Significance Translation requires aminoacyl-tRNAs that are mainly formed by acylating tRNAs with the corresponding amino acids. Methanogenic archaea synthesize Cys-tRNA in an unusual indirect fashion. They attach a precursor amino acid, phosphoserine, to tRNA Cys , which is then converted to cysteine. This study shows that the indirect Cys-tRNA formation is carried out in a multienzyme complex assembled by a translation factor. Complex formation markedly promotes reaction efficiency. Because the indirect Cys-tRNA formation is the ancestral pathway of Cys biosynthesis in archaea, this complex may represent a remnant of a primordial machinery for Cys coding.

Initial evaluations of large microbial libraries for potential producers of novel antimicrobial proteins require both qualitative and quantitative methods to screen for target enzymes prior to investing greater research effort and resources. The goal of this protocol is to demonstrate two complementary assays for conducting these initial evaluations. The microslide diffusion assay provides an initial or simple detection screen to enable the qualitative and rapid assessment of proteolytic activity against an array of both viable and heat-killed bacterial target substrates. As a counterpart, the increased sensitivity and reproducibility of the dye-release assay provides a quantitative platform for evaluating and comparing environmental influences affecting the hydrolytic activity of protein antimicrobials. The ability to label specific heat-killed cell culture substrates with Remazol brilliant blue R dye expands this capability to tailor the dye-release assay to characterize enzymatic activity of interest.

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Contact sites between the mitochondria and endoplasmic reticulum (ER) are irregulates the exchange of lipids, Ca2+, and reactive oxygen species (ROS) across the two organelles. Mitofusin 2 (Mfn2) has been shown as one of the major components tethering these two organelles. Several post-translational modifications (PTMs) of Mfn2 have been identified to modulate canonical (i.e., mitochondrial fusion) and non-canonical functions, such as mitophagy and activation of ER stress signaling, however there is little information whether any PTMs can regulate mitochondrial and ER tethering. Basal tyrosine phosphorylation of Mfn2 was detected by mass spectroscopy, but it is unknown whether Mfn2 is a substrate of mitochondria-localized tyrosine kinases. Here we show that the mitochondria-localized Src family tyrosine kinases including c-Src can phosphorylate Mfn2, which decreases distance between the mitochondria and ER, and increases Ca2+ transfer from the ER to mitochondria, followed by changes in ROS generation and mitochondrial bioenergetics. Our findings suggest that tyrosine phosphorylation of Mfn2 may uniquely work to fine-tune ER-mitochondrial Ca2+ transport under physiological conditions, without activating mitophagy or ER stress signaling.

Introduction Contact sites between the endoplasmic reticulum (ER) and mitochondria (i.e., mitochondria‐associated membranes: MAMs) have important roles for the exchange of lipids, Ca2+, and reactive oxygen species (ROS), and greatly influence mitochondrial bioenergetics and cell fate. Mitofusin 2 (Mfn2), a mitochondrial fusion protein, is critical for MAM formation by tethering two organelles together to initiate contact. Although several post‐translational modifications (PTMs) of Mfn2 have been identified, including serine/threonine phosphorylation and ubiquitination, it remains unclear whether the PTMs of Mfn2 regulate its tethering function. In addition, while basal tyrosine phosphorylation (P‐Tyr) of Mfn2 was reported from mass spectroscopy data, the signaling pathways that regulate P‐Tyr levels of Mfn2 are completely unknown. Objective To determine whether P‐Tyr of Mfn2 modulates MAM functions. Methods Biochemical (mitochondrial fractionation), cell biological (Foster resonance energy transfer [FRET] efficiency between the outer mitochondrial membrane (OMM)‐targeted cyan fluorescent protein and ER membrane‐targeted yellow fluorescent protein), and physiological (imaging of mitochondrial Ca2+ [mtCa2+], ROS, and membrane potential [Δψm] in live cells) assays were performed in HEK293T cells. Results Endogenous expression of several tyrosine kinases, including proto‐oncogene tyrosine protein kinase (Src), C‐Terminal Src Kinase (CSK), and proline‐rich tyrosine kinase 2 (Pyk2), was found in the cytosolic and mitochondrial fractions of HEK293T cells. Overexpression of these proteins increased P‐Tyr levels of Mfn2, as detected by a general P‐Tyr antibody. Next, we found that CSK knockdown by shRNA in HEK293T cells enhances the physical coupling between the OMM and ER membrane compared to control cells, as determined by biochemical and live‐cell FRET assays. We also found that CSK knockdown induces mild, but significant, Δψm depolarization and increases basal mitochondrial ROS levels, which were quantified by a Δψm‐sensitive dye TMRE, and a mitochondria‐targeted H2O2 biosensor mt‐RoGFP2‐Orp1, respectively. Lastly, we observed mtCa2+ uptake in response to ER Ca2+ release induced by Gq protein‐coupled receptor stimulation, using a mitochondria‐targeted Ca2+ biosensor mt‐RCamp1h. Importantly, CSK‐knockdown enhanced mtCa2+ uptake in cells compared to control despite the mild Δψm depolarization. Conclusion Mfn2 has potential to be phosphorylated by tyrosine kinases in situ. P‐Tyr levels of Mfn2 may modulate the physical coupling and Ca2+ transport between organelles, which promotes mtCa2+‐dependent Δψm depolarization and mitochondrial ROS generation. Support or Funding Information A part of this research was supported by American Heart Association (AHA) 18CDA34110091(to B.S.J), NIH/NHLBI R01HL136757 (to J.O.‐U.), AHA 16SDG27260248 (to J.O.‐U.), and American Physiological Society (APS) 2017 Shih‐Chun Wang Young Investigator Award.