Tài liệu Báo cáo khoa học: Characterization of the interaction between the plasma membrane H+-ATPase

Characterization of the interaction between the plasma

membrane H+

-ATPase of Arabidopsis thaliana and a novel

interactor (PPI1)

Corrado Viotti, Laura Luoni, Piero Morandini and Maria Ida De Michelis

Dipartimento di Biologia ‘L. Gorini’, Universita` di Milano, CNR Istituto di Biofisica – Sezione di Milano, Italy

The H+-ATPase is the major electrogenic pump in the

plasma membrane (PM) of plant cells. By pumping

protons from the cytoplasm to the apoplast it gener￾ates an electrochemical proton gradient, which drives

the transport of mineral ions and organic solutes, and

plays a crucial role in cytoplasmic and apoplastic pH

homeostasis [1,2]. The PM H+-ATPase participates in

a variety of physiological processes such as phloem

loading, stomata opening, mineral nutrition, growth of

root hairs and pollen tubes, salt and osmotolerance,

leaf movements, and acid growth [1,2]. In vivo, its

activity is modulated by several signals such as hor￾mones (auxin, abscisic acid), light, water potential,

acid load, toxins like fusicoccin (FC) and pathogens,

but a molecular description of the mediators involved

is missing for most of these signals [1,2].

Plant genomes contain a large family of PM H+-

ATPase genes (12 in Arabidopsis thaliana, 10 in rice

and nine in Nicotiana plumbaginifolia), which can be

grouped in five clusters based on sequence alignments

and intron positions [3,4]. Individual isoforms exhibit

tissue- and developmental-specific expression patterns

and a number of quantitative differences in catalytic

and regulatory properties [1–4]. Thus, the first regula￾tion of proton pumping activity in different cells types

and physiological conditions takes place at both the

transcriptional and translational levels [1–4].

As to post-translational regulation, the best-known

mechanism described to date involves the auto-inhibi￾tory action of the C-terminal domain. The plant PM

H+-ATPase is a P-type ATPase with an extended

(approximately 100 amino acids) cytosolic C-terminus

containing two inhibitory regions. Proteolytic cleavage

or genetic deletion of the C-terminus has little effect

on enzyme activity at the acidic pH optimum (pH 6.4–

6.6), but markedly increases enzyme activity in the

physiological range of cytoplasmic pH values (pH 7.0–

7.5), resulting in an alkaline shift of the pH optimum

Keywords

Arabidopsis thaliana; H+

-ATPase; plasma

membrane; PPI1; 14-3-3 proteins

Correspondence

M. I. De Michelis, Dipartimento di Biologia

‘L. Gorini’, Universita` di Milano, CNR Istituto

di Biofisica – Sezione di Milano, via G.

Celoria 26, 20133 Milano, Italy

Fax: +39 02 50314815

Tel: +39 02 50314822

E-mail: [email protected]

(Received 28 July 2005, revised 13 September

2005, accepted 20 September 2005)

doi:10.1111/j.1742-4658.2005.04985.x

Proton pump interactor, isoform 1 (PPI1) is a novel interactor of the C-ter￾minus of Arabidopsis thaliana plasma membrane H+-ATPase (EC 3.6.3.6)

(Morandini P, Valera M, Albumi C, Bonza MC, Giacometti S, Ravera G,

Murgia I, Soave C & De Michelis MI (2002) Plant J 31, 487–497). We pro￾duced two fusion proteins consisting of, respectively, the first 88 amino

acids or the entire protein deleted of the last 24 hydrophobic amino acids,

and we show that the latter protein has a threefold higher affinity for the

H+-ATPase. PPI1-induced stimulation of H+-ATPase activity dramatically

decreased with the increase of pH above pH 6.8, but became largely

pH-independent when the enzyme C-terminus was displaced by fusicoccin￾induced binding of 14-3-3 proteins. The latter treatment did not affect

PPI1 affinity for the H+-ATPase. These results indicate that PPI1 can bind

the H+-ATPase independently of the C-terminus conformation, but is not

able to suppress the C-terminus auto-inhibitory action.

Abbreviations

Brij 58, polyoxyethilene 20 cethyl ether; BTP, bis tris propane {1,3-bis[tris(hydroxymethyl)methylamino]propane}; FC, fusicoccin; GST,

glutathione S-transferase; IPTG, isopropyl thio-b-D-galactoside; NTA, nitrilotriacetic acid; PM, plasma membrane.

5864 FEBS Journal 272 (2005) 5864–5871 ª 2005 FEBS