Prenylated proteins contain a covalently linked cholesterol intermediate near their carboxyl-termini. Maturation of most prenylated proteins involves proteolytic removal of the last three amino acids. Two genes in Saccharomyces cerevisiae, RCE1 and AFC1, were identified that appear to be responsible for this processing. The Afc1 protein is a zinc protease that participates in the processing of yeast a-factor mating pheromone. The Rce1 protein contributes to the processing of both Ras protein and a-factor. Deletion of both AFC1 and RCE1 resulted in the loss of proteolytic processing of prenylated proteins. Disruption of RCE1 led to defects in Ras localization and signaling and suppressed the activated phenotype associated with the allele RAS2val19.

Candida albicans is a ubiquitous opportunistic pathogen that is the most prevalent cause of hospital-acquired fungal infections. In mammalian hosts, C. albicans is engulfed by phagocytes that attack the pathogen with DNA-damaging reactive oxygen species (ROS). Acetylation of histone H3 lysine 56 (H3K56) by the fungal-specific histone acetyltransferase Rtt109 is important for yeast model organisms to survive DNA damage and maintain genome integrity. To assess the importance of Rtt109 for C. albicans pathogenicity, we deleted the predicted homolog of Rtt109 in the clinical C. albicans isolate, SC5314. C. albicans rtt109 −/− mutant cells lack acetylated H3K56 (H3K56ac) and are hypersensitive to genotoxic agents. Additionally, rtt109 −/− mutant cells constitutively display increased H2A S129 phosphorylation and elevated DNA repair gene expression, consistent with endogenous DNA damage. Importantly, C. albicans rtt109 −/− cells are significantly less pathogenic in mice and more susceptible to killing by macrophages in vitro than are wild-type cells. Via pharmacological inhibition of the host NADPH oxidase enzyme, we show that the increased sensitivity of rtt109 −/− cells to macrophages depends on the host's ability to generate ROS, providing a mechanistic link between the drug sensitivity, gene expression, and pathogenesis phenotypes. We conclude that Rtt109 is particularly important for fungal pathogenicity, suggesting a unique target for therapeutic antifungal compounds.acetylation | chromatin | fungal pathogenesis | DNA damage resistance | macrophage

Many proteins that contain a carboxyl-terminal CaaX sequence motif, including Ras and yeast a-factor, undergo a series of sequential posttranslational processing steps. Following the initial prenylation of the cysteine, the three C-terminal amino acids are proteolytically removed, and the newly formed prenylcysteine is carboxymethylated. The specific amino acids that comprise the CaaX sequence influence whether the protein can be prenylated and proteolyzed. In this study, we evaluated processing of a-factor variants with all possible single amino acid substitutions at either the a 1 , the a 2 , or the X position of the a-factor Ca 1 a 2 X sequence, CVIA. The substrate specificity of the two known yeast CaaX proteases, Afc1p and Rce1p, was investigated in vivo. Both Afc1p and Rce1p were able to proteolyze a-factor with A, V, L, I, C, or M at the a 1 position, V, L, I, C, or M at the a 2 position, or any amino acid at the X position that was acceptable for prenylation of the cysteine. Eight additional a-factor variants with a 1 substitutions were proteolyzed by Rce1p but not by Afc1p. In contrast, Afc1p was able to proteolyze additional a-factor variants that Rce1p may not be able to proteolyze. In vitro assays indicated that farnesylation was compromised or undetectable for 11 a-factor variants that produced no detectable halo in the wild-type AFC1 RCE1 strain. The isolation of mutations in RCE1 that improved proteolysis of a-factor-CAMQ, indicated that amino acid substitutions E139K, F189L, and Q201R in Rce1p affected its substrate specificity.

The B10.Q͞J strain of mice was serendipitously discovered to be highly susceptible to infection by the intracellular protozoan parasite, Toxoplasma gondii but markedly resistant to induction of autoimmune arthritis. We have previously shown that the B10.

We have used a novel quantitative trait locus model to study the genetics of survival of F2 progeny of susceptible BALB/cByJ and resistant C57BL/6ByJ mice that have been infected with Listeria monocytogenes. This allowed us to map modifiers of L. monocytogenes susceptibility to chromosomes 5 and 13.