| This thesis focuses on the Saccharomyces cerevisiae prenylcysteine carboxyl methyltransferase, Ste14p. Prenylcysteine carboxyl methyltransferases (pcCMTs) modify proteins which terminate in a CaaX motif (C is a cysteine, a is an aliphatic amino acid, and X is one of several amino acids). CaaX proteins undergo a series of post-translational modifications that include isoprenylation, C-terminal proteolytic cleavage, and carboxyl methylation. The latter step is mediated by Ste14p.; Ste14p is the founding member of the pcCMT family. Though they methylate proteins with important cellular roles, little is known about the structure, mechanism, or cell biology of these methyltransferases. In this thesis, we have characterized the intracellular localization and topology of Ste14p and generated mutations in Ste14p to identify residues that are important for its function.; Chapter 1 introduces the CaaX processing pathway, the family of pcCMTs, and the potential roles of CaaX protein methylation.; In chapter 2, we identify multiple Ste14p species and demonstrate that they resulted from aberrant transcription due to the absence of sufficient 5'UTR sequences.; In chapter 3, we describe the use of antibodies raised against a Ste14p-GST fusion to investigate the intracellular localization of Ste14p. We demonstrate by indirect immunofluorescence and subcellular fractionation that Ste14p resides at the endoplasmic reticulum (ER), leading us to propose that CaaX processing occurs on the cytosolic face of the ER membrane. We also demonstrated that the Schizosaccharomyces pombe homologue, mam4p, complements the mating defect of a Deltaste14 S. cerevisiae strain.; In chapter 4, we investigate the topology of Ste14p using protease treatment of epitope tagged versions of Ste14p and endoglycosidase H treatment of Ste14p-invertase fusions. These studies led us to favor a six membrane span model for Ste14p with its major hydrophilic regions, including the N and C terminus, located on the cytosolic side of the membrane.; In chapter 5, we identify residues important for Ste14p function. Database searches revealed a C-terminal region of Ste14p, which is conserved with phosopholipid methyltransferases, ergosterol biosynthesis enzymes, and several bacterial orfs. Site-directed and random mutagenesis demonstrated that residues in this conserved motif were important for Ste14p function. Interestingly, the ste14 mutations are all found in residues predicted to be on the cytosolic side of the membrane or within the membrane spans. |
