The Candida albicans CHS4 gene complements a Saccharomyces cerevisiae skt5/chs4 mutation and is involved in chitin biosynthesis

Sudoh, Masayuki; Tatsuno, Kenji; Ono, Naoki; Ohta, Atsunori; Chibana, Hiroji; Yamada-Okabe, Hisafumi; Arisawa, Mikio

doi:10.1099/13500872-145-7-1613

Cited by 24 publications

(27 citation statements)

References 35 publications

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“…Although Chs4p contains a possible farnesyl attachment site at the C-terminal end (the CVIM motif) which is conserved between Chs4p of S. cerevisiae and a number of other fungi, the role of farnesylation of Chs4p in chitin biosynthesis has been questioned by several reports (5,11,32,39,40). Since previously wt or mutated Chs4p was expressed from a plasmid, we suspected that the effect of the mutation might have been masked by variations in the protein expression level; therefore, in this study we used only yeast strains expressing Chs4p from the genome.…”

Section: Resultsmentioning

confidence: 99%

“…In previous reports, no phenotype related to chitin synthesis was attributed to loss of the potential prenylation site (CVIM) in Chs4p (5,11,32,39,40). Two reasons led us to reinvestigate the role of Chs4p farnesylation.…”

Section: Discussionmentioning

confidence: 99%

“…Thus, Chs4p is predicted to be prenylated by the Prenylation Prediction Suite (http://mendel.imp.ac.at/sat/PrePS/). However, the role of prenylation of Chs4p in chitin biosynthesis, or even the occurrence of this modification, has been questioned in several reports (5,11,32,39,40).…”

mentioning

confidence: 99%

“…It has been demonstrated that Chs4p interacts directly with Chs3p and is responsible for the localization of Chs3p to the septin ring thorough interaction with the scaffolding protein Bni4p (11). Chs4p contains a possible farnesyl attachment site at the C-terminal end (CaaX motif), which is conserved among Saccharomyces cerevisiae Chs4p and other fungi, including the human pathogens Candida albicans (39) and Cryptococcus neoformans (1). The possibility that Chs4p is prenylated is enhanced by the fact that the CVIM motif is preceded by a lysine-rich amino acid stretch (29).…”

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confidence: 99%

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Prenylation of Saccharomyces cerevisiae Chs4p Affects Chitin Synthase III Activity and Chitin Chain Length

2007

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Chs4p (Cal2/Csd4/Skt5) was identified as a protein factor physically interacting with Chs3p, the catalytic subunit of chitin synthase III (CSIII), and is indispensable for its enzymatic activity in vivo. Chs4p contains a putative farnesyl attachment site at the C-terminal end (CVIM motif) conserved in Chs4p of Saccharomyces cerevisiae and other fungi. Several previous reports questioned the role of Chs4p prenylation in chitin biosynthesis. In this study we reinvestigated the function of Chs4p prenylation. We provide evidence that Chs4p is farnesylated by showing that purified Chs4p is recognized by anti-farnesyl antibody and is a substrate for farnesyl transferase (FTase) in vitro and that inactivation of FTase increases the amount of unmodified Chs4p in yeast cells. We demonstrate that abolition of Chs4p prenylation causes a ϳ60% decrease in CSIII activity, which is correlated with a ϳ30% decrease in chitin content and with increased resistance to the chitin binding compound calcofluor white. Furthermore, we show that lack of Chs4p prenylation decreases the average chain length of the chitin polymer. Prenylation of Chs4p, however, is not a factor that mediates plasma membrane association of the protein. Our results provide evidence that the prenyl moiety attached to Chs4p is a factor modulating the activity of CSIII both in vivo and in vitro.

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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confidence: 99%

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Prenylation of Saccharomyces cerevisiae Chs4p Affects Chitin Synthase III Activity and Chitin Chain Length

2007

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“…In both C. albicans and S. cerevisiae, Chs4p is involved directly or indirectly in the activation of Chs3p (Sudoh et al, 1999;Ono et al, 2000). ScChs5p is required for targeting of ScChs3p to polarized growth sites and for cell fusion during mating (Santos et al, 1997;Santos & Snyder, 2003 plasma membrane (Trilla et al, 1999).…”

Section: Introductionmentioning

confidence: 99%

Candida albicans mutants in the BNI4 gene have reduced cell-wall chitin and alterations in morphogenesis

2004

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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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Proteomic profiling of yeast‐ and hyphal‐specific responses ofCandida albicansto the antifungal agent, HWY‐289

Kim

Shin

Kang

et al. 2009

Proteomics Clinical Apps

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Virulence of Candida albicans is attributable to its unique dimorphic transition from nonpathogenic yeast cells to pathogenic hyphal cells. We previously discovered a novel antifungal agent, known as HWY-289. To characterize the mechanism underlying HWY-289 antifungal activity, we performed 2-DE to identify proteins that were differentially expressed during yeast-to-hyphal transition and in response to HWY-289. Twenty-four differentially expressed protein spots were identified in HWY-289-treated yeast. Most differentially expressed proteins were involved in carbohydrate-derived energy metabolism, cellular detoxification, and antioxidant defenses. Two proteins were involved in cell cycle regulation and DNA processing, and both were downregulated by HWY-289, suggesting that this agent might promote cell death by weakening cellular defense systems. HWY-289 inhibited yeast-to-hyphal transition in a dose-dependent manner. 2-DE analysis of hyphae uncovered several proteins that were induced during yeast-to-hyphal transition. Of these, aconitase and phosphatidylinositol transfer protein were downregulated by HWY-289, suggesting that they mediate the antifungal effects of HWY-289. Finally, RT-PCR analysis revealed that HWY-289 induced expression of three RAS-related genes (CcCST20, CaHST7, and CaCPH1) in yeast cells, but suppressed their expression in hyphae. Thus, the antifungal action of HWY-289 may be attributable to its ability to disrupt prohyphal RAS signaling.

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The Candida albicans CHS4 gene complements a Saccharomyces cerevisiae skt5/chs4 mutation and is involved in chitin biosynthesis

Cited by 24 publications

References 35 publications

Prenylation of Saccharomyces cerevisiae Chs4p Affects Chitin Synthase III Activity and Chitin Chain Length

Prenylation of Saccharomyces cerevisiae Chs4p Affects Chitin Synthase III Activity and Chitin Chain Length

Candida albicans mutants in the BNI4 gene have reduced cell-wall chitin and alterations in morphogenesis

Proteomic profiling of yeast‐ and hyphal‐specific responses ofCandida albicansto the antifungal agent, HWY‐289

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