The ability of to switch between yeast to hyphal form is a property that is primarily associated with the invasion and virulence of this human pathogenic fungus. Several glycosylphosphatidylinositol (GPI)-anchored proteins are expressed only during hyphal morphogenesis. One of the major pathways that controls hyphal morphogenesis is the Ras-signaling pathway. We examine the cross-talk between GPI anchor biosynthesis and Ras signaling in We show that the first step of GPI biosynthesis is activated by Ras in This is diametrically opposite to what is reported in Of the two Ras proteins, CaRas1 alone activates GPI-GnT activity; activity is further stimulated by constitutively activated CaRas1. CaRas1 localized to the cytoplasm or endoplasmic reticulum (ER) is sufficient for GPI-GnT activation. Of the six subunits of the GPI--acetylglucosaminyltransferase (GPI-GnT) that catalyze the first step of GPI biosynthesis, CaGpi2 is the key player involved in activating Ras signaling and hyphal morphogenesis. Activation of Ras signaling is independent of the catalytic competence of GPI-GnT. This too is unlike what is observed in where multiple subunits were identified as inhibiting Ras2. Fluorescence resonance energy transfer (FRET) studies indicate a specific physical interaction between CaRas1 and CaGpi2 in the ER, which would explain the ability of CaRas1 to activate GPI-GnT. CaGpi2, in turn, promotes activation of the Ras-signaling pathway and hyphal morphogenesis. The mutant is also more susceptible to macrophage-mediated killing, and macrophage cells show better survival when co-cultured with .

Restricting the localization of the evolutionarily conserved centromeric histone H3 variant CENP-A to centromeres prevents chromosomal instability (CIN). The mislocalization of CENP-A to non-centromeric regions contributes to CIN in yeasts, flies, and human cells. Even though overexpression and mislocalization of CENP-A have been reported in cancers the mechanisms responsible for its mislocalization remain poorly understood. Here, we used an imaging-based high-throughput RNAi screen to identify factors that prevent mislocalization of overexpressed YFP-tagged CENP-A (YFP-CENP-A) in HeLa cells. Amongst the top five lead candidates of the screen that showed increased nuclear YFP-CENP-A fluorescence were depletions of histone chaperones (CHAF1B/p60 and CHAF1A/p150). Follow-up validation and characterization experiments showed that CHAF1B-depleted cells exhibit CENP-A mislocalization, CIN phenotypes, and increased enrichment of CENP-A in the chromatin fraction. The depletion of DAXX, a histone H3.3 chaperone, suppressed CENP-A mislocalization and CIN in CHAF1B-depleted cells. We propose that in CHAF1B-depleted cells, DAXX promotes mislocalization of the overexpressed CENP-A to non-centromeric regions, resulting in CIN. In summary, we have identified regulators of CENP-A localization and defined a role for CHAF1B in preventing DAXX-dependent CENP-A mislocalization and CIN.

Candida albicans N-acetylglucosaminylphosphatidylinositol de-N-acetylase (CaGpi12) recognises N-acetylglucosaminylphosphatidylinositol (GlcNAc-PI) from Saccharomyces cerevisiae and is able to complement ScGPI12 function. Both N- and C-terminal ends of CaGpi12 are important for its function. CaGpi12 was biochemically characterised using rough endoplasmic reticulum microsomes prepared from BWP17 strain of C. albicans. CaGpi12 is optimally active at 30°C and pH 7.5. It is a metal-dependent enzyme that is stimulated by divalent cations but shows no preference for Zn2+ unlike the mammalian homologue. It irreversibly loses activity upon incubation with a metal chelator. Two conserved motifs, HPDDE and HXXH, are both important for its function in the cell. CaGPI12 is essential for growth and viability of C. albicans. Its loss causes reduction of GlcNAc-PI de-N-acetylase activity, cell wall defects and filamentation defects. The filamentation defects could be specifically correlated to an upregulation of the HOG1 pathway.