Evaluation of substrate and inhibitor binding to yeast and human isoprenylcysteine carboxyl methyltransferases (Icmts) using biotinylated benzophenone-containing photoaffinity probes

Abstract

Isoprenylcysteine carboxyl methyltransferases (Icmts) are a class of integral membrane protein methyltransferases localized to the endoplasmic reticulum (ER) membrane in eukaryotes. The Icmts from human (hIcmt) and Saccharomyces cerevisiae (Ste14p) catalyze the α-carboxyl methyl esterification step in the post-translational processing of CaaX proteins, including the yeast a-factor mating pheromones and both human and yeast Ras proteins. Herein, we evaluated synthetic analogs of two well-characterized Icmt substrates, N-acetyl-S-farnesyl-L-cysteine (AFC) and the yeast a-factor peptide mating pheromone, that contain photoactive benzophenone moieties in either the lipid or peptide portion of the molecule. The AFC based-compounds were substrates for both hIcmt and Ste14p, whereas the a-factor analogs were only substrates for Ste14p. However, the a-factor analogs were found to be micromolar inhibitors of hIcmt. Together, these data suggest that the Icmt substrate binding site is dependent upon features in both the isoprenyl moiety and upstream amino acid composition. Furthermore, these data suggest that hIcmt and Ste14p have overlapping, yet distinct, substrate specificities. Photocrosslinking and neutravidin-agarose capture experiments with these analogs revealed that both hIcmt and Ste14p were specifically photolabeled to varying degrees with all of the compounds tested. Our data suggest that these analogs will be useful for the future identification of the Icmt substrate binding sites.

Figure 1. Structures of Photoactive Analogs of AFC and the a-factor peptide from S

cerevisiae.

Figure 2. Immumoblot of Analyses of His-Ste14p (A) or His-hIcmt (B) photocrosslinked with benzophenone-containing analogs

One hundred μg of crude membrane protein were incubated with each substrate for 40 min on ice under UV irradiation (365nm). The samples were extracted and re-solubilized in RIPA/10% SDS before enrichment using neutravidin-agarose beads. Proteins were eluted, resolved by 10% SDS-PAGE, and visualized as described in Materials and Methods. A: His-Ste14p: lane 1: 200 μM 1, lane 2:100 μM 2, lane 3:100 μM 3, lane 4: 50 μM 7b, lane 5: 100 μM 7a , lane 6: 50 μM probe 8b, lane 7: 100 μM 8a, lane 8: empty vector (Δste14) + 100 μM 2. B: His-hIcmt: lane 1: 200 μM 1, lane 2: 50 μM 2, lane 3: 50 μM 3, lane 4: 200 μM 7b, lane 5: 200 μM 7a, lane 6: 200 μM 8b, lane 7: 200 μM 8a, lane 8: empty vector (Δste14) + 50 μM 2.