The Candida albicans BNI4 gene was identified by homology to the Saccharomyces cerevisiae orthologue and encodes a predicted 1655 amino acid protein. In S. cerevisiae most cell-wall chitin is associated with primary septum formation and Bni4p is involved in tethering the Chs3p chitin synthase enzyme to the mother-bud neck by forming a bridge between a regulatory protein Chs4p and the septin Cdc10p. CaBni4p shows 20 % overall identity to the ScBni4p, with 73 % identity over the C-terminal 63 amino acids, which includes a putative protein phosphatase type 1 (PP1) binding domain. Northern blot analysis revealed a transcript of the expected size that was expressed in both yeast and hyphal growth forms. C. albicans has more chitin in its cell wall than S. cerevisiae, and again most chitin is synthesized by CaChs3p. The function of CaBNI4 was investigated by performing a targeted gene disruption using the 'Ura-blaster' method to delete amino acids 1120-1611 that are essential for function. The resulting Cabni4D/Cabni4D null mutants formed lemon-shaped yeast cells and had a 30 % reduction in cell-wall chitin, reduced hyphal formation on solid serum-containing medium and increased sensitivity to SDS and increased resistance to Calcofluor White. The Cabni4D/Cabni4D null mutants were unaffected in chitin ring formation, but often exhibited displaced bud sites with more obvious but flattened birth scars. Therefore, unlike in S. cerevisiae, the Cabni4 mutant apparently alters chitin distribution throughout the cell wall and not exclusively at the bud-neck region. INTRODUCTIONChitin is an essential polysaccharide in the fungal cell wall that is important in determining cell shape, for cytokinesis and as a possible antifungal drug target ). In the human fungal pathogen Candida albicans, chitin is synthesized by four enzymes encoded by CaCHS1, CaCHS2, CaCHS3 and CaCHS8. CaChs3p is responsible for the synthesis of most chitin in distinct locations of the cell wall including the lateral cell wall, chitin ring and bud scar (Bulawa et al., 1995). In Saccharomyces cerevisiae, Bni4p is involved in localizing Chs3p to the bud-neck region where the septum is formed. The mechanism of localization of Chs3p by CaBni4p was the subject of this investigation.In C. albicans, CaChs3p expression is increased in hyphae, which have more chitin than yeast cells (Munro et al., 1998).CaChs1p synthesizes the chitin of the primary septum, is expressed at a low level in both yeast and hyphal cells, and has a potential role in lateral cell-wall stability (Munro et al., 1998(Munro et al., , 2003. CaChs2p is a non-essential enzyme and its expression is increased during the yeast-to-hyphal transition (Gow et al., 1994). CaChs8p also contributes to in vitro chitin synthase activity in yeast and hyphal cells and has highest identity to CaChs2p (Munro et al., 2003). Therefore, chitin synthesis is regulated temporally and spatially at the transcriptional and post-translational levels (Roncero, 2002;).The spatial and temporal regulation of Chs3p has been stu...
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