Impact of Protein Palmitoylation on the Virulence Potential of

The localization and specialized function of Ras-like proteins are largely determined by posttranslational processing events. In a highly regulated process, palmitoyl groups may be added to C-terminal cysteine residues, targeting these proteins to specific membranes. In the human fungal pathogen Cryptococcus neoformans, Ras1 protein palmitoylation is essential for growth at high temperature but is dispensable for sexual differentiation. Ras1 palmitoylation is also required for localization of this pro-tein on the plasmamembrane. Together, these results support a model in which specific Ras functions are mediated from differ-ent subcellular locations. We therefore hypothesize that proteins that activate Ras1 or mediate Ras1 localization to the plasma membrane will be important for C. neoformans pathogenesis. To further characterize the Ras1 signaling cascade mediating high-temperature growth, we have identified a family of protein S-acyltransferases (PATs), enzymes that mediate palmitoyl-ation, in the C. neoformans genome database. Deletion strains for each candidate gene were generated by homogenous recombi-nation, and eachmutant strain was assessed for Ras1-mediated phenotypes, including high-temperature growth, morphogene-sis, and sexual development. We found that full Ras1 palmitoylation and function required one particular PAT, Pfa4, and deletion of the PFA4 gene in C. neoformans resulted in altered Ras1 localization to membranes, impaired growth at 37°C, and reduced virulence. S-palmitoylation involves the reversible attachment of palmiticacid moieties to proteins. This process of posttranslational modification is catalyzed by the protein S-acyltransferase (PAT