Cyclic AMP pathway activation and extracellular zinc induce rapid intracellular zinc mobilization in Candida albicans

LK was supported by Innovation Fund Denmark, DK (4019-00019B). Pcovery ApS received funding from Wellcome Trust, Research Councils, UK (100480/Z/12), Novo Seeds, DK and Boehringer Ingelheim Venture Fund, D. DW is supported by a Sir Henry Dale Fellowship jointly funded by the Wellcome Trust and the Royal Society (102549/Z/13/Z), the Medical Research Council and University of Aberdeen (MR/N006364/1) and received support from a Wellcome Trust Strategic Award for Medical Mycology and Fungal Immunology (097377/Z/11/Z). The funders had no part in study design, data collection and interpretation, or the decision to submit the work for publication.Peer reviewedPublisher PD

Assessment of phosphopeptide enrichment/precipitation method for LC-MS/MS based phosphoproteomic analysis of plant tissue

  IntroductionMass spectrometry (MS) is a powerful technology for study of PTMs, including protein phosphorylation. Due to the low abundance of many phosphoproteins and the relatively poor ionization efficiency of phosphopeptides, specific enrichment of phosphopeptides prior to MS analysis is necessary. At present, numerous phosphopeptide enrichment approaches have been established and applied to complex biological samples. We and others have reported that multi-step phosphopeptide purification methods enable better recovery of phosphopeptide and achieve higher selectivity and sensitivity than stardand sample preparation protocols. Here, we combine 3 phosphpeptide enrichment methods (IMAC, TiO2 and Calcium Phosphate Precipitation (CPP)), and apply them to phosphoproteomic analysis of Arabidopsis thaliana plasma membrane preparation.MethodPlant plasma membranes were isolated from Arabidopsis thaliana (Col-0) leaves using a two-phase partitioning system. The concentration of plasma membrane protein was determined by Bradford assay. Protein was digested with Lys-C for 4 hours and then by trypsin overnight. The peptide mixture was purified with IMAC, TiO2, CPP, SIMAC (IMAC+TiO2), the combination of CPP and IMAC, and the combination of CPP and TiO2, respectively.Nano-LC-MS was performed using LTQ-Orbitrap XL and LTQ-Orbitrap-XL/ETD mass spectrometer (Thermo Electron, Bremen, Germany) connected to an EASY nano-LC system (Proxeon Biosystems, Odense, Denmark). In CID mode, multi-stage activation (MSA) method was used for phosphopeptide fragmentation. The resulting fragment ion spectra were processed with Proteome Discoverer software (Thermo Electron, Bremen, Germany).ResultsWe first investigated the global phosphorylation profile of plant plasma membrane proteins by enriching the phosphopeptides with IMAC, TiO2 enrichment methods prior to LTQ-Orbitrap MS analysis. 100 ug plant plasma membrane protein was used for each enrichment experiment. The data was searched against NCBI database on MASCOT server, and the results were validated by in home bioinformatic software using the A-score algorithm. Among 890 unique peptides, 389 of them were identified as phosphopeptides from IMAC enrichment. From TiO2 enrichment, 131 of 240 identified peptides were phosphopeptides. Since the results are not so satisfactory, we further investigated these samples using the combination of CPP and TiO2 enrichment methods. 1024 phosphopeptides were identified from the combined method, with a efficiency of 90% in this combined method. The results produced from the 3 enrichment experiments were carefully analyzed, and we conclude that the combined method gives better phosphopeptide recovery and higher selectivity. The overlap between the 3 enrichment experiments was quite small. We are currently investigating further combination of enrichment methods: SIMAC enrichment and the combination of CPP and IMAC enrichment. Samples will be analyzed by LTQ-Orbitrap-ETD MS, and the behavior of phosphopeptides on CID mode and ETD mode will be compared. Innovative aspects Combination of different phosphopeptide enrichment methods</p

Assessment of phosphopeptide enrichment/precipitation method for LC-MS/MS based phosphoproteomic analysis of plant tissue

  IntroductionMass spectrometry (MS) is a powerful technology for study of PTMs, including protein phosphorylation. Due to the low abundance of many phosphoproteins and the relatively poor ionization efficiency of phosphopeptides, specific enrichment of phosphopeptides prior to MS analysis is necessary. At present, numerous phosphopeptide enrichment approaches have been established and applied to complex biological samples. We and others have reported that multi-step phosphopeptide purification methods enable better recovery of phosphopeptide and achieve higher selectivity and sensitivity than stardand sample preparation protocols. Here, we combine 3 phosphpeptide enrichment methods (IMAC, TiO2 and Calcium Phosphate Precipitation (CPP)), and apply them to phosphoproteomic analysis of Arabidopsis thaliana plasma membrane preparation.MethodPlant plasma membranes were isolated from Arabidopsis thaliana (Col-0) leaves using a two-phase partitioning system. The concentration of plasma membrane protein was determined by Bradford assay. Protein was digested with Lys-C for 4 hours and then by trypsin overnight. The peptide mixture was purified with IMAC, TiO2, CPP, SIMAC (IMAC+TiO2), the combination of CPP and IMAC, and the combination of CPP and TiO2, respectively.Nano-LC-MS was performed using LTQ-Orbitrap XL and LTQ-Orbitrap-XL/ETD mass spectrometer (Thermo Electron, Bremen, Germany) connected to an EASY nano-LC system (Proxeon Biosystems, Odense, Denmark). In CID mode, multi-stage activation (MSA) method was used for phosphopeptide fragmentation. The resulting fragment ion spectra were processed with Proteome Discoverer software (Thermo Electron, Bremen, Germany).ResultsWe first investigated the global phosphorylation profile of plant plasma membrane proteins by enriching the phosphopeptides with IMAC, TiO2 enrichment methods prior to LTQ-Orbitrap MS analysis. 100 ug plant plasma membrane protein was used for each enrichment experiment. The data was searched against NCBI database on MASCOT server, and the results were validated by in home bioinformatic software using the A-score algorithm. Among 890 unique peptides, 389 of them were identified as phosphopeptides from IMAC enrichment. From TiO2 enrichment, 131 of 240 identified peptides were phosphopeptides. Since the results are not so satisfactory, we further investigated these samples using the combination of CPP and TiO2 enrichment methods. 1024 phosphopeptides were identified from the combined method, with a efficiency of 90% in this combined method. The results produced from the 3 enrichment experiments were carefully analyzed, and we conclude that the combined method gives better phosphopeptide recovery and higher selectivity. The overlap between the 3 enrichment experiments was quite small. We are currently investigating further combination of enrichment methods: SIMAC enrichment and the combination of CPP and IMAC enrichment. Samples will be analyzed by LTQ-Orbitrap-ETD MS, and the behavior of phosphopeptides on CID mode and ETD mode will be compared. Innovative aspects Combination of different phosphopeptide enrichment methods</p

RIN4 Functions with Plasma Membrane H+-ATPases to Regulate Stomatal Apertures during Pathogen Attack

In plants, the protein Rin4 acts with the plasma membrane H+-ATPase to regulate pathogen entry and the innate immune response, in part, through the regulation of stomatal closure

Assessment of phosphopeptide enrichment/precipitation method for LC-MS/MS based phosphoproteomic analysis of plant tissue

  IntroductionMass spectrometry (MS) is a powerful technology for study of PTMs, including protein phosphorylation. Due to the low abundance of many phosphoproteins and the relatively poor ionization efficiency of phosphopeptides, specific enrichment of phosphopeptides prior to MS analysis is necessary. At present, numerous phosphopeptide enrichment approaches have been established and applied to complex biological samples. We and others have reported that multi-step phosphopeptide purification methods enable better recovery of phosphopeptide and achieve higher selectivity and sensitivity than stardand sample preparation protocols. Here, we combine 3 phosphpeptide enrichment methods (IMAC, TiO2 and Calcium Phosphate Precipitation (CPP)), and apply them to phosphoproteomic analysis of Arabidopsis thaliana plasma membrane preparation.MethodPlant plasma membranes were isolated from Arabidopsis thaliana (Col-0) leaves using a two-phase partitioning system. The concentration of plasma membrane protein was determined by Bradford assay. Protein was digested with Lys-C for 4 hours and then by trypsin overnight. The peptide mixture was purified with IMAC, TiO2, CPP, SIMAC (IMAC+TiO2), the combination of CPP and IMAC, and the combination of CPP and TiO2, respectively.Nano-LC-MS was performed using LTQ-Orbitrap XL and LTQ-Orbitrap-XL/ETD mass spectrometer (Thermo Electron, Bremen, Germany) connected to an EASY nano-LC system (Proxeon Biosystems, Odense, Denmark). In CID mode, multi-stage activation (MSA) method was used for phosphopeptide fragmentation. The resulting fragment ion spectra were processed with Proteome Discoverer software (Thermo Electron, Bremen, Germany).ResultsWe first investigated the global phosphorylation profile of plant plasma membrane proteins by enriching the phosphopeptides with IMAC, TiO2 enrichment methods prior to LTQ-Orbitrap MS analysis. 100 ug plant plasma membrane protein was used for each enrichment experiment. The data was searched against NCBI database on MASCOT server, and the results were validated by in home bioinformatic software using the A-score algorithm. Among 890 unique peptides, 389 of them were identified as phosphopeptides from IMAC enrichment. From TiO2 enrichment, 131 of 240 identified peptides were phosphopeptides. Since the results are not so satisfactory, we further investigated these samples using the combination of CPP and TiO2 enrichment methods. 1024 phosphopeptides were identified from the combined method, with a efficiency of 90% in this combined method. The results produced from the 3 enrichment experiments were carefully analyzed, and we conclude that the combined method gives better phosphopeptide recovery and higher selectivity. The overlap between the 3 enrichment experiments was quite small. We are currently investigating further combination of enrichment methods: SIMAC enrichment and the combination of CPP and IMAC enrichment. Samples will be analyzed by LTQ-Orbitrap-ETD MS, and the behavior of phosphopeptides on CID mode and ETD mode will be compared. Innovative aspects Combination of different phosphopeptide enrichment methods</p

Measurements of intracellular ATP provide new insight into the regulation of glycolysis in the yeast Saccharomyces cerevisiae

Glycolysis in the yeast Saccharomyces cerevisiae exhibits temporal oscillation under anaerobic or semianaerobic conditions. Previous evidence indicated that at least two membrane-bound ATPases, the mitochondrial F0F1 ATPase and the plasma membrane P-type ATPase (Pma1p), were important in regulating the glycolytic oscillation. Measurements of intracellular ATP provide a unique tool to understand the role of these membrane ATPases and how their activities are regulated. We have constructed a new nanobiosensor that can perform time-resolved measurements of intracellular ATP in intact cells. Measurements of the temporal behaviour of intracellular ATP in a yeast strain with oscillating glycolysis showed that, in addition to oscillation in intracellular ATP, there is an overall slow decrease in intracellular ATP because the ATP consumption rate exceeds the ATP production in glycolysis. Measurements of the temporal behaviour of intracellular ATP in yeast strains lacking either of the two membrane bound ATPases have confirmed that F0F1 ATPase and Pma1p contribute significantly to the ATP consumption in the cell and to the regulation of glycolytic oscillation. Furthermore, our measurements also demonstrate that ATPase activity is under strict control. In the absence of glucose ATPase activity is switched off, and the intracellular ATP concentration is high. When glucose is added to the cells the ATP concentration starts to decrease, because ATP consumption exceeds ATP production by glycolysis. Finally, when glucose is used up, the ATP consumption stops immediately. Thus, glucose or some compound derived from glucose must be involved in controlling the activity of these two ATPases

Proton and calcium pumping P-type ATPases and their regulation of plant responses to the environment

Plant plasma membrane H(+)-ATPases and Ca(2+)-ATPases maintain low cytoplasmic concentrations of H(+) and Ca(2+), respectively, and are essential for plant growth and development. These low concentrations allow plasma membrane H(+)-ATPases to function as electrogenic voltage stats, and Ca(2+)-ATPases as “off” mechanisms in Ca(2+)-based signal transduction. Although these pumps are autoregulated by cytoplasmic concentrations of H(+) and Ca(2+), respectively, they are also subject to exquisite regulation in response to biotic and abiotic events in the environment. A common paradigm for both types of pumps is the presence of terminal regulatory (R) domains that function as autoinhibitors that can be neutralized by multiple means, including phosphorylation. A picture is emerging in which some of the phosphosites in these R domains appear to be highly, nearly constantly phosphorylated, whereas others seem to be subject to dynamic phosphorylation. Thus, some sites might function as major switches, whereas others might simply reduce activity. Here, we provide an overview of the relevant transport systems and discuss recent advances that address their relation to external stimuli and physiological adaptations

Proton and calcium pumping P-type ATPases and their regulation of plant responses to the environment

Abstract Plant plasma membrane H+-ATPases and Ca2+-ATPases maintain low cytoplasmic concentrations of H+ and Ca2+, respectively, and are essential for plant growth and development. These low concentrations allow plasma membrane H+-ATPases to function as electrogenic voltage stats, and Ca2+-ATPases as “off” mechanisms in Ca2+-based signal transduction. Although these pumps are autoregulated by cytoplasmic concentrations of H+ and Ca2+, respectively, they are also subject to exquisite regulation in response to biotic and abiotic events in the environment. A common paradigm for both types of pumps is the presence of terminal regulatory (R) domains that function as autoinhibitors that can be neutralized by multiple means, including phosphorylation. A picture is emerging in which some of the phosphosites in these R domains appear to be highly, nearly constantly phosphorylated, whereas others seem to be subject to dynamic phosphorylation. Thus, some sites might function as major switches, whereas others might simply reduce activity. Here, we provide an overview of the relevant transport systems and discuss recent advances that address their relation to external stimuli and physiological adaptations.</jats:p