ABSTRACT A pea actin isoform PEAc1 with green fluorescent protein (GFP) fusion to its C-terminus and His-tag to its N-terminus, was expressed in prokaryotic cells in soluble form, and highly

purified with Ni-Chelating Sepharose™ Fast Flow column. The purified fusion protein (PEAc1-GFP) efficiently inhibited DNase I activities before polymerization, and activated the myosin

Mg-ATPase activities after polymerization. The PEAc1-GFP also polymerized into green fluorescent filamentous structures with a critical concentration of 0.75 μM. These filamentous structures

were labeled by TRITC-phalloidin, a specific agent for staining actin microfilaments, and identified as having 9 nm diameters by negative staining. These results indicated that PEAc1

function _in vivo_. SIMILAR CONTENT BEING VIEWED BY OTHERS BINDING OF S100A6 TO ACTIN AND THE ACTIN–TROPOMYOSIN COMPLEX Article Open access 30 July 2020 ALLOSTERIC REGULATION CONTROLS

ACTIN-BUNDLING PROPERTIES OF HUMAN PLASTINS Article 19 May 2022 AUTOREGULATION AND DUAL STEPPING MODE OF MYA2, AN _ARABIDOPSIS_ MYOSIN XI RESPONSIBLE FOR CYTOPLASMIC STREAMING Article Open

access 24 February 2022 INTRODUCTION Actin is one of the major cytoskeleton proteins and plays important roles in a variety of subcellular processes, such as cytoplasmic streaming 1, 2, 3,

organellar and nuclear positioning 4, 5, 6, cellular morphogenesis 7, 8, 9, and cell division 10, 11, 12. The diversity of these functional roles reflects the diversity in plant _actin_ gene

families 13, 14. In plants, actins are encoded by gene families that are much more diverse than those in other eukaryotes. Eight actin homologues in _Arabidopsis_ are grouped into two major

phylogenetic classes (vegetative and reproductive) and five subclasses were found 13, 14. Three kinds of actin isoform genes (_PEAc1, PEAcII, PEAcIII_) were found in pea (_Pisum sativum L_)

15. _PEAc1_ has the highest identity about 97.3% with _ACT7_ among all the actin isoforms of _Arabidopsis_ (DNA Star). Actin isovariants in plants differ in their expression patterns 16,

17, 18, and their dynamics are thought to expand the biochemical processes that buffer the responses of plant cells to internal or external signals14. Recently, Kandasarmy _et al_ 19

demonstrated that a plant actin isovariant (ACT7) was induced by auxin and was required for normal callus formation. Further understanding of the specificity in biochemical and cell

biological characteristics among actins will require a detailed analysis of each kind of actin isoform. To elucidate the biochemical properties of plant actin, previous studies have focused

on actin purification directly from plant materials 20, 21. The low abundance of actin and the presence of proteases in most plant materials made it difficult to obtain sufficient highly

purified actin for _in vitro_ studies 22, 23. Although some methods have been developed to get relatively high yields of actin from plant pollens 24, 25, it is still very difficult to purify

actin (especially actin isoforms) from most of the plants 26. Expression of proteins in _E. coli_ has greatly facilitated our understanding of physicochemical properties of proteins

including plant actin binding protein 27, 28. Unfortunately, there is still no successful example of the expression of active eukaryotic actin in _E. coli_. The failure is generally

considered to be due to the lack of appropriate molecular chaperones for proper actin folding 29, 30 and that actin is unable to spontaneously refold into a functional form after

denaturation. Microinjection of fluorescent phalloidin as a specific probe for F-actin has shown to be an effective method for _in vivo_ labeling of actin filaments in living plant cells 10,

31, but unsuitable for labeling specific actin isoforms. Since GFP was used as a non-invasive labeling marker to localize specific proteins in living cells 32, GFP fusion has been

successfully applied to light up microfilaments in animal cells 33, 34, 35. Kost _et al_ 36 observed the distri-bution of microfilaments in tobacco pollens and suspension cells with the

assistance of GFP-labeled talin, one of the microfilament binding proteins, and found GFP-fused talin satisfactorily revealed the location of microfilaments in cells. It is challenging to

elucidate the biochemical characte-ristics and dynamic distributions for each kind of plant actin isoform. Here we report that PEAc1, a pea tendril actin isoform, was expressed with GFP

fusion in soluble form in _E. coli_, and the product was purified and physico- and biochemically characterized. The fusion protein was also expressed in tobacco suspension cells. MATERIALS

AND METHODS MATERIALS The _PEAc1_ cDNA was cloned from pea (_Pisum sativum L_.) tendril 37 (GenBank accession number X67666). The prokaryotic expression vector pET-30 (a+) was from Novagen

Inc. (Madison, WI) and the plant binary vector pBI121 was from Clontech (Palo Alto, CA). Primers were synthesized by the Gene Center of Institute of Microbiology, Chinese Academy of Science

(Beijing, China). Ni-Chelating Sepharose™ Fast Flow was purchased from Amersham Pharmacia (Uppsala, Sweden). Monoclonal mouse anti-GFP and anti-His-tag antibodies, and HRP-conjugated goat

anti-mouse (or anti-rabbit) IgG were from Sigma (St. Louis, MO). The rabbit anti-serum against PEAc1 was prepared in our lab using PEAc1 whole protein. DNA restriction enzymes, ligase and

polymerase were from Bio-Lab (Hercules, CA). DNA purification kit was from Shanghai Sangon Company (Shanghai, China). PLASMID CONSTRUCTION The coding region (1131 bp) of _PEAc1_ was

amplified from _PEAc1_ full length cDNA 37 using two primers containing restriction sites (_Bam_HI-forward, 5′-gcggatccatggccgatgctgaggatat-3′; _Kpn_ I-reverse,

5′-gcggtaccgaagcattttctgtggacaat-3′). The GFP coding region (723 bp) was PCR amplified from psm-RS-GFP (donated by American _Arabidopsis_ Biological Resource Center) using two primers (_Kpn_

I-forward, 5′-gcggtaccatgagtaaaggagaagaact-3′; _Sac_I-reverse, 5′-gcgagctcttatttgtatagtt-3′). The restriction fragments corresponding to PEAc1 and GFP were inserted into _Bam_HI/_Sac_I site

of pET30 (a+). The recombined plasmid was transformed into _E.coli_ BL21 cells. pET-GFP vector containing the fragment of GFP served as a control. All constructs were verified by

sequencing. For the expre-ssion in tobacco BY-2 cells, the _PEAc1-GFP_ fusion gene was inserted into _Bam_HI_/Sac_I site of pBI121 to yield pBAc1-GFP vector. PROKARYOTIC EXPRESSION AND

PURIFICATION OF RECOMBINANT PROTEIN Transformed _E. coli_ BL21 cells were cultured in liquid LB medium (with 50 μg/ml kanamycin) at 37°C to OD600= 0.6, then at 25°C to OD600= 0.8. The

expression of PEAc1-GFP was induced by addition of 0.25 mM isopropyl-β-D-thiogalactoside (IPTG) and a further shaking at 22°C for 20 h. After identification under the fluorescence

microscope, the cells were harvested and sonicated on ice in buffer A (2 mM Tris, pH 8.0, 0.2 mM CaCl2, 0.2 mM ATP, 0.5 mM PMSF, 0.2 mM NaN3). All subsequent steps were performed at 4°C. The

cell extracts were centrifuged at 10,000 g for 20 min. The resultant supernatants were applied to a self-packed 1 ml Ni-resins pre-equilibrated with buffer A. Following serial washing with

10, 50 and 100 mM imidazole (in buffer A, 5 ml each), the His-tagged fusion protein was eluted with 5 ml 300 mM imidazole (in buffer A). To obtain the GFP protein, the cells containing

pET-GFP vector were induced by 0.1 mM IPTG and cultured at 37°C for 3 h. Based on SDS-PAGE and western blot analysis, the fractions containing purified fusion protein were pooled and

dialyzed against buffer B (buffer A plus 0.5 mM DTT). The concentration of protein extracts was determined using the Bio-Rad protein assay kit with bovine serum albumin (BSA) as the

standard. POLYMERIZATION _IN VITRO_ AND CRITICAL CONCENTRATION DETERMI-NATION Actin polymerization and steady state polymer levels were measured by 90o light scattering 38. Fluorescence

changes were monitored in a fluorescence spectrophotometer (F-4500, Hitachi) with the excitation monochromator set at 450 nm and the emission monochromator set at the same wavelength. Before

polymerization, the freshly purified PEAc1-GFP was clarified by centrifugation at 200,000 g for 90 min at 4°C. The polymerization of 80 μg/ml (about 1.1 μM) PEAc1-GFP monomers was initiated

by the addition of KCl to 50 mM and MgCl2 to 2 mM in buffer B at 21°C. For green fluorescence detection of polymerized products, each sample was directly observed under a fluorescent

microscope (LEICA, DMIRB) equipped with Cooled CCD (Photometrix, KAF1400-G2, Sensys). For critical concentration determination, samples containing 1-6 μM chicken muscle actin or PEAc1-GFP in

8 ml of buffer B were prepared. Then at time zero, salts were added to give a final concentration of 2 mM MgCl2 and 50 mM KCl in 7 ml of reaction systems. The remaining 1 ml of each sample

without these two salts was used as control. The polymerization was allowed to proceeding at 4°C overnight (approximately 16 h). The amount of polymerized filaments of each reaction sample

at steady state equilibrium was measured in F-4500. INHIBITION OF DNASE I WITH PEAC1-GFP MONOMERS The inhibition assay of DNase I (Roche) activities by PEAc1-GFP monomers was according to

the procedure of Fox _et al_ 39. 40 μM PEAc1-GFP monomers or chick muscle G-actin was prepared in 1 ml reaction system (125 mM Tris, pH 7.5, 5 mM MgCl2, 2 mM CaCl2, 3 mM NaN3). The

substrate, salmon sperm DNA (Sigma), was added to a final concentration of 0.1 mg/ml. The reaction was started by the addition of an equal molecular amount DNase I at 21°C. The optical

absorption was recorded at 260 nm for 30 min with a Nucleic Acid and Protein Analyzer (Beckman, DU640). ACTIVATION OF MUSCLE MYOSIN MG-ATPASE ACTIVITY BY FILAMENTOUS P EAC1-GFP ATPase

activity of myosin prepared from the chick muscle, was measured according to the method of Pollard 40 with some modifications. The reaction mixtures for the assay of Mg-ATPase activity

contained 38 mM imidazole, pH 7.0, 25 mM KCl, 0.8 mM MgCl2, 0.1 mM CaCl2, and 0.06 mg/ml myosin, in the presence or absence of 0.5 μM filamentous PEAc1-GFP or muscle F-actin. Reactions were

started by the addition of ATP (2 mM). The inorganic phosphate in reaction mixtures was determined as described by Le Bel _et al_ 41. Chick muscle F-actin was prepared by the methods of

Pardee and Spudich 42. SDS-PAGE AND WESTERN BLOT ANALYSIS Proteins were separated on 10% SDS-PAGE 43, and analyzed by western blot according to Towbin _et al_ 44. Mouse anti-His-tag (with

1:6000 dilute) and anti-GFP (with 1:8000 dilute) monoclonal antibodies and rabbit anti-PEAc1 serum (with 1:4000 dilute) were used as the primary antibodies, while goat anti-mouse (or

anti-rabbit) IgG-conjugated with HRP (with 1:4000 dilute) were used as the secondary antibodies. The membranes were visualized by using an enhanced Lumi-Light western blotting Substrate kit

(Roche, Indianapolis, IN), following the manufacturer's instructions. PHALLOIDIN-STAINING OF FILAMENTOUS PEAC1-GFP The polymerized samples were dropped on poly-L-lysine (Sigma) coated

slides, stained with tetramethyrhodamine isothiocyanate (TRITC)-phalloidin (5 μg/ml in buffer B, plus 50 mM KCl and 2 mM MgCl2) for 30 min at room temperature in a wet-atmosphere. Then, the

same volume of fluorescence antifade reagent (Bio-Rad) was added. The polymerization was monitored with a microscope as described above. EUKARYOTIC EXPRESSION OF PEAC1-GFP IN TOBACCO BY2

CELLS The pBAc1-GFP plasmid was transformed into tobacco suspension BY2 cells by microprojected bombardment with a Bio-Rad PDS-1000/He. The cells were cultured in MS medium supplemented with

0.6 μg/ml 2, 4-D, pH 5.8, and 100 μg/ml Kanamycin at 25°C. The GFP fluorescence from PEAc1-GFP fusion proteins was monitored. ELECTRON MICROSCOPY For the measurement of filament diameter,

the samples containing polymerized PEAc1-GFP were applied to formvar grids and negatively stained with 2% uranyl acetate. The specimens were visualized by using a Hitachi H7500 electron

microscope. RESULTS PROKARYOTIC SOLUBLE EXPRESSION AND PURIFICATION OF PEAC1-GFP FUSION PROTEIN The coding region of _PEAc1_ was PCR amplified, with GFP fused to its C-terminus, and a

6×his-tag fused to its N-terminus.The prokaryotic expression plasmid was introduced into _E. coli_ cells. For the expression of the fusion gene, different inducing conditions were tested.

The strong green fluorescence could be detected after the cells were induced with 0.25 mM IPTG at 22°C for 20 h (data not shown). Protein analysis by SDS-PAGE and western blot showed that

74.3 kD fusion proteins were mainly expressed in soluble form (Fig. 1A, shown by arrow). The results show that the soluble fusion protein could be highly purified in large amounts (milligram

fusion quantities of protein from a liter liquid culture) by an affinity column (Fig. 1A, lane 7, shown by arrow). Western blot analysis demonstrated that the purified 74.3 kD polypeptides

could be recognized by anti-GFP monoclonal antibodies, anti-his-tag, and anti-PEAc1 polyclonal antibody (Fig. 1B, lanes 1-3); No polypeptides from bacteria before inducing (Fig. 1A, lane l)

could be recognized by any antibodies above (Fig. 1B, lanes 4-6), only the induced 74.3 kD polypeptides in lane 3 (Fig. 1A) could react with these antibodies (data not shown) giving the same

results as Fig. 1B (lanes 1-3). PEAC1-GFP FUSION PROTEINS CAN POLYMERIZE INTO A FILAMENTOUS STRUCTURE _IN VITRO_ Under actin polymerization condition, purified PEAc1-GFP fusion proteins

polymerized to form green fluorescence filamentous structures (blue light excitation) as shown in Fig. 2A; and the filamentous structures could be specifically stained by TRITC-phalloidin

(green light excitation) as shown in Fig. 2B. The Electron micrographs from negatively staining demonstrated that the diameter of the filamentous PEAc1-GFP is about 9 nm (Fig. 3), similar as

F-actin (7-9 nm) 45, 46. These results above indicate that the prokaryotic expressed PEAc1-GFP fusion proteins were able to polymerize into F-actin-like structures _in vitro_. PEAC1-GFP

FUSION PROTEINS POLYMERIZED WITH ESSENTIALLY NORMAL POLYMERIZATION DYNAMICS The polymerization dynamics and the critical concentration determination of PEAc1-GFP were monitored at 450 nm.

The light scattering curves indicated that the amount of filaments at steady state equilibrium was dependent on the initial protein monomer concentration, and that the polymerization

dynamics of PEAc1-GFP were similar to that of animal muscle actin (Fig. 4A). Under this condition, the polymerization usually took more than 3 h to attain equilibrium (data not shown). As

shown in Fig. 4B, the point at which a line through these data intersected the X-axis is a indication of critical concentration. The average critical concentration for PEAc1-GFP is 0.75 ±

0.02 μM (n=7), a little higher than chicken muscle actin critical concentration 0.56 ± 0.02 μM measured under the same conditions. As a control, we also purified GFP fused with 6×his-tag

(about 30 kD, data not shown), and determined its spectrum characteristics: the absorb peak is at 489 nm (with UV640); the maximum fluorescence exciting wavelength is 490 nm; the emission

wavelength is 510 nm (with F-4500), all of which are far away from 450 nm. At the same polymerization condition, the value of light scattering of GFP protein at 450 nm did not change (data

not shown). So we could draw a conclusion that the method used to determine the polymerization of actin could also be adapted to PEAc1-GFP, and the polymerization characters of PEAc1-GFP

came from PEAc1, but not GFP. THE PURIFIED PEAC1-GFP FUSION PROTEINS INHIBIT DNASE I ACTIVITY AND STIMULATE MYOSIN MG-ATPASE ACTIVITY The inhibition of DNase I activities by active G-actin

monomer through binding to DNase I and forming a high-affinity 1:1 complex is an important character of G-actin. In our assay, the inhibition of DNase I activity was obvious when purified

PEAc1-GFP fusion protein monomers were added to a DNase I solution at about a 1:1 molecular proportion (Fig. 5A middle). The dynamic profile of PEAc1-GFP is similar to chick muscle G-actin.

The GFP alone did not show an obvious effect on DNase I activity at the same condition (data not shown). The result indicates that the fusion protein of actin isoform PEAc1 still maintains

its property to inhibit DNase I activity. Stimulating myosin Mg-ATPase activity is another important characteristic of actin in filaments 47. To know if PEAc1-GFP filamentous structures have

this ability, the Mg-ATPase activities of purified chick muscle myosin were measured in the presence or absence of polymerized PEAc1-GFP filaments, using muscle F-actin as a positive

control. The results shown in Fig. 5B demonstrated that the basic Mg-ATPase activity of chick muscle myosin was about 23.32 ± 1.76 (n=3) nM Pi/mg protein·min (black column) in our

experimental conditions, and 49.42 ± 8.23 (n=3) after the addition of polymerized PEAc1-GFP filaments to the reaction (gray column), and 119.49 ± 23.32 (n=3) for polymerized chicken muscle

actin (white column). The 2-fold stimulation of activity of myosin Mg-ATPase indicates that the polymerized filaments of the actin fusion proteins are able to interact with myosin motor

molecules (_P_<0.005). PEAC1-GFP FUSION PROTEIN WAS EXPRESSED IN TOBACCO CELL SUSPENSION BY2 LINE The recombinant plasmid pBAc1-GFP was transformed into the tobacco cell suspension BY2

line for eukaryotic expression. Stable expression was observed in cells selected with kanamycin. After several subcultures, PEAc1-GFP fluorescence was mainly found surrounding the nuclei of

normal cells (Fig. 6A-C), and concentrated near the tip and cell wall of extending cells (Fig. 6D-F). The distribution of PEAc1-GFP in tobacco cells suggests that the GFP fusion does not

affect the expression of PEAc1 in eukaryotic cells, which makes a foundation for a detailed study into the distribution or function of PEAc1 _in vivo_. DISCUSSION In this study, we have

successfully purified the soluble expression product of PEAc1, one isoform of pea tendril actins, using GFP fusion. Evidences from fluorescence study, dynamic and biochemical analysis showed

that the fusion proteins retain the basic physical and biochemical properties of actin. Ni-Chelating Sepharose™ Fast Flow chromatography and gradient washing with buffers containing

different levels of immidazole purified a specific His-tagged actin PEAc1fusion protein (Fig. 1). Specifically, more than 1 milligram of actin fusion protein with high purity could be

rapidly obtained from 1 liter of bacteria culture. Notably the purified product is a kind of plant actin isoform that is very difficult to obtain by normal biochemical isolation 26. The

soluble expression and rapid purification of pea actin isoform PEAc1 provides a relatively simple method for comparing the biochemical properties among actin isoforms _in vitro_.

Furthermore, the GFP labeling of actin isoform might be optimal for motility assays _in vitro_ and for investigating the dynamic distribution _in vivo_. The ability to bind to DNase I

molecules is a basic physicochemical property of G-actin monomers, whereas the ability to stimulate myosin activity and to bind to phalloidin is the property of polymeric actin. We have

shown that the purified monomeric PEAc1-GFP could bind to DNase I and inhibit DNase I activity using salmon sperm DNA as substrate (Fig. 5A). Also, polymerized PEAc1-GFP filaments stimulated

muscle myosin ATPase activity up to two-fold (Fig. 5B). Under physiological ionic conditions, PEAc1-GFP polymerized with kinetics similar to those of skeleton muscle actin (Fig. 4A), and

with a critical concentration of 0.75 μM (Fig. 4B). The critical concentration was a little higher than that of muscle α-actin, but consistent with the result of 0.6 μM of pollen actin 25.

The filamentous structures could be seen directly under the fluorescence microscope because of the fusion with GFP and could be specifically stained with TRITC-phalloidin (Fig. 2A-B). The

filamentous PEAc1-GFP was further viewed ultrastructurally by negative staining, showing long straight filaments with an average diameter of about 9 nm (Fig. 3). Although it is known that

the prokaryotic cytosol is unable to provide a post-translational modification found on most forms of actin: the methylation of histidine 73 48, our results indicated that the bacteria

expressed PEAc1-GFP proteins still retained their basic physicochemical properties, which was consistent with previous foundings that purified _Dictyostelium discoideum_ actin from highly

insoluble aggregates expressed in _E. coli_ held the ability both to polymerize and to bind to DNase I 48. Our results in this paper also provided new evidence that GFP fusion neither

disturbs the polymerization ability of actin isoform PEAc1 monomers, nor influences its interaction with DNase I and myosin motor molecules. Actin isotypes encoded by a relatively ancient

and highly divergent multigene family differ in their functions and expression patterns, and play different roles in different tissues and development stages 13, 14, 17, 18. Although the

tissue-specific expression of actin isoforms can be detected by anti-peptides antibodies against specific peptides of each kind of actin isoforms 49, their dynamic distribution patterns in

living cells keep relatively unknown. GFP has been extensively used as a reporter gene and a protein label for its ability to reveal real time dynamics of proteins _in vivo_ without

destroying the cell structure. Some successful examples have been shown on the distribution and dynamics of microfilaments in _Dictyostelium discoideum_50, mammalian cells 34 and yeast 51.

In our study, GFP was sucessfully used as a molecular “flag” to label PEAc1 and thus allow further studies on its characterizaion. The presence of the expressed fusion proteins in cells

indicated that the GFP fusion did not affect the expression of PEAc1 in eukaryotic cells. Furthermore, in different cell lines or same cells at different stages, the distribution and state

of actin proteins are always in changing, sometimes in filaments, sometimes in bundles even networks, to play different roles in cell activities 11. Direct observation of the dynamic state

of each actin isoform in plant cells could help us in further understanding the detailed roles of plant actins. Our results suggest that it is promising to use GFP fusion to analyze the

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USA_ 1996; 93: 3886–91. Article  CAS  Google Scholar  Download references ACKNOWLEDGEMENTS We are most grateful to Dr. Richard B. Meagher (The Genetics Department, University of Georgia) for

his valuable suggestions. We thank Drs. N. Ronald Morris and Richard S Nowakowski (UMDNJ-Robert Wood Johnson Medical School) for their critical reading the manuscript. This research was

supported by grants from the National Natural Science Foundation of China (No. 30170457, 39970358 to Guo Qin LIU, and No. 30270664 to Dong Tao REN) and from The Education Ministry of China

(No. 2000001911) to Guo Qin LIU. AUTHOR INFORMATION Author notes * Ai Xiao LIU and Shao Bin ZHANG: These authors contributed equally to this work. AUTHORS AND AFFILIATIONS * State Key

Laboratory of Plant Physiology and Biochemistry, College of Biological Science, China Agricultural University, Beijing, 100094, China Ai Xiao LIU, Shao Bin ZHANG, Xiao Jing XU, Dong Tao REN 

& Guo Qin LIU Authors * Ai Xiao LIU View author publications You can also search for this author inPubMed Google Scholar * Shao Bin ZHANG View author publications You can also search for

this author inPubMed Google Scholar * Xiao Jing XU View author publications You can also search for this author inPubMed Google Scholar * Dong Tao REN View author publications You can also

search for this author inPubMed Google Scholar * Guo Qin LIU View author publications You can also search for this author inPubMed Google Scholar CORRESPONDING AUTHOR Correspondence to Guo

Qin LIU. RIGHTS AND PERMISSIONS Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE LIU, A., ZHANG, S., XU, X. _et al._ Soluble expression and characterization of a GFP-fused pea

actin isoform (PEAc1). _Cell Res_ 14, 407–414 (2004). https://doi.org/10.1038/sj.cr.7290241 Download citation * Received: 08 December 2003 * Revised: 28 May 2004 * Accepted: 08 June 2004 *

Issue Date: 01 October 2004 * DOI: https://doi.org/10.1038/sj.cr.7290241 SHARE THIS ARTICLE Anyone you share the following link with will be able to read this content: Get shareable link

Sorry, a shareable link is not currently available for this article. Copy to clipboard Provided by the Springer Nature SharedIt content-sharing initiative KEYWORDS * actin isoform *

polymerization * DNase I inhibition * myosin Mg-ATPase activation * expression