| Project/Area Number |
11680697
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Cell biology
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| Research Institution | Kyoto University |
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Principal Investigator |
AKIYAMA Yoshinori Institute of Virus Research, Kyoto University, Associate Professor, ウイルス研究所, 助教授 (10192460)
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| Project Period (FY) |
1999 – 2001
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| Project Status |
Completed (Fiscal Year 2001)
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| Budget Amount *help |
¥3,600,000 (Direct Cost: ¥3,600,000)
Fiscal Year 2001: ¥1,400,000 (Direct Cost: ¥1,400,000)
Fiscal Year 2000: ¥1,400,000 (Direct Cost: ¥1,400,000)
Fiscal Year 1999: ¥800,000 (Direct Cost: ¥800,000)
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| Keywords | E. coli / FtsH / ATPase / Protein degradation / membrane protein / AAA family / Protein degradation / プロテアーゼ / 膜ポテンシャル / 膜貫通配列 |
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| Research Abstract |
E. coli FtsH is a membrane-bound and ATP-dependent protease. The N-terminal region of FtsH mediates membrane association as well as homo-oligomeric interaction of this enzyme. Previously we studied in vivo functionality of FtsH derivatives, in which the N-terminal membrane region was either deleted (FtsH(ΔTM)), replaced by a leucine-zipper (Zip-FtsH(ΔTM)), or replaced by a lactose permease transmembrane segment (LacY-FtsH)(1). It was indicated that homo-oligomerization is required for the minimum proteolytic activity, whereas a transmembrane sequence is required for membrane protein degradation. We purified and characterized these proteins in vitro. LacY-FtsH degraded both soluble and membrane proteins, but Zip-FtsH(ΔTM) only degraded soluble proteins. These proteins also exhibited significant ATPase activities. However, FtsH(ΔTM) remained inactive both in ATPase and protease activities, although it retained ATP-binding as well as denatured protein-binding abilities. These results indi
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cates that subunit association is important for the ATP hydrolytic activity of FtsH, and that the transmembrane sequence must exist to degrade a membrane protein even under detergent-solubilized conditions. We showed that this enzyme can also initiate proteolysis at a C-terminal cytosolic tail. This mode of degradation is also processive, which can be aborted by a tightly folded periplasmic domain. These results suggest that a same enzyme can exhibits either N to C or C to N processivity depending. Sequence alignment of FtsH indicates that glutamic acid residues are conserved at the positions corresponding to Glu 479 of E. coli FtsH. Mutations at this position compromised the proteolytic functions of FtsH in vivo. In vitro proteolytic activities of the mutant enzymes were low but significantly stimulated by high concentration of zinc ion. The mutations did not cause gross conformational changes in FtsH. The mutant proteins exhibited reduced zinc contents upon purification. From these results, we conclude that Glu 479 is a zinc-coordinating residue. Less
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