In vitro farnesylation

In vitro farnesylation assays were carried out as described^29,30 in 50 mM Tris–HCl, pH 8.0, 20 mM KCl, 5 mM MgCl₂, 10 μM ZnCl₂ and 10 mM dithiothreitol. A reaction volume of 10 ml with 100 μM PEX19 was incubated with 130 μM farnesyl pyrophosphate (Sigma Aldrich) and 250 nM farnesyl transferase for 1 h at 37 °C. A subsequent Ni²⁺ affinity chromatography (nickel-nitrilotriacetic acid) removed the FTase. The farnesylated PEX19 was further purified by size exclusion chromatography on a HiLoad 16/60 Superdex 75 (GE Healthcare) and stored in 20 mM potassium phosphate, pH 6.5 and 50 mM NaCl. The completeness of the farnesylation was analysed by SDS gel electrophoresis and ¹H,¹⁵N HSQC NMR spectra.