Ras signaling activates glycosylphosphatidylinositol (GPI) anchor biosynthesis via the GPI-N-acetylglucosaminyltransferase (GPI-GnT) in Candida albicans.

The ability of Candida albicans 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 C. albicans. We show that the first step of GPI biosynthesis is activated by Ras in C. albicans This is diametrically opposite to what is reported in Saccharomyces cerevisiae Of the two C. albicans 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-N-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 S. cerevisiae 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 Cagpi2 mutant is also more susceptible to macrophage-mediated killing, and macrophage cells show better survival when co-cultured with Cagpi2.

Generating anchors only to lose them: The unusual story of glycosylphosphatidylinositol anchor biosynthesis and remodeling in yeast and fungi.

Glycosylphosphatidylinositol (GPI)-anchored proteins (GPI-APs) are present ubiquitously at the cell surface in all eukaryotes. They play a crucial role in the interaction of the cell with its external environment, allowing the cell to receive signals, respond to challenges, and mediate adhesion. In yeast and fungi, they also participate in the structural integrity of the cell wall and are often essential for survival. Roughly four decades after the discovery of the first GPI-APs, this review provides an overview of the insights gained from studies of the GPI biosynthetic pathway and the future challenges in the field. In particular, we focus on the biosynthetic pathway in Saccharomyces cerevisiae, which has for long been studied as a model organism. Where available, we also provide information about the GPI biosynthetic steps in other yeast/ fungi. Although the core structure of the GPI anchor is conserved across organisms, several variations are built into the biosynthetic pathway. The present Review specifically highlights these variations and their implications. There is growing evidence to suggest that several phenotypes are common to GPI deficiency and should be expected in GPI biosynthetic mutants. However, it appears that several phenotypes are unique to a specific step in the pathway and may even be species-specific. These could suggest the points at which the GPI biosynthetic pathway intersects with other important cellular pathways and could be points of regulation. They could be of particular significance in the study of pathogenic fungi and in identification of new and specific antifungal drugs/ drug targets. © 2018 IUBMB Life, 70(5):355-383, 2018.

First step of glycosylphosphatidylinositol (GPI) biosynthesis cross-talks with ergosterol biosynthesis and Ras signaling in Candida albicans.

Candida albicans is a leading cause of fungal infections worldwide. It has several glycosylphosphatidylinositol (GPI)-anchored virulence factors. Inhibiting GPI biosynthesis attenuates its virulence. Building on our previous work, we explore the interaction of GPI biosynthesis in C. albicans with ergosterol biosynthesis and hyphal morphogenesis. This study is also the first report of transcriptional co-regulation existing between two subunits of the multisubunit enzyme complex, GPI-N-acetylglucosaminyltransferase (GPI-GnT), involved in the first step of GPI anchor biosynthesis in eukaryotes. Using mutational analysis, we show that the accessory subunits, GPI2 and GPI19, of GPI-GnT exhibit opposite effects on ergosterol biosynthesis and Ras signaling (which determines hyphal morphogenesis). This is because the two subunits negatively regulate one another; GPI19 mutants show up-regulation of GPI2, whereas GPI2 mutants show up-regulation of GPI19. Two different models were examined as follows. First, the two GPI-GnT subunits independently interact with ergosterol biosynthesis and Ras signaling. Second, the two subunits mutually regulate one another and thereby regulate sterol levels and Ras signaling. Analysis of double mutants of these subunits indicates that GPI19 controls ergosterol biosynthesis through ERG11 levels, whereas GPI2 determines the filamentation by cross-talk with Ras1 signaling. Taken together, this suggests that the first step of GPI biosynthesis talks to and regulates two very important pathways in C. albicans. This could have implications for designing new antifungal strategies.

Author Correction: Ras hyperactivation versus overexpression: Lessons from Ras dynamics in Candida albicans.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has not been fixed in the paper.

Ras hyperactivation versus overexpression: Lessons from Ras dynamics in Candida albicans.

Ras signaling in response to environmental cues is critical for cellular morphogenesis in eukaryotes. This signaling is tightly regulated and its activation involves multiple players. Sometimes Ras signaling may be hyperactivated. In C. albicans, a human pathogenic fungus, we demonstrate that dynamics of hyperactivated Ras1 (Ras1G13V or Ras1 in Hsp90 deficient strains) can be reliably differentiated from that of normal Ras1 at (near) single molecule level using fluorescence correlation spectroscopy (FCS). Ras1 hyperactivation results in significantly slower dynamics due to actin polymerization. Activating actin polymerization by jasplakinolide can produce hyperactivated Ras1 dynamics. In a sterol-deficient hyperfilamentous GPI mutant of C. albicans too, Ras1 hyperactivation results from Hsp90 downregulation and causes actin polymerization. Hyperactivated Ras1 co-localizes with G-actin at the plasma membrane rather than with F-actin. Depolymerizing actin with cytochalasin D results in faster Ras1 dynamics in these and other strains that show Ras1 hyperactivation. Further, ergosterol does not influence Ras1 dynamics.