The Ste14p isoprenylcysteine carboxyl methyltransferase catalyzes the final methylation step in the post-translational processing of CaaX proteins in S. cerevisiae.  Our goal is to identify the key residues in Ste14p that form the binding site for the isoprene moiety and peptide/protein portions of methyl-accepting substrates.  We evaluated synthetic analogs of two Ste14p substrates, N-acetyl-S-farnesyl-L-cysteine (AFC) and a -factor peptide mating pheromone, that contain photoactive benzophenone or diazirine moieties in the lipid or peptide portion of the molecule.  Both types of analogs were substrates for Ste14p.  Photocrosslinking and neutravidin-agarose capture experiments revealed that Ste14p was specifically photolabeled with all of the compounds to varying extents.  Using a cysteine-specific cleavage reagent, we demonstrated that an AFC analog containing a benzophenone moiety adjacent to the cysteine photolabeled a peptide containing the last 83 residues of the protein.  These data suggest that these analogs will be useful in fully elucidating the Ste14p substrate binding site.

Elucidation of the isoprenylated substrate binding site in ras carboxyl methyltransferase using biotinylated benzophenone-containing photoaffinity probes

245th National Meeting (2013)

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.