Date

2014

Author

Yıldırım, Volkan

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In this study, time-dependent heavy metal response of the white rot fungus P. chrysosporium was analyzed by using 2D-PAGE-MS for soluble cytosolic fraction and GeLC-MS approach for membrane enriched fraction. After the 2D-PAGE-MS analysis, a total of 123 protein spots were detected as differentially expressed for Cu-exposed samples and 89 of them were identifed. Further analysis revealed that the 89 protein spots are the products of the 58 distinct ORFs. Overall, strongly up-regulated proteins included (i) Ras and NAC signalling, (ii) defense against oxidative damage and redox metabolism, (iii) translation and transcription and (iv) yet unknown function represented by putative proteins. For the cadmium-exposed cells, a total of 130 spots were detected as differentially expressed after Cd exposure for 1h, 2h, 4h and 8h in P. chrysosporium proteome. MALDI-TOF analysis of these 130 protein spots identified 89 of them. Further analysis revealed that these 89 protein spots are the products of 58 distinct ORFs. Similar to Cu exposed samples, strongly up-regulated protein groups included (i) Ras and NAC signalling, (ii) defense against oxidative damage (iii) heat shock proteins (HSPs) and chaperons, (iv) transcription and carbohydrate metabolism and (v) poorly characterized hypothetical proteins. Membrane proteins were successfully obtained by disrupting the cells with the help of lysing enzymes and enriched by employing PEG/Dextran aqueous two phase separation method. For quantification of the membrane enriched proteins, 15N based SILAC method was utilized. GeLC-MS analysis of the Cu-exposed samples resulted in identification and quantification of more than 700 proteins for the Cu exposure of 2h, 4h and 8h. Subcellular location prediction analysis revealed that 20% of the identified proteins were plasma membrane proteins. Among the identified proteins, 83 were found to be as upregulated and 55 were found to be down-regulated. In addition to these, 32 proteins were only detected in Cu exposed samples and 18 of the proteins could not be detected after Cu treatment.

Subject Keywords

Phanerochaete chrysosporium., Heavy metal stress., Proteomics., Proteins.

URI

http://etd.lib.metu.edu.tr/upload/12618199/index.pdf
https://hdl.handle.net/11511/24209

Collections

Graduate School of Natural and Applied Sciences, Thesis