Cell surface mannan is implicated in almost every aspect of pathogenicity of Candida albicans. In Saccharomyces cerevisiae, the Vrg4 protein acts as a master regulator of mannan synthesis through its role in substrate provision. The substrate for mannosylation of proteins and lipids in the Golgi apparatus is GDP-mannose, whose lumenal transport is catalyzed by Vrg4p. This nucleotide sugar is synthesized in the cytoplasm by pathways that are highly conserved in all eukaryotes, but its lumenal transport (and hence Golgi apparatusspecific mannosylation) is a fungus-specific process. To begin to study the role of Golgi mannosylation in C. albicans, we isolated the CaVRG4 gene and analyzed the effects of loss of its function. CaVRG4 encodes a functional homologue of the S. cerevisiae GDP-mannose transporter. CaVrg4p localized to punctate spots within the cytoplasm of C. albicans in a pattern reminiscent of localization of Vrg4p in the Golgi apparatus in S. cerevisiae. Like partial loss of ScVRG4 function, partial loss of CaVRG4 function resulted in mannosylation defects, which in turn led to a number of cell wall-associated phenotypes. While heterozygotes displayed no growth phenotypes, a hemizygous strain, containing a single copy of CaVRG4 under control of the methioninerepressible MET3 promoter, did not grow in the presence of methionine and cysteine, demonstrating that CaVRG4 is essential for viability. Mutant Candida vrg4 strains were defective in hyphal formation but exhibited a constitutive polarized mode of pseudohyphal growth. Because the VRG4 gene is essential for yeast viability but does not have a mammalian homologue, it is a particularly attractive target for development of antifungal therapies.Candida albicans is the most important human fungal pathogen. It causes infections that range from superficial colonization of oral and vaginal tissues to life-threatening infections in severely immunocompromised hosts. An essential step for the colonization and infection of host tissues is adhesion, which is initiated by the outermost components of the fungal cell wall. Cell wall-associated mannosylated proteins (mannans) on the external layer of the cell wall have been implicated as key determinants that mediate these initial and critical interactions between the fungus and its host (for reviews, see references 8, 10, and 11). The mannose branches on these glycoproteins, attached by N-and O-glycosidic linkages, are the structures recognized during the immune response against the pathogen (for reviews, see references 3, 14, and 15). Therefore, the enzymes that regulate addition of the mannose molecules are the focus of intense research for understanding fungal biology and mechanisms of host defense and as potential antifungal drug targets.Biogenesis of mannoproteins in Saccharomyces cerevisiae has been well characterized (for a review see reference 40). Following the initial glycosylation steps in the endoplasmic reticulum, yeast mannoproteins are elongated by addition of mannose or mannosylphosphate in the Golgi...

Rapid and long-distance secretion of membrane components is critical for hyphal formation in filamentous fungi, but the mechanisms responsible for polarized trafficking are not well understood. Here, we demonstrate that in Candida albicans, the majority of the Golgi complex is redistributed to the distal region during hyphal formation. Randomly distributed Golgi puncta in yeast cells cluster toward the growing tip during hyphal formation, remain associated with the distal portion of the filament during its extension, and are almost absent from the cell body. This restricted Golgi localization pattern is distinct from other organelles, including the endoplasmic reticulum, vacuole and mitochondria, which remain distributed throughout the cell body and hypha. Hyphal-induced positioning of the Golgi and the maintenance of its structural integrity requires actin cytoskeleton, but not microtubules. Absence of the formin Bni1 causes a hyphal-specific dispersal of the Golgi into a haze of finely dispersed vesicles with a sedimentation density no different from that of normal Golgi. These results demonstrate the existence of a hyphal-specific, Bni1-dependent cue for Golgi integrity and positioning at the distal portion of the hyphal tip, and suggest that filamentous fungi have evolved a novel strategy for polarized secretion, involving a redistribution of the Golgi to the growing tip. INTRODUCTIONThe establishment of axes of polarization is crucial for the growth and division of both unicellular and multicellular organisms. A striking example of polarized morphogenesis is the process of hyphal formation in Candida albicans. In response to a number of inductive signals, C. albicans switches from an ovoid yeast form to a highly elongated hyphal form, and its capacity to switch between these forms is postulated to be related to its virulence as a fungal pathogen (Lo et al., 1997;Gow et al., 2002). Within minutes of encountering the inducing signal, growth is restricted to the germ tube, which rapidly extends into a filament that becomes Ͼ10 times the length of the cell body within hours.The mechanism that establishes and maintains rapid apical growth during hyphal formation in C. albicans is not well understood. However, based on our knowledge of how polarized growth occurs in other systems, it is generally accepted to involve the asymmetric deposition of membrane and cell wall at the growing tip. Studies from Saccharomyces cerevisiae have established that in response to certain spatial or positional cues, Golgi-derived secretory vesicles that fuse with the plasma membrane at random sites during isotropic growth of the mother cell are retargeted to fuse at a specified site, resulting in bud emergence and apical growth (for review, see Lew and Reed, 1995;Finger and Novick, 1998). The process of budding requires many proteins that regulate site selection, reorganization of the actin cytoskeleton, and polarization of the secretory apparatus (for review, see Pruyne et al., 2004b). Hyphal growth in C. albicans presents an additi...

The early steps of N-linked glycosylation involve the synthesis of a lipid-linked oligosaccharide, Glc(3)Man(9)GlcNAc(2)-PP-dolichol, on the endoplasmic reticulum (ER) membrane. Prior to its lumenal translocation and transfer to nascent glycoproteins, mannosylation of Man(5)GlcNAc(2)-PP-dolichol is catalyzed by the Alg1, Alg2, and Alg11 mannosyltransferases. We provide evidence for a physical interaction between these proteins. Using a combination of biochemical and genetic assays, two distinct complexes that contain multiple copies of Alg1 were identified. The two Alg1-containing complexes differ from one another in that one complex contains Alg2 and the other contains Alg11. Alg1 self-assembles through a C-terminal domain that is distinct from the region required for its association with Alg2 or Alg11. Missense mutations affecting catalysis but not Alg1 protein stability or assembly with Alg2 or Alg11 were also identified. Overexpression of these catalytically inactive alleles resulted in dominant negative phenotypes, providing genetic evidence for functional Alg1-containing complexes in vivo. These data suggest that an additional level of regulation that ensures the fidelity of complex oligosaccharide structures involves the physical association of the related catalytic enzymes in the ER membrane.