探討第三穿膜區於綠豆質子傳送焦磷酸水解酶扮演的功能性角色

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研究生：	李慶宏 Lee, Ching-Hung
論文名稱：	探討第三穿膜區於綠豆質子傳送焦磷酸水解酶扮演的功能性角色 Functional Investigation of Transmembrane Helix 3 in Mung Bean H+-translocating Pyrophosphatase
指導教授：	潘榮隆 Pan, Rong-Long
口試委員:	林彩雲 Lin, Tsai-Yun 簡麗鳳 Chien, Lee-Feng 蘇士哲 Sue, Shih-Che 張文綺 Chang, Wen-Chi
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：	2013
畢業學年度：	102
語文別：	英文
論文頁數：	41
中文關鍵詞：	質子運送焦磷酸水解酶、質子運送、穿膜區、定點突變、耦合效率、GxxxG-like 區位
外文關鍵詞：	Proton-translocating pyrophosphatase, Proton transport, Transmembrane helix, Site-directed mutagenesis, Coupling efﬁciency, GxxxG-like motif
相關次數：	點閱：1 下載：0
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植物質子傳送焦磷酸水解酶藉由水解焦磷酸將質子運送至液泡內，在液泡酸化上扮演重要角色。綠豆質子傳送焦磷酸水解酶包含16個穿膜區，其中第3穿膜區的親水性較其他大部分穿膜區都來得高，而且其上的胺基酸在植物中高度保守。此外對此酵素功能相當重要之內圈上的第5及6穿膜區鄰近第3穿膜區，因此第3穿膜區可能和質子傳送焦磷酸水解酶的活性相關，為了驗證此可能性，本研究利用定點突變法探討第3穿膜區在質子傳送焦磷酸水解酶上所扮演的角色。丙胺酸(絲胺酸)定點取代實驗結果顯示邊鏈朝向內圈的T138及S142可能和質子傳送效率相關。位在2個GxxxG-like功能區重要端點位置的G149/S153及G160/A164，對維持酵素活性及構形穩定是不可或缺的。此外G149鄰近區域的穩定度對蛋白質有效表達是很重要的。S153、M161、A164對於鉀離子促進質子傳送焦磷酸水解酶活性亦很重要。本研究結果顯示第3穿膜區對於質子傳送焦磷酸水解酶的焦磷酸水解、質子運送、蛋白表現及鉀離子刺激佔有必要地位。

H+-translocating pyrophosphatase (H+-PPase; EC 3.6.1.1) is critical for acidifying lumens by translocating protons across membranes at the expense of pyrophosphate (PPi) hydrolysis. Vigna radiata H+-PPase (VrH+-PPase) contains 16 transmembrane helices (TMs). The amino acids in TM3 are highly conserved in plants, and the hydrophobicity of TM3 is relatively lower than most other TMs, and. Moreover, TM5 and 6, which are the core TMs involving in H+-PPase functions are in the vicinity of TM3. It is thus speculated that TM3 is related with the H+-PPase activities. To address this possibility, site-directed mutagenesis was applied in this study to determine the role of TM3 in VrH+-PPase. Upon alanine/serine substitution, T138 and S142, whose functional groups face toward the center TMs were shown to be involved in efficient proton transport. G149/S153 and G160/A164 pairs at the critical termini of the two GxxxG-like motifs are necessary in maintaining the enzymatic activities and conformational stability. Moreover, stability in the vicinity surrounding G149 is vital for efficient expression. S153, M161, and A164 are crucial for the K+-mediated stimulation of H+-PPase. Taken together, this study demonstrates that TM3 plays essential roles in PPi hydrolysis, proton transport, expression, and K+ stimulation of H+-PPase.

Abbreviations ………………………………………………………...1
Introduction …………………………………………………………..2
Experimental Procedures
Mutagenesis and DNA construction ………………………………..4 
Expression in Yeasts and Preparation of Microsomes ……………..4
Assay of Protein Concentration and Enzymatic Activities …………6
SDS-PAGE and Western Blotting Analysis ………………………...6
Trypsin Proteolysis …………………………………………………7
Determination of Background Ion Concentrations ………………...7
Results and Discussion
Expression and Enzymatic Activities of TM3 Mutants ……………..8
Ion Effects …………………………………………………………10
Leucine Substitution at the Termini of GxxxG-like Motifs ………..11
Trypsinolysis of Leucine mutants at the Termini of GxxxG-like Motifs ……………………………………………………………..14
Conclusion ………………………………………...…………………16
References ……………………………………………………………17
Figures 
Fig.    1.    The multiple sequence alignment of various H+-PPases and X-ray crystal structure of VrH+-PPase ………………................23
Fig.    2.    The expression and enzymatic activities of the H+-PPase variants ………………………………………………………….25
Fig. 3.    Ion effects on H+-PPase …………………………………..27
Fig. 4.    Expression and activities of alanine and leucine-substituted variants mutated at the termini of the GxxxG-like motifs………………………………………………………….....29
Fig. 5.    Trypsinolysis of alanine and leucine-substituted mutants at the termini of the GxxxG-like motifs…………………………….31
Fig. 6.    Helix wheel of TM3 showing residues related to the enzymatic activities and proton transport of H+-PPase ……......32
Appendix
Table S1. The parameters for the enzymatic activities of the WT and    variants mutated at amino acids in TM3………………...33
Table S2. PPi hydrolysis and proton pumping activities of variants mutated at the termini of the putative GxxxG and GxxxG-like motifs in TM5 and 6, respectively…………...36
Table S3. PPi hydrolysis activities of mutants mutated at residues along the two GxxxG-like motifs in TM3 and their putative interacting residues in TM5 and 6……………………….37
Fig S1.   Hydropathic plot of H+-PPase ………………………….38
Fig S2.    The dynamic cross-correlation of each four putative proton transport residues (R242, D294, E301, K742) to the other residues in VrH+-PP……………………………………...39
Fig S3.    X-ray crystal structure of H+-PPase showing possible interactions between its TM3 and TM5 and 6….………...41

                                

Bakan A, Meireles LM, Bahar I (2011) ProDy: protein dynamics inferred from theory and experiments. Bioinformatics 27:1575–1577

Barik S, (1997) In: White BA (ed) PCR cloning protocols: from molecular cloning to genetic engineering. Humana Press, New Jersey, pp 173–182

Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye binding. Anal Biochem 72:248–254

Curran AR, Engelman DM (2003) Sequence motifs, polar interactions and conformational changes in helical membrane proteins. Curr Opin Struct Biol 13:412–417

Dawson JP, Weinger JS, Engelman DM (2002) Motifs of serine and threonine can drive association of transmembrane helices. J Mol Biol 316:799–805

Drozdowicz YM, Kissinger JC, Rea PA (2000) AVP2, a sequence-divergent, K+- insensitive H+-translocating inorganic pyrophosphatase from Arabidopsis. Plant Physiol 123:353–362

Drozdowicz YM, Rea PA (2001) Vacuolar H+-pyrophosphatases: from the evolutionary backwaters into the mainstream. Trends Plant Sci 6:206–211

Duan P, Wu J, You G (2011) Mutational analysis of the role of GXXXG motif in the function of human organic anion transporter 1 (hOAT1). Int J Biochem Mol Biol 2:1–7

Faingold O, Cohen T, Shai Y (2012) A GxxxG-like motif within HIV-1 fusion peptide is critical to its immunosuppressant activity, structure, and interaction with the transmembrane domain of the T-cell receptor. J Biol Chem 287:33503–33511

Gasteiger E, Hoogland C, Gattiker A, Duvaud S, Wilkins MR, Appel RD, Bairoch A (2005) In: Walker JM (ed) The proteomics protocols handbook. Humana Press, New Jersey, pp 571–607

Gietz RD, Schiestl RH, Willems AR, Woods RA (1995) Studies on the transformation of intact yeast cells by the LiAc/SS-DNA/PEG procedure. Yeast 11:355–360

Gordon-Weeks R, Steele SH, Leigh RA (1996) The role of magnesium, pyrophosphate, and their complexes as substrates and activators of the vacuolar H+-pumping inorganic pyrophosphatase (studies using ligand protection from covalent inhibitors). Plant Physiol 111:195–202

Hsiao YY, Van RC, Hung SH, Lin HH, Pan RL (2004) Roles of histidine residues in plant vacuolar H+-pyrophosphatase. Biochim Biophys Acta 1608:190–199

Hsiao YY, Pan YJ, Hsu SH, Huang YT, Liu TH, Lee CH, Lee CH, Liu PF, Chang WC, Wang YK, Chien LF, Pan RL (2007) Functional roles of arginine residues in mung bean vacuolar H+-pyrophosphatase. Biochim Biophys Acta 1767:965–973

Huang YT, Liu TH, Chen YW, Lee CH, Chen HH, Huang TW, Hsu SH, Lin S M, Pan YJ, Lee CH, Hsu IC, Tseng FG, Fu CC, Pan RL (2010) Distance variations between active sites of H+-pyrophosphatase determined by fluorescence resonance energy transfer. J Biol Chem 285:23655–23664

Hung SH, Chiu SJ, Lin LY, Pan RL (1995) Vacuolar H+-pyrophosphatase cDNA (Accession No. U31467) (PGR 95-082) from etiolated mung bean seedlings. Plant Physiol 109:1125–1127

Kim EJ, Zhen RG, Rea PA (1994) Heterologous expression of plant vacuolar pyrophosphatase in yeast demonstrates sufficiency of the substrate-binding subunit for proton transport. Proc Natl Acad Sci USA 91:6128–6132

Kim EJ, Zhen RG, Rea PA (1995) Site-directed mutagenesis of vacuolar H+-pyrophosphatase. Necessity of Cys634 for inhibition by maleimides but not catalysis. J Biol Chem 270:2630–2635

Kirsch RD, Joly E (1998) An improved PCR-mutagenesis strategy for two-site mutagenesis or sequence swapping between related genes. Nucleic Acids Res 26:1848–1850

Kyte J, Doolittle RF (1982) A simple method for displaying the hydropathic character of a protein. J Mol Biol 157:105–132

Laemmli UK (1970) Cleavage of structure proteins during the assembly of the head of bacteriophage T4. Nature 222:680–685

Lee CH, Pan YJ, Huang YT, Liu TH, Hsu SH, Lee CH, Chen YW, Lin SM, Huang LK, Pan RL (2011) Identification of essential lysines involved in substrate binding of vacuolar H+-pyrophosphatase. J Biol Chem 286:11970–11976

Lezon TR, Bahar I (2012) Constraints imposed by the membrane selectively guide the alternating access dynamics of the glutamate transporter GltPh. Biophys J 102:1331–1340

Lin SM, Tsai JY, Hsiao CD, Huang YT, Chiu CL, Liu MH, Tung JY, Liu TH, Pan RL, Sun YJ (2012) Crystal structure of a membrane-embedded H+-translocating pyrophosphatase. Nature 484:399–403

Liu TH, Hsu SH, Huang YT, Lin SM, Huang TW, Chuang TH, Fan SK, Fu CC, Tseng FG, Pan RL (2009) The proximity between C-termini of dimeric vacuolar H+-pyrophosphatase determined using atomic force microscopy and a gold nanoparticle technique. FEBS J 276:4381–4394

Lo A, Cheng CW, Chiu YY, Sung TY, Hsu WL (2011) TMPad: an integrated structural database for helix-packing folds in transmembrane proteins. Nucleic Acids Res 39:D347–355

MacKenzie KR, Prestegard JH, Engelman DM (1997) A transmembrane helix dimer: structure and implications. Science 276:131–133

Maeshima M (1991) H+-translocating inorganic pyrophosphatase of plant vacuoles. Inhibition by Ca2+, stabilization by Mg2+ and immunological comparison with other inorganic pyrophosphatases. Eur J Biochem 196:11–17

Maeshima M (2000) Vacuolar H+-pyrophosphatase. Biochim Biophys Acta 1465:37–51

Maeshima M (2001) Tonoplast transporters: organization and function. Annu Rev Plant Physiol Plant Mol Biol 52:469–497

Mimura H, Nakanishi Y, Hirono M, Maeshima M (2004) Membrane topology of the H+-pyrophosphatase of Streptomyces coelicolor determined by cysteine-scanning mutagenesis. J Biol Chem 279:35106–35112

Nakanishi Y, Saijo T, Wada Y, Maeshima M (2001) Mutagenic analysis of functional residues in putative substrate-binding site and acidic domains of vacuolar H+-pyrophosphatase. J Biol Chem 276:7654–7660

Pan YJ, Lee CH, Hsu SH, Huang YT, Lee CH, Liu TH, Chen YW, Lin SM, Pan RL (2011) The transmembrane domain 6 of vacuolar H+-pyrophosphatase mediates protein targeting and proton transport. Biochim Biophys Acta 1807:59–67

Polgar O, Robey RW, Morisaki K, Dean M, Michejda C, Sauna ZE, Ambudkar SV, Tarasova N, Bates SE (2004) Mutational analysis of ABCG2: role of the GXXXG motif. Biochemistry 43:9448–9456

Russ WP, Engelman DM (2000) The GxxxG motif: a framework for transmembrane helix-helix association. J Mol Biol 296:911–919

Schneider D, Engelman DM (2004) Involvement of transmembrane domain interactions in signal transduction by α/β integrins. J Biol Chem 279:9840–9846

Senes A, Engel DE, DeGrado WF (2004) Folding of helical membrane proteins: the role of polar, GxxxG-like and proline motifs. Curr Opin Struct Biol 14:465–479

Serrano A, Perez-Castineira JR, Baltscheffsky H, Baltscheffsky M (2004) Proton-pumping inorganic pyrophosphatases in some archaea and other extremophilic prokaryotes. J Bioenerg Biomembr 36:127–133

Subramaniam S (1998) The biology workbench-a seamless database and analysis environment for the biologist. Proteins 32:1–2

Tirion MM (1996) Large amplitude elastic motions in proteins from a single parameter, atomic analyses. Phys Rev Lett 77:1905–1908

Unterreitmeier S, Fuchs A, Schaffler T, Heym RG, Frishman D, Langosch D (2007) Phenylalanine promotes interaction of transmembrane domains via GxxxG motifs. J Mol Biol 374:705–718

Van RC, Pan YJ, Hsu SH, Huang YT, Hsiao YY, Pan RL (2005) Role of transmembrane segment 5 of the plant vacuolar H+-pyrophosphatase. Biochim Biophys Acta 1709:84–94

Yang SJ, Jiang SS, Kuo SY, Hung SH, Tam MF, Pan RL (1999) Localization of carboxylic residues possible involved in the inhibition of vacuolar H+-pyrophosphatase by N, N′-dicyclohexylcarbodiimide. Biochem J 342:641–646

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