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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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Identification of sumoylated proteins in C. albicans using a proteomic screen. (A) Cells expressing FLAG-Smt3 (MLC01) were grown for 5 h and then exposed to stress for 1 h. Protein extracts were prepared, run on replicate 2-D gels, and either stained with Coomassie blue or subjected to Western blotting with an α-FLAG antibody: Western blots of no-stress control, peroxide-treated cells (50 mM H2O2), and heat-shocked cells (30°C to 42°C). Autoradiographs were aligned with the Coomassie-stained gels, spots chosen for analysis, and the corresponding proteins identified by tryptic digestion and LC-MS/MS. The identities of some sumoylation targets are shown. (B) THE1 cells (FLAG-Smt3 −) and MLC01 cells (FLAG-Smt3 +) were heat shocked for 1 h, analyzed by Western blotting with an anti-Hsp104 antibody, and compared with untreated cells. Membranes were then reprobed with an anti-FLAG antibody. The band corresponding to the molecular mass of Hsp104 is highlighted. (C) Untreated and heat-shocked MLC01 cells were immunoprecipitated with an anti-FLAG antibody, and lysates were analyzed by Western blotting with the anti-Hsp104 antibody. The highlighted band corresponds to the molecular mass of Hsp104.
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Figure 2: Identification of sumoylated proteins in C. albicans using a proteomic screen. (A) Cells expressing FLAG-Smt3 (MLC01) were grown for 5 h and then exposed to stress for 1 h. Protein extracts were prepared, run on replicate 2-D gels, and either stained with Coomassie blue or subjected to Western blotting with an α-FLAG antibody: Western blots of no-stress control, peroxide-treated cells (50 mM H2O2), and heat-shocked cells (30°C to 42°C). Autoradiographs were aligned with the Coomassie-stained gels, spots chosen for analysis, and the corresponding proteins identified by tryptic digestion and LC-MS/MS. The identities of some sumoylation targets are shown. (B) THE1 cells (FLAG-Smt3 −) and MLC01 cells (FLAG-Smt3 +) were heat shocked for 1 h, analyzed by Western blotting with an anti-Hsp104 antibody, and compared with untreated cells. Membranes were then reprobed with an anti-FLAG antibody. The band corresponding to the molecular mass of Hsp104 is highlighted. (C) Untreated and heat-shocked MLC01 cells were immunoprecipitated with an anti-FLAG antibody, and lysates were analyzed by Western blotting with the anti-Hsp104 antibody. The highlighted band corresponds to the molecular mass of Hsp104.

Mentions: Proteins were extracted from untreated MLC01 (FLAG-Smt3) cells and from equivalent cells that were exposed to a 30–42°C heat shock or to 50 mM H2O2 for 1 h. These protein extracts were subjected to two-dimensional (2-D) gel electrophoresis, and replicate gels were stained with Coomassie blue or subjected to Western blotting using an anti-FLAG antibody. Sumoylated proteins were identified by aligning the Western blots and Coomassie-stained gels (Figure 2A). Spots that displayed reproducible effects in two independent replicate experiments were then cut from the Coomassie-stained gels, and the corresponding proteins were identified by tryptic digestion followed by tandem mass spectrometry (Materials and Methods).


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)

Identification of sumoylated proteins in C. albicans using a proteomic screen. (A) Cells expressing FLAG-Smt3 (MLC01) were grown for 5 h and then exposed to stress for 1 h. Protein extracts were prepared, run on replicate 2-D gels, and either stained with Coomassie blue or subjected to Western blotting with an α-FLAG antibody: Western blots of no-stress control, peroxide-treated cells (50 mM H2O2), and heat-shocked cells (30°C to 42°C). Autoradiographs were aligned with the Coomassie-stained gels, spots chosen for analysis, and the corresponding proteins identified by tryptic digestion and LC-MS/MS. The identities of some sumoylation targets are shown. (B) THE1 cells (FLAG-Smt3 −) and MLC01 cells (FLAG-Smt3 +) were heat shocked for 1 h, analyzed by Western blotting with an anti-Hsp104 antibody, and compared with untreated cells. Membranes were then reprobed with an anti-FLAG antibody. The band corresponding to the molecular mass of Hsp104 is highlighted. (C) Untreated and heat-shocked MLC01 cells were immunoprecipitated with an anti-FLAG antibody, and lysates were analyzed by Western blotting with the anti-Hsp104 antibody. The highlighted band corresponds to the molecular mass of Hsp104.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 2: Identification of sumoylated proteins in C. albicans using a proteomic screen. (A) Cells expressing FLAG-Smt3 (MLC01) were grown for 5 h and then exposed to stress for 1 h. Protein extracts were prepared, run on replicate 2-D gels, and either stained with Coomassie blue or subjected to Western blotting with an α-FLAG antibody: Western blots of no-stress control, peroxide-treated cells (50 mM H2O2), and heat-shocked cells (30°C to 42°C). Autoradiographs were aligned with the Coomassie-stained gels, spots chosen for analysis, and the corresponding proteins identified by tryptic digestion and LC-MS/MS. The identities of some sumoylation targets are shown. (B) THE1 cells (FLAG-Smt3 −) and MLC01 cells (FLAG-Smt3 +) were heat shocked for 1 h, analyzed by Western blotting with an anti-Hsp104 antibody, and compared with untreated cells. Membranes were then reprobed with an anti-FLAG antibody. The band corresponding to the molecular mass of Hsp104 is highlighted. (C) Untreated and heat-shocked MLC01 cells were immunoprecipitated with an anti-FLAG antibody, and lysates were analyzed by Western blotting with the anti-Hsp104 antibody. The highlighted band corresponds to the molecular mass of Hsp104.
Mentions: Proteins were extracted from untreated MLC01 (FLAG-Smt3) cells and from equivalent cells that were exposed to a 30–42°C heat shock or to 50 mM H2O2 for 1 h. These protein extracts were subjected to two-dimensional (2-D) gel electrophoresis, and replicate gels were stained with Coomassie blue or subjected to Western blotting using an anti-FLAG antibody. Sumoylated proteins were identified by aligning the Western blots and Coomassie-stained gels (Figure 2A). Spots that displayed reproducible effects in two independent replicate experiments were then cut from the Coomassie-stained gels, and the corresponding proteins were identified by tryptic digestion followed by tandem mass spectrometry (Materials and Methods).

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