensure accurate transfer of information from mRNA to protein according to the genetic code. As an essential translation factor responsible for the first biochemical reaction in protein biosynthesis, ARSs control protein production by catalyzing aminoacylation, and by editing of mischarged aminoacyl-tRNAs to maintain translational fidelity. In addition to their primary enzymatic activities, many ARSs have noncanonical functions unrelated to their catalytic activity in protein synthesis. Among the ARSs with “moonlighting” activities, several, including GluProRS (or EPRS), LeuRS, LysRS, SerRS, TyrRS, and TrpRS, exhibit cell signaling-related activities that sense environmental signals, regulate gene expression, and modulate cellular functions. ARS signaling functions generally depend on catalytically-inactive, appended domains not present in ancient enzyme forms, and are activated by stimulus-dependent post-translational modification. Activation often results in cellular re-localization and gain of new interacting partners. The newly formed ARS-bearing complexes conduct a host of signal transduction functions, including immune response, mTORC1 pathway signaling, and fibrogenic and angiogenic signaling, among others. Because noncanonical functions of ARSs in signal transduction are uncoupled from canonical aminoacylation functions, function-specific inhibitors can be developed, thus providing promising opportunities and therapeutic targets for treatment of human disease.
Selective Export into Extracellular Vesicles and Function of tRNA Fragments during T Cell Activation
2018, Cell Reports
Citation Excerpt :
Both miR-223 and some of the known YBX1-associated tRFs were enriched in EVs in an activation-induced manner in T cells, making YBX1 a candidate for tRF sorting into EVs upon T cell activation. tRNA synthetases that associate with ceramide or the lysosome may also play a role in the observed selectivity of tRF processing and secretion (Bidlingmaier et al., 2016; Choi et al., 2017; Han et al., 2012). Our findings indicate that removal of cellular tRFs via sorting into MVBs and EVs may be an important mechanism to avert inhibitory functions of tRFs during T cell activation.
The discovery of microRNA (miRNA) sorting into extracellular vesicles (EVs) revealed a novel mode of intercellular communication and uncovered a link between cellular endomembrane compartments and small RNAs in EV-secreting cells. Using a two-step ultracentrifugation procedure to isolate EVs released by T cells, we found that 45% of tRNA fragments (tRFs), but fewer than 1% of miRNAs, were significantly enriched in EVs compared with the corresponding cellular RNA. T cell activation induced the EV-mediated release of a specific set of tRFs derived from the 5′ end and 3′-internal region of tRNAs without variable loops. Inhibition of EV biogenesis pathways specifically led to the accumulation of these activation-induced EV-enriched tRFs within multivesicular bodies (MVBs). Introducing antisense oligonucleotides to inhibit these tRFs enhanced T cell activation. Taken together, these results demonstrate that T cells selectively release tRFs into EVs via MVBs and suggest that this process may remove tRFs that repress immune activation.
Three human aminoacyl-tRNA synthetases have distinct sub-mitochondrial localizations that are unaffected by disease-associated mutations
2018, Journal of Biological Chemistry
Citation Excerpt :
As another example, the human cytosolic LeuRS is translocated to the lysosome membrane upon leucine addition. In this situation, LeuRS acts as an intracellular leucine sensor and activates the mammalian target of rapamycin-signaling pathway, regulating translation, cell size, and autophagy (51, 52). Finally, in cyanobacteria, four aaRSs are anchored to the thylakoid membranes thanks to an additional protein domain named CAAD (for cyanobacterial aminoacyl-tRNA synthetases appended domain) (53).
Human mitochondrial aminoacyl-tRNA synthetases (mt-aaRSs) are key enzymes in the mitochondrial protein translation system and catalyze the charging of amino acids on their cognate tRNAs. Mutations in their nuclear genes are associated with pathologies having a broad spectrum of clinical phenotypes, but with no clear molecular mechanism(s). For example, mutations in the nuclear genes encoding mt-AspRS and mt-ArgRS are correlated with the moderate neurodegenerative disorder leukoencephalopathy with brainstem and spinal cord involvement and lactate elevation (LBSL) and with the severe neurodevelopmental disorder pontocerebellar hypoplasia type 6 (PCH6), respectively. Previous studies have shown no or only minor impacts of these mutations on the canonical properties of these enzymes, indicating that the role of the mt-aaRSs in protein synthesis is mostly not affected by these mutations, but their effects on the mitochondrial localizations of aaRSs remain unclear. Here, we demonstrate that three human aaRSs, mt-AspRS, mt-ArgRS, and LysRS, each have a specific sub-mitochondrial distribution, with mt-ArgRS being exclusively localized in the membrane, LysRS exclusively in the soluble fraction, and mt-AspRS being present in both. Chemical treatments revealed that mt-AspRs is anchored in the mitochondrial membrane through electrostatic interactions, whereas mt-ArgRS uses hydrophobic interactions. We also report that novel mutations in mt-AspRS and mt-ArgRS genes from individuals with LBSL and PCH6, respectively, had no significant impact on the mitochondrial localizations of mt-AspRS and mt-ArgRS. The variable sub-mitochondrial locations for these three mt-aaRSs strongly suggest the existence of additional enzyme properties, requiring further investigation to unravel the mechanisms underlying the two neurodegenerative disorders.
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2023, Basic and Clinical Pharmacology and Toxicology
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View all citing articles on Scopus

¹: Equally contributed.

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Leucine-induced localization of Leucyl-tRNA synthetase in lysosome membrane

Highlights

Abstract

Introduction

Section snippets

Cell preparation

Co-localization of LRS and LAMP2 through CLSM showed leucine-dependent LRS translocation to the lysosome

Discussion

Acknowledgements

Trends Mol. Med.

Cell

Mol. Cell. Neurosci.

Biophysical J.

Methods Enzymol.

Pattern Recognit.

Cell

Cell

Mol. Cell

Mol. Cell

J. Biol. Chem.

J. Biol. Chem.

Cell Rep.

Bioorg. Med. Chem.

Bioorg. Med. Chem. Lett.

mTOR and lysosome regulation

F1000Prime Rep.