Biochemical and Biophysical Research Communications
Leucine-induced localization of Leucyl-tRNA synthetase in lysosome membrane
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 RagDGTP to RagDGDP [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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Equally contributed.