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.
Glycosylphosphatidylinositol (GPI)-anchored proteins are important for virulence of many pathogenic organisms including the human fungal pathogen, Candida albicans . GPI biosynthesis is initiated by a multi-subunit enzyme, GPI- N- acetylglucosaminyltransferase (GPI-GnT). We showed previously that two GPI-GnT subunits, encoded by CaGPI2 and CaGPI19 , are mutually repressive. CaGPI19 also co-regulates CaERG11 , the target of azoles while CaGPI2 controls Ras signaling and hyphal morphogenesis. Here, we investigated the role of a third subunit. We show that CaGpi15 is functionally homologous to Saccharomyces cerevisiae Gpi15. CaGPI15 is a master activator of CaGPI2 and CaGPI19 . Hence, CaGPI15 mutants are azole-sensitive and hypofilamentous. Altering CaGPI19 or CaGPI2 expression in CaGPI15 mutant can elicit alterations in azole sensitivity via CaERG11 expression or hyphal morphogenesis, respectively. Thus, CaGPI2 and CaGPI19 function downstream of CaGPI15 . One mode of regulation is via H3 acetylation of the respective GPI-GnT gene promoters by Rtt109. Azole sensitivity of GPI-GnT mutants is also due to decreased H3 acetylation at the CaERG11 promoter by Rtt109. Using double heterozygous mutants, we also show that CaGPI2 and CaGPI19 can independently activate CaGPI15 . CaGPI15 mutant is more susceptible to killing by macrophages and epithelial cells and has reduced ability to damage either of these cell lines relative to the wild type strain, suggesting that it is attenuated in virulence.
GPI2 encodes for one of the six accessory subunits of the GPI-N-acetylglucosaminyltransferase (GPI-GnT) complex that catalyzes the first step of GPI biosynthesis in S. cerevisiae and C. albicans. It has been previously reported in S. cerevisiae that this subunit physically interacts with and negatively modulates Ras signaling. On the other hand, studies from our lab have shown that the homologous subunit in C. albicans is a positive modulator of Ras signaling. Are the functions of this subunit therefore strictly species dependent? We present here functional complementation studies on GPI2 from S. cerevisiae and C. albicans that were carried out to address this issue. Expression of CaGPI2 in a ScGPI2 conditional lethal mutant could not restore its growth defects. Likewise, ScGPI2 overexpression in a CaGPI2 heterozygous mutant could not restore its deficient GPI-GnT activity or reverse defects in its cell wall integrity and could only poorly restore filamentation. However, interestingly, ScGPI2 could restore lanosterol demethylase (CaERG11) levels and reverse azole resistance of the CaGPI2 heterozygote. It appeared to do this by regulating levels of another GPI-GnT subunit, CaGPI19, which we have previously shown to be involved in cross-talk with CaERG11. Thus, the effect of CaGPI2 on sterol biosynthesis in C. albicans is independent of its interaction with the GPI-GnT complex and Ras signaling pathways. In addition, the interaction of Gpi2 with other subunits of the GPI-GnT complex as well as with Ras signaling appears to have evolved differently in the two organisms.
CaGpi14 is the catalytic subunit of the first mannosyltransferase that is involved in the glycosylphosphatidylinositol (GPI) biosynthetic pathway in Candida albicans. We show that CaGPI14 is able to rescue a conditionally lethal gpi14 mutant of Saccharomyces cerevisiae, unlike its mammalian homologue. The depletion of this enzyme in C. albicans leads to severe growth defects, besides causing deficiencies in GPI anchor levels. In addition, CaGpi14 depletion results in cell wall defects and upregulation of the cell wall integrity response pathway. This in turn appears to trigger the osmoticstress dependent activation of the HOG1 pathway and an upregulation of HOG1 as well as its downstream target, SKO1, a known suppressor of expression of hyphae-specific genes. Consistent with this, mutants of CaGPI14 are unable to undergo hyphal transformations in different hyphae-inducing media, under conditions that produce abundant hyphae in the wild-type cells. Hyphal defects in the CaGPI14 mutants could not be attributed either to reduced protein kinase C activation or to defective Ras signalling in these cells but appeared to be driven by perturbations in the HOG1 pathway.
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