Probing the functional oligomerization state of the isoprenylcysteine carboxylmethyltransferase, Ste14p, from Saccharomyces cerevisiae

Some proteins, including the Ras superfamily, the nuclear lamins, and the yeast a-factor mating pheromone, possess a CaaX motif at their C-terminus that signals a sequential series of three post-translational modifications. First, the cysteine residue is isoprenylated which directs the protein to the endoplasmic reticulum where the second step, endoproteolysis of the terminal three residues, occurs. The newly exposed α-carboxyl group of the isoprenylated cysteine residue is then methylesterified by isoprenylcysteine carboxylmethyltransferase (Icmt), known as Ste14p in Saccharomyces cerevisiae. Because Ste14p is an integral membrane protein, it remains extremely difficult to obtain a three-dimensional crystal structure. This thesis focuses on using biochemical techniques to further understand how Ste14p dimerizes and determining the residues needed for activity, stability, and folding. By utilizing cysteine scanning mutagenesis we have discovered that T26, S27, Y28, L30, G31, G35, and G39 are important for methyltransferase activity and our trypsin sensitivity experiments have revealed that Y28C, G31C, G35C, and G39C are mis-folded. We have demonstrated through the use of an amine reactive chemical cross-linker, co-immunoprecipitation experiments, and co-purification techniques that Ste14p forms a homodimer. Furthermore, by co-expressing an inactive Ste14p mutant with an untagged-Ste14p protein we have determined that Ste14p may function as a dimer. We have mapped a portion of the dimerization interface to contain Y28, G31, G35, I36, G39, P42, and Q43 through the use of two different sulfhydryl specific chemical cross-linkers. And finally by optimizing a S-adenosyl-methionine (SAM) filter binding assay, we have been able to identify residues, R171, P173, and Y175 to be essential for the binding of SAM