a, The GXXXG packing motif between helices M1 and M3 of ICMT. Residues on M1 and M3 that make contacts between these helices are drawn as sticks and coloured magenta. Because the M1 and M2 helices are not present in S. cerevisiae ICMT, we hypothesize that the GXXXG motif in the first transmembrane helix of yeast ICMT (equivalent to M3 of beetle ICMT) may cause dimerization of the yeast enzyme through packing of these helices , whereas ICMT enzymes that contain M1 and M2, which include human and beetle ICMT, are monomeric. The location of a PreScission protease cleavage site (PS site) that was introduced at Asn58, in the connection between the M2 and M3 helices, for experiments outlined in this figure, is indicated. 40 b, Anti-His western blot showing that this cleavage site can be cleaved by PreScission protease. In this experiment, ICMT was expressed in HEK293 cells with an N-terminal His–GFP tag (His–GFP–ICMT), with or without the cleavage site. The addition of PreScission protease to detergent-solubilized sample 4 (His–GFP–ICMT with the cleavage site), but not control samples 1–3, results in a cleavage product consisting of His–GFP followed by amino acids 1–58 of ICMT (His–GFP–ICMT 1–58), which is detected by anti-His western blot. This confirms that the loop connecting the M2 and M3 helices is cut by the protease. Samples 1–3 are control experiments, as indicated. For gel source data, see Supplementary Fig. 1. c, FSEC profiles of the samples evaluated by western blot in b and numbered accordingly. Elution volumes for the void, His–GFP–ICMT and free GFP are indicated on the plot for sample 1. The cleavage of the M2–M3 loop by PreScission protease does not alter the elution profile (sample 4) in comparison to the other samples, which indicates that the cleaved portion (His–GFP–ICMT 1–58) is associated with the remainder of the enzyme via non-covalent interactions. No replicates of these experiments were performed.