- 學位論文
篩選大腸桿菌熱休克蛋白ClpY I domain之突變蛋白與其辨識專一性基質之研究
The mutantion studies of ClpY I domain and idenitification of its function for specific substrates recognition
並列摘要
Recognition of the appropriate molecular targets is critical for most biological activity in the cells. For irreversible processes such as protein degradation, recognizing of the correct substrates are important because cleavage of the wrong targets might be damaging to the cells or even lethal. It is required for cells to degrade proper targets like misfolded or abnormal proteins and to control the levels of critical short-lived regulatory proteins. The ATP-dependent proteases play a role in phenomena described above. In bacteria and other organisms, many of intracellular proteases have to hydrolyze ATP to degrade complex substrates；including peptide enzymes, such as Lon, and two-component protease, such as ClpXP, ClpQY, ClpAP. In ClpQY, the small subunit ClpQ (19kDa) is a peptidase, and the larger subunit ClpY (49kDa) exhibits both ATPase and chaperone activities. ClpY can recognize, unfold, and translocate the specific substrates. It is unclear about the mechanisms of how the ClpY recognizes, binds and translocates the specific substrates to ClpQ and the ClpQY degrades the substrates. In this study, the yeast two-hybrid system was used to screen the ClpY mutants either interact or not with the specific substrates. To test the abilities of interaction of ClpY mutants with substrates, lacZ and leu2 expressions was used in yeast system；cps-lacZ expression was used to detect the RcsA degraded by the ClpY mutants in the presence of ClpQ, MMS test was used to detect the SulA degradation.In addition, the ClpYmutants were tested of their influences on cell growth while overproducted.We show that the loop of I domain(175-209) is necessary for substrate recognition and the altered specific amino acid residues on the loop have an influence on ClpY cellular activities.
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