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Identification of sumoylation targets, combined with inactivation of SMT3, reveals the impact of sumoylation upon growth, morphology, and stress resistance in the pathogen Candida albicans.

Leach MD, Stead DA, Argo E, Brown AJ - Mol. Biol. Cell (2011)

Bottom Line: Smt3/smt3 cells also displayed sensitivity to thermal, oxidative, and cell wall stresses as well as to the antifungal drug caspofungin.Furthermore, signaling via the cell integrity pathway was defective in C. albicans smt3/smt3 cells.Clearly sumoylation plays key roles in fundamental cellular processes that underpin the pathogenicity of this medically important fungus.

View Article: PubMed Central - PubMed

Affiliation: School of Medical Sciences, Institute of Medical Sciences, University of Aberdeen, Foresterhill, Aberdeen AB25 2ZD, United Kingdom.

ABSTRACT
Posttranslational modifications of proteins play critical roles in the control of cellular differentiation, development, and environmental adaptation. In particular, the covalent attachment of the small ubiquitin-like modifier, SUMO, to target proteins (sumoylation) regulates cell cycle progression, transcription, nucleocytoplasmic transport, and stress responses. Here we combine proteomic, molecular, and cellular approaches to examine the roles of sumoylation in the major fungal pathogen of humans, Candida albicans. Using an N-terminally FLAG-tagged SUMO, 31 sumoylated proteins were identified in C. albicans with roles in stress responses (e.g., Hsp60, Hsp70 family members, Hsp104), the cytoskeleton and polarized growth (e.g., Tub1, Cct7, Mlc1), secretion, and endocytosis (e.g., Lsp1, Sec24, Sec7). The output from this proteomic screen was entirely consistent with the phenotypes of C. albicans mutants in which the single SUMO-encoding locus (SMT3) was inactivated or down-regulated. C. albicans smt3/smt3 cells displayed defects in growth, morphology, cell separation, nuclear segregation, and chitin deposition, suggesting important roles for sumoylation in cell cycle control. Smt3/smt3 cells also displayed sensitivity to thermal, oxidative, and cell wall stresses as well as to the antifungal drug caspofungin. Mutation of consensus sumoylation sites in Hsp60 and Hsp104 affected the resistance of C. albicans to thermal stress. Furthermore, signaling via the cell integrity pathway was defective in C. albicans smt3/smt3 cells. These observations provide mechanistic explanations for many of the observed phenotypic effects of Smt3 inactivation upon C. albicans growth and environmental adaptation. Clearly sumoylation plays key roles in fundamental cellular processes that underpin the pathogenicity of this medically important fungus.

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Related in: MedlinePlus

Mkc1 activation is compromised in C. albicans following inactivation of Smt3. Western blotting of the phosphorylated forms of Mkc1 and Cek1 in C. albicans following treatment with caspofungin for 5 min or calcofluor white (CFW) for 2 h: SMT3/SMT3 (BWP17), SMT3/smt3 (MLC33), and smt3/smt3 (MLC37). The Cek1 blot is a longer exposure of the same blot used to detect Mkc1.
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Figure 8: Mkc1 activation is compromised in C. albicans following inactivation of Smt3. Western blotting of the phosphorylated forms of Mkc1 and Cek1 in C. albicans following treatment with caspofungin for 5 min or calcofluor white (CFW) for 2 h: SMT3/SMT3 (BWP17), SMT3/smt3 (MLC33), and smt3/smt3 (MLC37). The Cek1 blot is a longer exposure of the same blot used to detect Mkc1.

Mentions: Wild-type (BWP17), SMT3/smt3 (MLC33), and smt3/smt3 (MLC37) cells were either untreated (controls), exposed to caspofungin (0.032 μg/ml for 5 min), or incubated with calcofluor white (100 μg/ml for 2 h). These concentrations were chosen because they are known to activate Pkc1 signaling in C. albicans (Munro et al., 2007). Interestingly, the inactivation of Smt3 led to an attenuation of Mkc1 phosphorylation in response to both caspofungin and calcofluor white (Figure 8). Indeed, no significant Mkc1 activation was observed in response to the antifungal drug caspofungin.


Identification of sumoylation targets, combined with inactivation of SMT3, reveals the impact of sumoylation upon growth, morphology, and stress resistance in the pathogen Candida albicans.

Leach MD, Stead DA, Argo E, Brown AJ - Mol. Biol. Cell (2011)

Mkc1 activation is compromised in C. albicans following inactivation of Smt3. Western blotting of the phosphorylated forms of Mkc1 and Cek1 in C. albicans following treatment with caspofungin for 5 min or calcofluor white (CFW) for 2 h: SMT3/SMT3 (BWP17), SMT3/smt3 (MLC33), and smt3/smt3 (MLC37). The Cek1 blot is a longer exposure of the same blot used to detect Mkc1.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3046064&req=5

Figure 8: Mkc1 activation is compromised in C. albicans following inactivation of Smt3. Western blotting of the phosphorylated forms of Mkc1 and Cek1 in C. albicans following treatment with caspofungin for 5 min or calcofluor white (CFW) for 2 h: SMT3/SMT3 (BWP17), SMT3/smt3 (MLC33), and smt3/smt3 (MLC37). The Cek1 blot is a longer exposure of the same blot used to detect Mkc1.
Mentions: Wild-type (BWP17), SMT3/smt3 (MLC33), and smt3/smt3 (MLC37) cells were either untreated (controls), exposed to caspofungin (0.032 μg/ml for 5 min), or incubated with calcofluor white (100 μg/ml for 2 h). These concentrations were chosen because they are known to activate Pkc1 signaling in C. albicans (Munro et al., 2007). Interestingly, the inactivation of Smt3 led to an attenuation of Mkc1 phosphorylation in response to both caspofungin and calcofluor white (Figure 8). Indeed, no significant Mkc1 activation was observed in response to the antifungal drug caspofungin.

Bottom Line: Smt3/smt3 cells also displayed sensitivity to thermal, oxidative, and cell wall stresses as well as to the antifungal drug caspofungin.Furthermore, signaling via the cell integrity pathway was defective in C. albicans smt3/smt3 cells.Clearly sumoylation plays key roles in fundamental cellular processes that underpin the pathogenicity of this medically important fungus.

View Article: PubMed Central - PubMed

Affiliation: School of Medical Sciences, Institute of Medical Sciences, University of Aberdeen, Foresterhill, Aberdeen AB25 2ZD, United Kingdom.

ABSTRACT
Posttranslational modifications of proteins play critical roles in the control of cellular differentiation, development, and environmental adaptation. In particular, the covalent attachment of the small ubiquitin-like modifier, SUMO, to target proteins (sumoylation) regulates cell cycle progression, transcription, nucleocytoplasmic transport, and stress responses. Here we combine proteomic, molecular, and cellular approaches to examine the roles of sumoylation in the major fungal pathogen of humans, Candida albicans. Using an N-terminally FLAG-tagged SUMO, 31 sumoylated proteins were identified in C. albicans with roles in stress responses (e.g., Hsp60, Hsp70 family members, Hsp104), the cytoskeleton and polarized growth (e.g., Tub1, Cct7, Mlc1), secretion, and endocytosis (e.g., Lsp1, Sec24, Sec7). The output from this proteomic screen was entirely consistent with the phenotypes of C. albicans mutants in which the single SUMO-encoding locus (SMT3) was inactivated or down-regulated. C. albicans smt3/smt3 cells displayed defects in growth, morphology, cell separation, nuclear segregation, and chitin deposition, suggesting important roles for sumoylation in cell cycle control. Smt3/smt3 cells also displayed sensitivity to thermal, oxidative, and cell wall stresses as well as to the antifungal drug caspofungin. Mutation of consensus sumoylation sites in Hsp60 and Hsp104 affected the resistance of C. albicans to thermal stress. Furthermore, signaling via the cell integrity pathway was defective in C. albicans smt3/smt3 cells. These observations provide mechanistic explanations for many of the observed phenotypic effects of Smt3 inactivation upon C. albicans growth and environmental adaptation. Clearly sumoylation plays key roles in fundamental cellular processes that underpin the pathogenicity of this medically important fungus.

Show MeSH
Related in: MedlinePlus