The transmembrane domain 6 of vacuolar H+-pyrophosphatase mediates protein targeting and proton transport

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研究生：	潘羿娟 Pan, Yih-Jiuan
論文名稱：	The transmembrane domain 6 of vacuolar H+-pyrophosphatase mediates protein targeting and proton transport 液泡焦磷酸水解酶第六穿膜區參與蛋白質定位與質子傳送
指導教授：	潘榮隆 Pan, Rong-Long
口試委員:
學位類別：	博士 Doctor
系所名稱：	生命科學暨醫學院 - 生物資訊與結構生物研究所 Institute of Bioinformatics and Structural Biology
論文出版年：	2011
畢業學年度：	99
語文別：	英文
論文頁數：	54
中文關鍵詞：	Vacuolar H + -pyrophosphatase 、Site-directed mutagenesis 、Proton translocation 、Tonoplast 、Vacuole 、protein targeting
相關次數：	點閱：3 下載：0
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Vacuolar H+-pyrophosphatase (H+-PPase; EC 3.6.1.1) plays a significant role in the maintenance of the pH in cytoplasm and vacuoles via proton translocation from the cytosol to the vacuolar lumen at the expense of PPi hydrolysis. The topology of H+-PPase as predicted by TopPred II suggests that the catalytic site is putatively located in loop e and exposed to the cytosol. The adjacent transmembrane domain 6 (TM6) is highly conserved and believed to participate in the catalytic function and conformational stability of H+-PPase. In this study, alanine-scanning mutagenesis along TM6 of the mung bean H+-PPase was carried out to identify its structural and functional role. Mutants Y299A, A306S and L317A exhibited gross impairment in both PPi hydrolysis and proton translocation. Meanwhile, mutations at L307 and N318 completely abolished the targeting of the enzyme, causing broad cytosolic localization and implicating a possible role of these residues in protein translocation. The location of these amino acid residues was on the same side of the helix wheel, suggesting their involvement in maintaining the stability of enzyme conformation. G297A, E301A and A305S mutants showed declines in proton translocation but not in PPi hydrolysis, consequently resulting in decreases in the coupling efficiency. These amino acid residues cluster at one face of the helix wheel, indicating their direct/indirect participation in proton translocation. Taken together, these data indicate that TM6 is crucial to vacuolar H+-pyrophosphatase, probably mediating protein targeting, proton transport, and the maintenance of enzyme structure.

Abbreviations………………………………………………………………………….1
Introduction…………………………………………………………………………...2
Materials and Methods
1. Microorganisms and plasmids……………………………………………...6
2. Mutagenesis and DNA manipulation………………………………………7
3. Preparation of microsomes and protein determination………………....10
4. Enzyme assay－PPi hydrolysis activity………………………………...…11
5. Measurement of PPi-dependent H+-translocation……..………………....11
6. SDS-PAGE and Western blotting analysis……………………………….12
7. Immunofluorescence……………………………………………………….13
8. Trypsin proteolysis………………………………………………………...14
9. Control over K+, Na+, and Ca2+ contamination…………………………..14
Results
1. Expression and function of TM6 mutants………………………………..15
2. Subcellular localization of H+-PPase in mutants………………………...18
3. Proteolytic analysis of H+-PPase…………………………………….....….19
4. The characteristics of TM6 mutants……………………………………...20
Discussions………………...…………………………………………………………24
References……………………………………………………………………………30
Table and Figures
Table 1. Kinetic parameters of mutant H+-PPases…….…………………...37
Fig. 1. The predicted topology of H+-PPase…………..……………………..39
Fig. 2. The multiple sequence alignment of various organisms……………40
Fig. 3. Heterologous expression of mung bean H+-PPase in yeast…………41
Fig. 4. Heterologous expression of mutant H+-PPases and their enzymatic activities……………………………………………………………….43
Fig. 5. The growth of mutated H+-PPases in selected agar plate………..…45
Fig. 6. Digestion of plasmid encoding wild-type and mutant H+-PPases
by restriction enzyme………………………………………………...46
Fig. 7. The expression pattern shown by SDS-PAGE………………………47
Fig.8. Relative coupling efficiencies of H+-PPase mutants………………....48
Fig. 9. The subcellular localization of mutant H+-PPases by indirect
immunofluorescence………………………………………………….49
Fig. 10. Recognition of mutant H+-PPase in soluble or membrane fractions
………………………………………………………………………….50
Fig. 11. The proteolytic analysis of H+-PPase……………..………………...51
Fig. 12. Ion effects on H+-PPase………..………………………………….....52
Fig. 13. Helix wheel of TM6………………………………………………….53

                                

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