Nec850e050uc

All plasmids were purified using the Nucleospin Mini kit (Macherey-Nagel, 740588.50) or the NucleoBond Xtra Midi Plus kit (Macherey-Nagel, 740412.50). All enzymes for molecular cloning and Gibson Isothermal assembly were from New England Biolabs or Thermo Fisher. Thin layer chromatography (TLC, Silica gel 60 aluminum foils) plates were from Sigma-Aldrich (56524-25EA) and high-performance TLC (HPTLC) (HPTLC Silica gel 60) from Merck (1.05547.0001). Silica gel 60 for lipid purification by chromatography was from Sigma-Aldrich (Merck, ref: 227196). Commercial ergosterol was from Sigma-Aldrich (45480-10G-F), as cholesterol (C8667). All reagents and solvents were of high-grade purity. The radiolabeled amino acid mixture was from PerkinElmer (NEC850E050UC), and [¹⁴C]Glycine (98 cpm/pmol, 600 μM) and [¹⁴C]Asp (280 cpm/pmol) were from Amersham Life Sciences. Anti-Afm ErgS antibodies were developed using purified Afm ErgS (fDUF) with the Covalab company (France). Secondary goat anti-rabbit antibodies were from Bio-Rad (170.6516). Anti-HA antibodies were purchased from Roche (1583816) and secondary rabbit anti-mouse antibodies from Jackson Immuno Research (11035003). Synthetic Erg-Asp and synthetic Erg-Gly were prepared as described in (10 (link), 11 (link)), respectively.

In the case of isotopic competitions, the exact same protocol was used, using a mixture of 15 [¹⁴C]aa (PerkinElmer, NEC850E050UC) in the presence of 6 μg and 160 μM total Sce tRNA, as to reach ∼ 50,000 cpm of [¹⁴C]aa-tRNA per vial at the plateau. For isotopic competition, 5 mM cold Gly was added, before Yli GlyRS was used to initiate the tRNA aminoacylation reaction, in order to compete specifically with [¹⁴C]Gly and [¹⁴C]Gly-tRNA synthesis. Then, at the plateau, 1 μM MBP-Yli ErgS served to initiate the transfer reaction.