質子傳送無機焦磷酸水解酶是植物液泡上維持酸鹼濃度穩定的關鍵蛋白。該酶透過代謝副產物焦磷酸的水解在細胞質和液泡腔之間產生質子梯度。 質子傳送焦磷酸水解酶在細胞蛋白質的調控已經引起許多工作者數十年的關注，但其機制仍不清楚。在此研究中，我們證明來自阿拉伯芥的質子傳送焦磷酸水解酶，AVP1，是液泡膜上14-3-3蛋白調控的目標蛋白，且分析了所有十二種亞型的14-3-3蛋白與AVP1之交互作用。發現在有14-3-3nu，-mu，-omicron和-iota的存在下，AVP1的酵素活性及其相關的質子傳送能力提高。而在這些14-3-3蛋白中，14-3-3mu對AVP1的酵素與氫離子傳送的偶合效率有最高刺激。再者14-3-3nu，-mu，-omicron和-iota在高濃度的焦磷酸下具保護AVP1的功能，可抑制高濃度自殺基質焦磷酸對AVP1的拮抗作用。而熱曲線則顯示當14-3-3omicron存在下提高了AVP1在高溫劣化的結構穩定性。此外我們發現14-3-3蛋白也可減輕鈉離子對AVP1的抑制，且確定了每個14-3-3蛋白對AVP1的結合位與模體基序。綜合以上研究，我們提出了一種工作模型來闡明14-3-3蛋白刺激AVP1酵素活性的交互作用。

Plant vacuolar H+-transporting inorganic pyrophosphatase (V-PPase; EC 3.6.1.1) is a crucial enzyme that exists on the tonoplast to maintain pH homeostasis across the vacuolar membrane. This enzyme generates proton gradient between cytosol and vacuolar lumen by hydrolysis of a metabolic byproduct, pyrophosphate (PPi). The regulation of V-PPase at protein level has drawn attentions of many workers for decades, but its mechanism is still unclear. In this work, we show that AVP1, the V-PPase from Arabidopsis thaliana, is a target protein for regulatory 14-3-3 proteins at the vacuolar membrane, and all twelve 14-3-3 isoforms were analyzed for their association with AVP1. In the presence of 14-3-3nu, -mu, -omicron, and -iota, both enzymatic activities and its associated proton pumping of AVP1 were increased. Among these 14-3-3 proteins, 14-3-3mu shows the highest stimulation on coupling efficiency. Furthermore, 14-3-3nu, -mu, -omicron, and -iota exerted protection of AVP1 against the inhibition of suicidal substrate PPi at high concentration. Moreover, the thermal profile revealed the presence of 14-3-3omicron improves the structural stability of AVP1 against high temperature deterioration. Additionally, the 14-3-3 proteins mitigate the inhibition of Na+ to AVP1. Besides, the binding sites/motifs of AVP1 were identified for each 14-3-3 protein. Taken together, a working model was proposed to elucidate the association of 14-3-3 proteins with AVP1 for stimulation of its enzymatic activity.

Maeshima M. (2001) Tonoplast transporters: organization and function. Annu Rev Plant Physiol Plant Mol Biol 52:469–497
Drozdowicz YM, Rea PA. (2001) Vacuolar H+-pyrophosphatases: from the evolutionary backwaters into the mainstream. Trends Plant Sci 6:206–211
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
Maeshima M (2000) Vacuolar H+-pyrophosphatase. Biochim Biophys Acta 1465:37–51
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
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 484:399–404
Gordon-Week 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. Plant Physiol 111:195–202
Gaxiola RA, Palmgren MG, Schumacher K (2007) Plant proton pumps. FEBS Lett 581:2204–2214
Hernandez A, Herrera-Palau R, Madronal JM, Albi T, Lopez-Lluch G, Perez-Castineira JR, Navas P, Valverde F, Serrano A (2016) Vacuolar H+-pyrophosphatase AVP1 is involved in amine fungicide tolerance in Arabidopsis thaliana and provides tridemorph resistance in yeast. Front Plant Sci 7:85
Yang H, Zhang X, Gaxiola RA, Xu G, Peer WA, Murphy AS (2014) Over-expression of the Arabidopsis proton-pyrophosphatase AVP1 enhances transplant survival, root mass, and fruit development under limiting phosphorus conditions. J Exp Bot 65:3045–3053
Gonzalez N, Bodt SD, Sulpice R, Jikumaru Y, Chae E, Dhondt S, Daele TV, Milde LD, Weigel D, Kumiya Y, Stitt M (2010) Increased leaf size: different means to an end. Plant Physiol 153:1261–1279
Gaxiola RA, Sanchez CA, Paez-Valencia J, Ayre BG, Elser, JJ (2012) Genetic manipulation of a ”vacuolar”H+-PPase: from salt tolerance to yield enhancement under phosphorus-deficient soils. Plant Physiol 159:3–11
Ferjani A, Segami S, Horiguchi G, Muto Y, Maeshima M, Tsukaya H (2011) Keep an eye on PPi: the vacuolar-type H+-pyrophosphatase regulates postgerminative development in Arabidopsis. Plant Cell 23:2895–2908
Pasapula V, Shen G, Kuppu S, Paez-Valencia J, Mendoza M, Hou P, Chen J, Qiu X, Zhu L, Zhang X, Auld D, Blumwald E, Zhang H, Gaxiola R, Payton P (2011) Expression of an Arabidopsis vacuolar H+-pyrophosphatase gene (AVP1) in cotton improves drought- and salt tolerance and increases fibre yield in the field conditions. Plant Biotechnol J 9:88–99
Gaxiola RA, Rao R, Sherman A, Grisafi P, Alper SL, Fink GR (1999) The Arabidopsis thaliana proton transporters, AtNhx1 and Avp1, can function in cation detoxification in yeast. Proc Natl Acad Sci USA 96:1480–1485
Gaxiola RA, Li J, Undurraga S, Dang LM, Allen GJ, Alper SL, Fink GR (2001) Drought- and salt-tolerant plants result from overexpression of the AVP1 H+-pump. Proc Natl Acad Sci USA 98:11444–11449
Dietz KJ, Tavakoli N, Kluge C, Mimura T, Sharma SS, Harris, GC, Chardonnens, AN, Golldack D (2001) Significance of the V-type ATPase for the adaptation to stressful growth conditions and its regulation on the molecular and biochemical level. J Exp Bot 52:1969–1980
Wang Y, Xu H, Zhang G, Zhu H, Zhang L, Zhang Z, Zhang C, Ma Z (2009) Expression and responses to dehydration and salinity stresses of V-PPase gene members in wheat. J Genet Genom 36:711–720
Zhang J, Li J, Wang X, Chen J (2011) OVP1, a vacuolar H+-translocating inorganic pyrophosphatase (V-PPase), overexpression improved rice cold tolerance. Plant Physiol Biochem 49:33–38
Fu H, Subramanian RR, Masters SC (2000) 14-3-3 proteins: structure, function, and regulation. Annu Rev Pharmacol Toxicol 40:617–647
Obsilova V, Kopecka M, Hosek D, Kacirova M, Kylarova S, Rezabkova L, Obsil T (2014) Mechanisms of the 14-3-3 protein function: regulation of protein function through conformational modulation. Physiol Res 63:(Suppl. 1) S155–164
Yang X, Lee WH, Sobott F, Papagrigoriou E, Robinson, CV, Grossmann JG, Sundstrom M, Doyle DA, Elkins JM (2006) Structural basis for protein-protein interactions in the 14-3-3 protein family. Proc Natl Acad Sci USA 103:17237–17242
Chevalier D, Morris ER, Walker JC (2009) 14-3-3 and FHA domains mediate phosphoprotein interactions. Annu Rev Plant Biol 60:67–91
Muslin AJ, Tanner JW, Allen PM, Shaw AS (1996) Interaction of 14-3-3 with signaling proteins is mediated by the recognition of phosphoserine. Cell 84:889–897
Yaffe MB, Rittinger K, Volinia S, Caron PR, Aitken A, Leffers H, Gambin SJ, Smerdon SJ, Cantley LC (1997) The structural basis for 14-3-3: phosphopeptide binding specificity. Cell 91:961–971
Coblitz B, Wu M, Shikano S, Li M (2006) C-terminal binding: an expanded repertoire and function of 14-3-3 proteins. FEBS Lett 580:1531–1535
Aitken A (2002) Functional specificity in 14-3-3 isoform interactions through dimer formation and phosphorylation. Chromosome location of mammalian isoforms and variants. Plant Mol Biol 50:993–1010
Sehnke PC, Delille JM, Ferl RJ (2002) Consummating signal transduction: the role of 14-3-3 protein in the completion of signal-induced transitions in protein activity. Plant Cell S339–S354
Alsterfjord M, Sehnke PC, Arkell A, Larsson H, Svennelid F, Rosenquist M, Ferl RJ, Sommarin M, Larsson C (2004) Plasma membrane H+-ATPase and 14-3-3 isoforms of Arabidopsis leaves: evidence for isoform specificity in the 14-3-3/H+-ATPase interaction. Plant Cell Physiol 45:1202–1210
Svennelid F, Olsson A, Piotrowski M, Rosenquist M, Ottman C, Larsson C, Oecking C, Sommarin M (1999) Phosphorylation of Thr-948 at the C terminus of the plasma membrane H+-ATPase creates a binding site for the regulatory 14-3-3 protein. Plant Cell 11:2379–2391
Camoni L, Iori V, Marra M, Aducci P (2000) Phosphorylation-dependent interaction between plant plasma membrane H+-ATPase and 14-3-3 proteins. J Biol Chem 275:9919–9923
Klychnikov OI, Li KH, Lill H, de Boer AH (2007) The V-ATPase from etiolated barley (Hordeum vulgare L.) shoots is activated by blue light and interacts with 14-3-3 proteins. J Exp Bot 58:1013–1023
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
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
Huang YT, Liu TH, Lin SM, Chen YW, Pan YJ, Lee CH, Sun YJ, Tseng FG, Pan RL (2013) Squeezing at entrance of proton transport pathway in proton-translocating pyrophosphatase upon substrate binding. J Biol Chem 288:19312–19320
Zheng J, Du GG, Anderson CT, Keller JP, Orem A, Dallos P, Cheatham M (2006) Analysis of the oligomeric structure of the motor protein prestin. J Biol Chem 281:19916–19924
Laemmli UK (1970) Cleavage of structure proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685
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
Yang SJ, Jiang SS, Hsiao YY, Van RC, Pan YJ, Pan RL (2004) Thermoinactivation analysis of vacuolar H+-pyrophosphatase. Biochim Biophys Acta 1656:88–95
Zhen RG, Kim EJ, Rea PA (1997) Acidic residues necessary for pyrophosphate-energized pumping and inhibition of the vacuolar H+-pyrophosphatase by N,N'-dicyclohexylcarbodiimide. J Biol Chem 272:22340–22348
Pathirana RD, O’Brien-Simpson NM, Veith PD, Riley PF, Reynolds EC (2006) Characterization of proteinase-adhesin complexes of Porphyromonas gingivalis. Microbiology 152, 2381–2394
Lo YY, Hsu, SH, Ko YC, Hung CC, Chang MY, Hsu HH, Pan MJ, Chen YW, Lee CH, Tseng FG, Sun YJ, Yang CW, Pan RL (2013) Essential calcium-binding cluster of Leptospira LipL32 protein for inflammatory responses through the toll-like receptor 2 pathway. J Biol Chem 288:12335–12344
Fullone MR, Visconti S, Marra M, Fogliano V, Aducci P (1998) Fusicoccin effect on the in vitro interaction between plant 14-3-3 proteins and plasma membrane H+-ATPase. J Biol Chem 273:7698–7702
Huber SC, MacKintosh C, Kaiser WM (2002) Metabolic enzymes as targets for 14-3-3 proteins. Plant Mol Biol 50:1053–1063
Jaspert N, Throm C, Oecking C (2011) Arabidopsis14-3-3 proteins: fascinating and less fascinating aspects. Front Plant Sci 2:96
Bunney TD, van Walraven HS, de Boer AH (2001) 14-3-3 protein is a regulator of the mitochondria and chloroplast ATP synthase. Proc Natl Acad Sci USA 98:4249–4254
Fuglsang AT, Borch J, Bych K, Jahn TP, Roepstorff P, Palmgren MG (2003) The binding site for regulatory 14-3-3 protein in plant plasma membrane H+-ATPase: involvement of a region promoting phosphorylation-independent interaction in addition to the phosphorylation-dependent C-terminal end. J Biol Chem 278:42266–42272
Al-Bataineh M, Li H, Marciszyn A, Bhalla V, Hallows K, Pastor-Soler N (2015) AMPK regulates the vacuolar H+-ATPase via 14­3-3 proteins. J FASEB 29: Supplement 969.23
Efendiev R, Chen Z, Krmar RT, Uhles S, Katz AI, Pedemonte CH, Bertorello AM (2005) The 14-3-3 protein translates the Na+, K+-ATPase 1-subunit phosphorylation signal into binding and activation of phosphoinositide 3-kinase during endocytosis. J Biol Chem 280:16272–16277