Essential GPI mannosyltransferases in Saccharomyces cerevisiae and the pathogenic fungus Candida albicans

Diverse glycoproteins are anchored on the outer face of the plasma membrane via a carboxy-terminal glycosylphosphatidylinositol (GPI). The conserved core of the GPI contains three alpha-linked mannoses, but a fourth side-branching alpha-mannose may sometimes be present. Interestingly, yeast GPIs contain at least four mannoses, whereas most mammalian GPIs normally contain only three. My research focused on the mannosyltransferase that adds the fourth GPI mannose in the model yeast Saccharomyces cerevisiae and the pathogenic fungus Candida albicans.; I show that the essential yeast Smp3 protein, a member of a new family of dolichol phosphate mannose-utilizing mannosyltransferases, is responsible for the addition of the fourth mannose. In vivo radiolabeling experiments using temperature-sensitive smp3 mutants, showed that loss of Smp3p function results in the accumulation of a GPI precursor lipid, whose glycan headgroup I structurally characterized as a trimannosyl-GPI. By creating double mutants of smp3 with other gpi mutants, I established that smp3 mutations prevent the formation of tetramannosyl-GPI precursors.; I cloned the C. albicans homologue of SMP3 and found that its product is the functional homologue of ScSmp3p, for it complements null and conditional smp3 mutations. Furthermore, CaSmp3p transfers a fourth mannose to a trimannosyl-GPI precursor in vivo. I determined that CaSMP3 is also an essential gene, as I was unable to recover homozygous null smp3 mutants from C. albicans, an obligate diploid. I created a conditional strain in which expression of CaSMP3 was controlled by the CaMAL2 promoter, and found that depletion of CaSmp3p results in the accumulation of a trimannosyl-GPI precursor and decreased viability. My characterization of this precursor is the first report of a GPI structure from C. albicans . The MAL2 promoter offers a tool to perform functional analysis studies on other C. albicans GPI assembly genes. Several C. albicans GPI anchored proteins have been implicated in virulence and disruption of GPI biosynthesis should globally impair the surface expression of these proteins. Because Smp3p is essential in fungi, yet dispensable in mammalian cells, its function could be exploited as a target for selective inhibitors of pathogenic fungi.