Functional Reconstitution of the Tonoplast Proton-ATPase from Higher Plants

Functional Reconstitution of the Tonoplast Proton-ATPase from Higher Plants Kunihiro Kasamo* and Hiroyasu Yamanishit

*Department of Plant physiology, National Institute of Agrobiological Resources, 2-1-2 Kannondai, Tsukuba, Ibaraki 305, Japan; and ?Department of Yamase, Agro-environment, Tohoku National Agricultural Experiment Station, Akahira 4,Shimo-Kuriyagawa, Morioka-City 020-01, Japan

Tonoplast proton ATPase (V-ATPase) is the most widely spread Ht pump in plants. The electrochemical gradient generated by the H+ pump provides the driving force for the secondary transport of amino acids, ions, sugars, and several metabolites. The V-ATPase has an apparent functional mass of 400-600 kDa and comprises at least 9 or 10 different subunits, of which the catalytic 67-73 kDa, the neucleotide-binding55-62 kDa, proteolipids 95-115 and 16-17 kDa, and 44-29 kDa required for activity and assembly are universal components. Reconstitutionof the V-ATPase complex into liposome has been successful. Reconstitutionis convenient to assess whether any set of subunits associated with the V-ATPase is sufficient to couple ATP hydrolysis to proton pumping. In this review, we describe practical approach of reconstitutionof the V-ATPase from mung bean hypocotyls into asolectin liposomes. KEY WORDS: Tonoplast, H+ pump, Reconstitution, Molecular cloning, Peripheral subunits, Integral subunits.

I. Introduction In plant cells, vacuoles play a fundamental role in the maintenance of pH and regulation of cell turgor and in the transport and storage of various ions and metabolites (Taiz, 1992). Several different electrogenic H+ pumps provide the energy required to take up and distribute essential mineral Internarional Review of Cytology, Vol. 174

0074-76%/97 $25.00

85

Copyright 8 1997 by Academic Press. All rights of reproduction in any form reserved.

86

KUNlHlRO KASAMO AND HIROYASU YAMANISHI

nutrients for growth and development. These H+ pumps are a plasma membrane H+-ATPase(PM-ATPase), a vacuolar H+-ATPase(V-ATPase), and vacuolar H+-pyrophosphatase(V-PPase). The electrochemical gradient generated by these H+ pumps provides the driving force for the secondary transport of numerous ions and metabolites (Sze, 1985). V-ATPases are the most widely spread H+ pumps in eukaryotic cells. They are present in plant vacuoles (Sze, 1985; Randall and Sze, 1986; Mandala and Taiz, 1985; Manolson et af., 1985), fungal vacuoles (Bowman and Bowman, 1986),Neurospora vacuoles (Bowman et al., 1989), the Golgi complex (Chanson and Taiz, 1985),clathrin-coated vesicles (Xie and Stone, 1986; Arai et al., 1987b), chromaffin granules (Njus et a!., 1986; Cidon and Nelson, 1986), kidney microsomes (Gluck and Caldwell, 1987), and lysosomes (Moriyama et al., 1984). All these V-ATPases appear to be a large molecular mass complex of 400-600 kDa (Mandala and Tab, 1985; Xie and Stone, 1986; Arai et al., 1988). They also have in common four subunits of 100, 70, 60, and 17 kDa and several subunits. To determine whether the V-ATPase complex transports protons actively, the purified enzyme or ATPase complex with a deleted subunit was incorporated into liposomes to make a functional proteoliposomes (Kasamo et al., 1991;Ward and Sze, 1992b;Yamanishi and Kasamo, 1994).The native properties of the reconstituted H+ pump were examined by bafilomycin, which completely inhibited H+ pumping at a concentration on the nanomole level and the H+-pumpingactivity was completely collapsed by the ionophores. Reconstitution is convenient to assess whether any set of subunits associated with the V-ATPase is sufficient to couple ATP hydrolysis to proton pumping. The characteristics of the H+ pumping and ATP hydrolysis in vacuolar membrane vesicles from plants have been reviewed by Sze (1985) and Sze et af. (1992b). The functional roles of the V-ATPase in growth and development of plants have been discussed (Sze et al., 1992a). This chapter presents the recent evidence concerning physiological functions, the subunit structure, molecular cloning of genes encoding the subunits, and functional reconstitution of V-ATPase from plant cells. Comparisons with V-ATPase subunits from animal tissues, yeast, and plants are discussed briefly.

II. Physiological Functions of V-ATPase Plant cells are unique in containing large acidic vacuoles that can encompass more than 80-90% of the cell volume in the case of mature mesophyll cells. The V-ATPase is the enzyme responsible for acidifying the central vacuole, although there is another H+-translocating electrogenic proton pump, namely V-PPase (Rea and Sanders, 1987; Maeshima and Yoshida, 1989). These two proton pumps actively transport protons across the vacuolar

FUNCTIONAL RECONSTITUTION OF PROTON-ATPASE

87

membrane using the free energy liberated by the hydrolysis of ATP or PPi to generate an inside acidic pH difference and an inside positive electrical potential difference. The electrochemical gradient across the vacuolar membrane generated by these HC pumps provides the driving force for the secondary transport of numerous ions and metabolites (Taiz, 1992; Sze et al., 1992a). The differences in physiological function between these two proton pumps are still controversial. V-PPase may operate as a backup system for metabolism under stress (Rea and Poole, 1993). The V-PPase may be particularly active in young, fast growing leaves in which synthetic pathways generate large amounts of PPi (Suzuki and Kasamo, 1993). VPPase may scavenge the PPi, which is produced as a by-product of RNA, protein, and polysaccharide syntheses. PPi is also produced in the process of @-oxidationof fatty acids. Indeed, V-PPase activity was about half that of the V-ATPase in the mature tap roots of radish, even though its activity was four times that of the V-ATPase in the young roots. The existence of the V-PPase in the tonoplast helps to conserve ATP, which is a universal energy source of many cellular activities, such as the synthesis and transport of cellular components (Maeshima et al., 1996). On the other hand, the concentration of cytoplasmic ATP is kept strictly at the millimolar level; thus, V-ATPase can operate as a fundamental proton pump in plant cells at any physiological stage. Further investigations using genetic and biochemical approaches are necessary to clarify the functional difference between the two proton pumps. The physiological functions of V-ATPase are very closely related to the functions of the vacuoles. We shall review the vacuolar functions briefly and the relation between the functions of V-ATPase and vacuole. Plant vacuoles play various functions as reviewed by Matile (1978), Marty et al. (1980), Boller and Wiemken (1986), Taiz (1992), and Wink (1993). The roles of plant vacuoles may be classified into two groups. The first role is a space filling one. Plant cells typically undergo a 10- to 20-fold increase in volume during cell expansion that is due to the formation of large water-filled vacuoles as reviewed by Maeshima et al. (1996). The other important role of the vacuole is the regulation of cellular metabolic processes. Taiz (1992) has classified the metabolic roles into six groups as follows: (i) storage, (ii) toxic avoidance, (iii) pH and ionic homeostasis, (iv) defense against microbial pathogens and herbivores, (v) pigmentation, and (vi) lysosomes. All these metabolic roles are closely related to the function of transport of solutes into the vacuoles across the vacuolar membranes.

A. Physiological Role of V-ATPase on the Space Filling of Cells Turgor regulation seems to be a basic property of all vacuoles, although specializations were detected in guard cells (Zeiger, 1983). It is argued that

88

KUNlHlRO KASAMO AND HIROYASU YAMANISHI

the formation of large water-filled vacuoles will be an economic way to fill the newly formed cell volume during cell growth (Taiz, 1992). Most of the water that increases cell size is shared in the vacuole and the cell expansion is mostly due to the increase in the volume of the vacuole because the volume of the cytoplasm is maintained relatively constant. Nevertheless, a large increase in the entire cell size occurs. The space-filling role of the vacuole within a cell makes it possible to maintain the concentrations of various metabolites in the cytoplasm during cell expansion. A variation of this scheme can be seen in succulent plants in which the vacuoles serve as water reservoirs for adaptation to dry and arid habitats. The larger the vacuoles grow, the more actively the vacuoles must take up protons because solutes must be accumulated in the growing vacuoles so that the osmolarity of its contents remains high for maintenance of the turgor pressure required for the continuous elongation of cells. The amount of proteins in the tonoplast, such as V-PPase and V-ATPase, increases with development of the vacuolar membrane on the basis of DNAcontent (Maeshima, 1990). The V-PPase has been shown to be immunoprecipitated by the V-PPasespecific antibody from the dividing and elongating region of mung bean seedlings. Proteins in the tonoplast in the mature cells may be actively synthesized even in elongating cells (Maeshima, 1990). An integral protein designated VM23 (Maeshima, 1992),which is a member of the y-TIP family (Chrispeels and Agre, 1994) and functions as a water channel, increases markedly during the growth of radish roots (Maeshima et al., 1996) and mung bean hypocotyls (Maeshima, 1990). Expansion of plant cells requires water channels together with the vacuolar proton pumps such as V-ATPase and V-PPase. It is difficult to obtain direct in vivo evidence to support the function of the V-ATPase in vacuolar solute accumulation, osmotic water uptake, and cell expansion. In yeast, which contains a single copy of the V-ATPase genes, it has been possible to directly assess the metabolic role of the V-ATPase by generating null mutants. Yeast null mutants obtained by disrupting the genes for the A, B, C, E, or c subunits are incapable of growth at neutral pH, are strongly inhibited by external calcium ions, fail to carry out endocytosis of lucifer yellow, and exhibit protein mistargeting (Rothman et al., 1989; Foury, 1990; Hirata et al., 1990; Nelson and Nelson, 1990; Umemoto et al., 1990; Beltran et al., 1992). These approaches are not yet feasible in higher plants; thus, carrot cells transformed with antisense DNA of the V-ATPase A subunit have been investigated to prove the physiological function of V-ATPase (Gogarten et al., 1992). The synthesis of the V-ATPase can be specifically inhibited with antisense DNA to the catalytic subunit; as a result, both proton-pumping activity and secondary transport activity are negligible in the transformant plants. The leaves of carrot plants transformed with antisense DNA are smaller and the tap roots are shorter than in normal plants because cell expansion is inhibited in the

FUNCTIONAL RECONSTITUTION OF PROTON-ATPASE

89

antisense mutant. These results demonstrate that V-ATPase does play an important role in facilitating cell expansion.

6.Physiological Role of V-ATPase on Metabolic Processes of Cells

The proton-motive force generated by V-ATPase consists of a ApH of about 1.5-2 and a A$ of about +30 mV relative to the cytoplasm (Sze, 1985). Although it is well established that the proton-motive force is used to transport various ions and primary metabolites (Wink, 1993), direct proof has been difficult to obtain because the V-ATPase null mutant that is frequently utilized for investigation of V-ATPase functions in yeast is not yet available in plants. In yeast, V-ATPase null mutants have been shown not to grow at neutral pH (Nelson and Nelson, 1990) or be lethal at a high external calcium concentration of 100 mM (Ohya et al., 1991). These mutants demonstrate directly the function of V-ATPase, which is the regulation of cytosolic pH and maintaining of intracellular Ca2+.Unfortunately, it is not possible to carry out these genetic approaches in plants. In the case of plants, biochemical approaches have been taken to characterize the transport system in relation to the V-ATPase function. The principal function of V-ATPase is to pump the protons into the lumen of the vacuole; thus, theV-ATPases contribute to maintain the pH of the acidic vacuoles and that of the neutral cytoplasm. Cold inactivation of V-ATPase (Moriyama and Nelson, 1989b; Matsuura-Endo et af.,1992) induces acidification of the cytoplasm in mung bean cells, which are sensitive to chilling (Yoshida, 1994). Under energy stress, such as chilling or anoxia, in which cytoplasmic ATP levels are reduced, V-PPase activity has been shown to be induced rapidly (Carystinos et al., 1995;Darley et af., 1995).V-PPase has been shown to be responsible for the maintenance of vacuolar ApH in cells treated with metabolic inhibitors (Macri et al., 1995). These facts indicate that V-ATPase is the fundamental proton pump under normal growth conditions, and V-PPase may be important in maintaining tonoplast energization under conditions of limited ATP supply. The vacuolar pH of a freshwater charophyte, Chara, increased in the presence of bafilomycin Al, known to be a specific inhibitor of V-ATPase (E. J. Bowman et a/., 1988a), suggesting that PPi-dependent H' pumping alone is not sufficient for pH regulation (Okazaki et af., 1992). Several antiporters have been demonstrated that utilize the proton motive force generated by V-ATPase to drive the uptake of anions, cations, amino acids, organic acids, and sugars in exchange for protons. The cytoplasmic Ca2+concentration is between 0.1 and 0.25 p M , whereas the vacuolar Ca2+concentration is 1 mM. The steep gradient in Ca2+has been thought to be maintained by a Ca2+/Ht antiporter (Hager

90

KUNlHlRO KASAMO AND HIROYASU YAMANISHI

et al., 1986; Schumaker and Sze, 1990; Blackford et al., 1990). On the other hand, Pfeiffer and Hager (1993) provided evidence that the H+ gradient is due to an Mg2+/H+exchanger. Thus, a Ca2+/H+antiporter across the tonoplast is active only at high Ca2f levels. In the case of low Ca2+concentrations, normally existing in the cytoplasm, Ca2' transport into the vacuoles cannot be influenced by the protonophore, which eliminates the Ht gradient across the tonoplast. The antiporter functions chiefly as an Mg2+transporter under physiological conditions because Mg2+ions in a physiologicalconcentration range trigger a fast efflux of Ht from acid-loaded vesicles. Instead of a Ca2+/H+antiport mechanism, an ATP-dependent, vanadate-sensitive Ca2+pumpin tonoplast vesicles has also been proposed (Pfeiffer and Hager, 1993). This type of Ca2+pump has been found in other plant low-density intracellular membrane vesicles, probably of vacuolar origin (Askerlund, 1996; DuPont and Morrissey, 1992). The Mg2+/Htantiporter has also been characterized in other plant vacuolar vesicles (Amalou et al., 1992). VATPase plays the principal role in energizing Nat/Ht antiport activity in cells accumulating significant quantities of NaCl (Barkla et al., 1995)because the activity of the V-PPase declines as a consequence of salt treatment (Bremberger and Luttge, 1992; Nakamura et al., 1992). A toxic pollutant metal ion, such as Cd2+,has also been proven to transport across the vacuolar membranes by a Cd2f/H+antiport mechanism that is inhibited by ionophores (Salt and Wagner, 1993). Accumulation of citrate or malate, which is inhibited by ionophores, indicates that the transport is also driven by the proton-motive force generated by the proton pump (Rentsch and Martinoia, 1991). The existence of an H+/N03- antiport system is also considered in plant tonoplasts (Miller and Smith, 1992). The inside positive membrane potential across the vacuolar membranes can drive the uptake of various anions such as C1-, Pi, and others through channels. Cations can also enter vacuoles through channels down the concentration gradient. The channels function during active proton pumping (Hedrich and Schroeder, 1989). However, the regulatory mechanism of cytoplasmic Pi homeostasis induced by export and import from the vacuole is not yet understood (Mimura et al., 1990). Amino acids such as aromatic amino acids are transported into vacuoles depending on the proton-motive forces generated by proton pumps in the tonoplast. However, arginine and lysine may be transported across the tonoplast by an ATP-stimulated permease specific for positively charged amino acids, and an ATP-dependent, but not energy-requiring, translocator is also postulated (Martinoia et al., 1991). Active glucose transport systems have been demonstrated in the tonoplast of maize coleoptiles, which are inhibited by antibodies against the catalytic subunit of the V-ATPase (Rausch et al., 1987). This fact indicates a direct coupling of 3-0-methylD-glucose transport to V-ATPase-dependent proton transport. The major

FUNCTIONAL RECONSTITUTION OF PROTON-ATPASE

91

sugar component of the vacuolar solution is frequently sucrose, particularly in the vacuoles of specialized storage tissues such as sugar beet tap root or sugarcane stalk tissue. Two conflicting results have been reported for the sucrose transport mechanisms. Evidence for a proton-sucrose antiport on the tonoplast in red beets (Getz and Klein, 1995b) and Japanese artichoke tubers (Greutert and Keller, 1993) was reported. In contrast to these reports, other published investigations of sucrose transport in sugarcane vacuoles and tonoplast vesicles have produced no evidence for protoncoupled sucrose translocation (Preisser et al., 1992; Preisser and Komor, 1991). Further investigations in this field will be necessary. It is known that V-ATPase activity itself is regulated by various factors such as plant hormones (Kasai et al., 1993) and phospholipids (Scherer et al., 1988; Martiny-Baron et al., 1992; Yamanishi and Kasamo, 1993). VATPases may be an integral component of the endomembrane system in plants (Sze et al., 1992b). Endocytosis and intracellular membrane traffic in plant cells similar to those of animal cells (Forgac, 1989) have been considered (Low and Chandra, 1994; Bassham and Raikhel, 1996). VATPases have been shown to be present on three organelles other than the vacuole of the endomembrane system of plant cells-and Golgi apparatus (Chanson and Taiz, 1985), coated vesicles (Fichmann et al., 1989; Depta et al., 1991), and the endoplasmic reticulum (Herman et al., 1994)-although the catalytic subunits of the Golgi apparatus and the tonoplast V-ATPases are encoded by different genes (Gogarten et al., 1992). These V-ATPases in various organelles may play a role in intracellular membrane traffic processes and protein sorting to the vacuolar membrane as in the case of animal cells. In animal cells, the process of recepter-mediated endocytosis begins by the binding of ligands to their corresponding receptors on the cell surface and then internalization of ligand-receptor complexes via clathrincoated pits and coated vesicles results in their delivery to an acidic compartment that is termed compartment of uncoupling of receptor and ligand (CURL). Various ligands dissociate from their receptors on exposure to mildly acidic pH (5.5-6.5). The CURL is a tubulovesicular compartment in which space is acidified by V-ATPase; thus, ligands dissociate from their receptors. V-ATPases are also found in the membrane of the endosome, Golgi apparatus, endoplasmic reticulum, and lysosomes; thus, the enzymes play an important role in the intracellular membrane traffic processes (Forgac, 1989). In plant cells, there is consensus that many internalized markers are initially captured at coated pits, engulfed quickly in coated vesicles, and transferred gradually to the partially coated reticulum that is analogous to the CURL in animal cells (Low and Chandra, 1994). The plant vacuoles accumulate soluble vacuolar proteins such as phytohemagglutinin (PHA) and tonoplast intrinsic proteins (TIP) (Gomez and Chrispeels, 1993). Monensin inhibits sorting in the trans-Golgi network by disrupting the proton

92

KUNlHlRO KASAMO AND HIROYASU YAMANISHI

gradient across the membrane and blocks the transport of PHA to the vacuole; however, this drug does not stop the arrival of TIP in the tonoplast. These facts indicate that PHA and TIP may be transported by different paths. However, at least for PHA, the proton gradient across the membrane is needed for protein transport. Strains of yeast with chromosomal disruptions of the genes encoding the V-ATPase subunits accumulate precursor forms of vacuolar membrane protein alkaline phosphatase and the soluble vacuolar hydrolases carboxypeptidase Y and proteinase A. These precursors have been shown to accumulate within the secretory pathway at some point before delivery to the vacuole but after transit to the Golgi complex (Yaver et al., 1993). These facts prove directly the role of V-ATPase in the membrane traffic and protein-sorting processes.

111. Molecular Structure and Function of the Tonoplast H+-ATPase Plant vacuoles are acidic organelles in which ions, sugars, amino acids, organic acids, and hydrolytic enzymes are stored. A proton-translocating ATPase (H+-ATPase)localized in the tonoplast generates an electrochemical gradient, which may be responsible for the observed accumulation of ions and solutes (Sze, 1985). Partially purified V-ATPases of oat (Randall and Sze, 1986), corn (Mandale and Taiz, 1985),beet (Manolson et al., 1985), and radish (Tognoli, 1985) retain the same characteristics as the tonoplast H’ pumps of Neurospora (Bowman, 1983), yeast (Uchida et al., 1985), and various animal cells including storage granules (Cidon and Nelson, 1983; Dean et al., 1984), lysosomes (Moriyama et al., 1984), and clathrin-coated vesicles (Forgac and Cantley, 1984). A similar ATP-dependent H+ pump is also present in the Golgi of maize (Chanson and Taiz, 1985). The FIFoATPase of the mitochondria/chloroplast/bacteria(Amzel and Pedersen, 1983) and PM-ATPase (O’Neill and Spanswick, 1984) are different from the V-ATPase. PM-ATPase is sensitive to vanadate (O’Neill and Spanswick, 1984). However, FIFo-ATPaseand V-ATPase are insensitive to vanadate. V-ATPase could be interpreted as a type of phosphohydrolase that does not form a covalent phosphoenzyme intermediate in a reaction mechanism similar to that of the FIFo-ATPase(Amzel and Pedersen, 1983; Wang and Sze, 1985). V-ATPase and FIFO-ATPaseshare common steps in their catalytic and vectorial reaction mechanism; however, sufficient differences exist to indicate that they are two distinct ATPases. Thus, V-ATPase was stimulated by C1- > HC03-, whereas FIFO-ATPase was stimulated by HC03- >>> C1-. V-ATPase was not inhibited by azide, but FIFO-ATPase was inhibited by it. FIFo-ATPase was 100 times more sensitive to N,N’-

FUNCTIONAL RECONSTITUTION OF PROTON-ATPASE

93

dicyclohexylcarbodiimide (DCCD) inhibition than was V-ATPase. VATPase from red beet has been found to consist of seven major subunits of 100,67,55,52,44,32,and 16-kDa and two minor components of 42 and 29-kDa (Parry et af.,1989) (Fig. 1).The H'-ATPase complex is composed of 67, 55, 44, 42, 32, 29, and 16 kDa subunits, in a probable stoichiometry of 3 : 3 : 1: 1: 1: 1: 6 (Arai et af., 1988; Bowman ef at., 1989). High-purity ATPase preparations from higher plant vacuolar membranes have a subunit composition that closely corresponds with the enzymes from clathrin-coated vesicles (Xie and Stone, 1986) and chromaffin granules (Cidon and Nelson, 1986). All five major subunits, 67, 55, 44, 42, and 29-kDa, are peripheral subunits and 100- and 16-kDa polypeptides are integral subunits because the former were removed from the membranes by a chaotropic salt such as KI (Lai et af., 1988; Rea et af., 1987a) and by cold treatment (Parry et af., 1989; Moriyama and Nelson, 1989b). The latter 16-kDa component can be purified to homogeneity by chloroform : methanol extraction of fast protein liquid chromatography-purified enzyme (Parry et af., 1989). Both 45- 28-kDa subunits are required for activity and/or assembly of the H'ATPase (Hirata et af., 1993; Ho et af., 1993b; Puopolo et af., 1992b). More recently, the 32-kDa subunit was reported to be an integral subunit (Nelson et af., 1995; Graham et af., 1995). Subunit compositions of VATPase are summarized in Table I. Only those subunits with welldefined kinetics of interaction with affinity labels have been identified.

FIG. 1 Structural model of a V-ATPase fromplants (reproduced with permission from Sze etal., 1992b). A large peripheral complex includes the 70-kDa (catalytic) and the 60-kDa (nucleotidebinding) subunits plus several accessory subunits of unknown function. Six copies of the DCCDbinding 16-kDa proteolipid together with other integral subunits are thought to form a proton pore. The 100-kDa subunit is an integral membrane protein but its location is still unknown.

94

KUNlHlRO KASAMO AND HIROYASU YAMANISHI

TABLE I Subunit Compositions of V-ATPase from Plant Tissues, Animal Sources, Neurospora, and Yeast

Source Plant vacuoles Oat Mung bean

Subunit composition ( X lo3)” 70 60

68 57

44 42 36 32 29 16 13 12 Ward and Sze (1992a) 44 38 37 32 16 13 12 Matsura-Endo er aL(1990) 45 42 34 32 17 13 12 DuPond and

Barley

115 68 53

Red beet

100 67 55 52 44 42

Kalanchoe

Reference

72 56 48

32 29 16

42

28 16

Animal Coated vesicles 100 73 58

40 38 34 33

17

116 70 58

40 38 34 33

17

115 72 57

39

17

68 58

40

Chromaffin granules Golgi (kidney) Neurospora vacuole

Yeast vacuole a

37

16

67 57

15

100 69 60

42 36 32 27 17

Morrissey (1992) Parry et al. (1989) Bremberger et al. (1988)

Arai et al. (1987b) Xie and Stone (1986) Cidon and Nelson (1986) Young et al. (1988) Bowman and Bowman (1986a) Uchid et al. (1985)

Subunits in bold have been shown to be peripheral.

Thus, [14C]N-ethylmaleimide(NEM) and 7-chloro-4-nitro [14C]benzo-2oxa-1,3-diazole(NBD-C1) prefentially labeled the 67- to 72-kDa subunit (Bowman et al., 1986; Mandala and Taiz, 1986; Randall and Sze, 1987; Yamanishi and Kasamo, 1992a), and labeling was prevented by ATP (Mandala and Taiz, 1986). [a-32P]3-0-(4-benzoyl)benzoyl-ATP binding prefentially labeled the 55- to 62-kDa subunit (Manolson et al., 1985). The hydrophobic carboxyl reagent [14C]DCCDbinds to the 16-kDa proteolipid (Rea et al., 1987a,b; Mandala and Taiz, 1986; Lai et al., 1988). These results implicate the 72-kDa subunit as the catalytic subunit of the tonoplast ATPase. The DCCD-binding 16-kDa subunit may comprise the proton channel. Bafilomycin, a membrane-permeant macrolide antibiotic isolated from Streptomyces griseus L. (Werner et al., 1984), has been found to be

FUNCTIONAL RECONSTITUTION OF PROTON-ATPASE

95

a potent and specific inhibitor of V-ATPase (E. J. Bowman et al., 1988a). An understanding of the mechanism by which bafilomycin inhibits V-ATPases would greatly facilitate its use as an inhibitor in both biochemical assays and physiological studies. Bafilomycin inhibits proton pumps by blocking proton conduction through a proton pore. Proton pores are involved in the hydrophobic membrane sector (V,). Thus, bafilomycin would bind to the 17-kDa subunit (Hanada et al., 1990; Rautiala et al., 1993), the 116-, 39- and 17-kDa components (Crider et al., 1994), and the 100-kDa subunit (Zang et al., 1994). Bafilomycin is highly lipophilic and is able to enter the cells; therefore, it can be used in intact cells for investigation of the role of acidification in various physiological processes (Okazaki et al., 1992). The presence of nucleotide-binding sites on the 62-kDa subunit suggests that it may function as a regulatory subunit. The 67-kDa subunit appears to be homologous to the p subunit of the FIFo-ATPase (Zimniak et al., 1988), and the 57-kDa subunit appears to be homologous to the a subunit (Bowman et al., 1988a; Xie and Stone, 1988; Manolson et al., 1988). The 116-kDa subunit present in H’-ATPase from clathrin-coated vesicles and chromaffin granules was not found in vacuolar proton pumps that were partially purified from fungi, yeast, and plants. However, the subunit in the vacuolar proton pumps purified from yeast (Kane et al., 1989) and plants (Parry et al., 1989) was found. In the absence of 116- and 38-kDa polypeptides, the enzyme is unable to pump protons and has no ATPase activity (Xie and Stone, 1988). These results show that the 116-kDa subunit has a function related to the coupling of ATP hydrolysis to proton translocation. The structure of the V-ATPase of Neurospora and plants was examined by a negative staining technique. Vacuolar membranes displayed “balland-stalk” particles, also called “knob-like” or “head-and-stalk” structures (Dschida and Bowman, 1992; Getz and Klein, 1995b). Two “arms,” in addition to about a 6.1-nm globular head and a 2.3-nm stalk, can be observed. Corresponding subunits of the arms are probably 52-, 44-, or 42-kDa subunits of beet root V-ATPase (Getz and Klein, 1995b). The respective dimensions of beet root V-ATPase are smaller than those determined for Neurospora crassa V-ATPase.

IV. Molecular Cloning of cDNA of the Tonoplast H+-ATPese Vacuolar H+-ATPases have an apparent functional mass of 400-600 kDa (Zimniak et al., 1988; Mandala and Taiz, 1985; Randall and Sze, 1986; Bowman et al., 1989) and comprise at least nine different subunits, including the catalytic subunit, 67-73 kDa (Mandala and Taiz, 1986; Randall and

96

KUNlHlRO KASAMO AND HIROYASU YAMANISHI

Sze, 1987), the nucleotide-binding, 55-62 kDa (Manolson et al., 1985), and two intrinsic membrane proteins-a 16- or 17-kDa subunit (Kaestner et al., 1988) and a 100-kDa subunit (Parry et al., 1989).

A. Molecular Cloning of the 67- t o 73-kDa Subunit Gene There is increasing evidence that the 70-kDa subunit contains the catalytic site. The covalent inhibitor, NBD-C1, which binds to the /3 subunit of FIFOATPase, also binds the 70-kDa subunit of the vacuolar ATPase in an ATPprotective manner (Bowman et al., 1986; Mandala and Taiz, 1986; Randall and SzeJ986; Arai et al., 1987b; Moriyama and Nelson, 1987b) and an antibody reactive manner (Bowman et al., 1986). The 70-kDa subunit is preferentially labeled by NEM (Wang and Sze, 1985). These data suggest that tyrosine and cysteine residues are in the catalytic site of the tonoplast ATPase. Furthermore, the antibody to the 70-kDa subunit strongly antagonized both ATP-hydrolytic activity (Mandala and Taiz, 1986) and protonpumping activity (Rausch et al., 1987). The gene encoding the 67- to 73-kDa subunit was cloned and sequenced from carrot (Zimniak et al., 1988), cotton (Wilkins, 1993), Neurospora (E. J. Bowman et al., 1988b), and bovine-coated vesicles (Puopolo et al., 1991).Using antibodies to the 70-kDa subunit of corn to screen a carrot root hgtll cDNA library, cDNA clones of the 69-kDa subunit were isolated. The complete primary structure of the 69-kDa subunit was then determined from the nucleotide sequence of its cDNA (Zimniaketal., 1988).The 69-kDa subunit from carrots consists of 623 amino acids and has a predicted molecular mass of 68,835 Da, with no obvious membrane-spanning regions. The protein lacks a hydrophobic leader sequence and contains no membrane-associated a-helixes, based on the method of Eisenberg et al. (1984). The carrot cDNA sequence was over 70% homologous with exons of a Neurospora 69-kDa genomic clone (E. J. Bowman et al., 1988b). The open reading frame from cotton ovules (Wilkins, 1993) consists of 623 amino acids with a predicted M,of 68,522 and an isoelectric point of 5.14. The ATP-binding site motif (GAFGCGKTV) located between amino acid residues 252-259 is absolutely conserved in cotton, carrots, and yeast. Amino acids 449-458 (PSVNWLISYS) were identified as ATP synthase (Y and /3 subunit signatures by computer analysis. The sequence of NBD-Cl binding site and the Mg*+-bindingregion of the p subunit of FIFO-ATPasewere conserved in the 69-kDa subunit (Zimniak et al., 1988). Recently, cold-regulated and ABA-induced cDNA was isolated from the leaves of winter Brassica nupus that corresponded to the transcript encoding the 70-kDa subunit of tonoplast ATPase (Orr et al., 1995). Similarly, an increase in tonoplast ATPase activities and accumulation of transcript(s)

FUNCTIONAL RECONSTITUTION OF PROTON-ATPASE

97

encoding the 70-kDa subunit of the tonoplast ATPase have been observed during salt adaptation and ABA treatment in tobacco cells (Narasimhan et al., 1991; Reuveni et al., 1990).

B. Molecular Cloning of the 55- to 62-kDa Subunit Gene The 57-kDa subunit has been shown to bind benzoyl ATP (Manolson et al., 1985), showing that this subunit contains a nucleotide binding site. The gene encoding the 57-kDa subunit was cloned from Arabidopsis (Manolson et al., 1988), cotton (Wan and Wilkins, 1994), barley (Berkelman et al., 1994), yeast (Nelson et al., 1989), N. crassa (B. Bowman et al., 1988), and bovine brain (Puopolo et al., 1992a). Two different cDNA clones for the 57-kDa subunit of the barley V-ATPase were isolated and cloned (Berkelman et al., 1994).The two clones were 98% identical. Two multigene families were also found in cotton ovules (Wan and Wilkins, 1994). This polypeptide of Neurospora has 513 amino acids with a molecular mass of 56,808 Da. Hydropathy plots of this subunit revealed no apparent transmembrane segment, suggesting that they constitute part of a peripheral membrane complex (Rea et al., 1987a). Analysis of the sequence for the 57-kDa subunit of Neurospora suggests that it may be the functional analog of the a subunit of F1. The a subunit and the 57-kDa polypeptide are very similar in molecular size; in fact, both Escherichia coli and Neurospora contain 513 amino acids. More important, a region of the a subunit shown to be essential for F, ATPase activity is highly conserved in the 57-kDa subunit of the vacuolar ATPase (E. J. Bowman et al., 1988b).

C. Molecular Cloning of the 16- or 17-kDa Subunit Gene The 16- or 17-kDa subunit is a major subunit of the membranous sector that binds DCCD, an inhibitor of the V-ATPase and a potential H t pore blocker (Kaestner et al., 1988; Arai et al., 1987a; Rea et al., 1987b). The DCCD-binding subunit can be extracted with chloroform/methanol, like the 8-kDa proteolipid of the FIFo-ATPase. The 16-kDa proteolipid was estimated to be present in about six copies in the V-ATPase complex, and the binding of DCCD to one copy caused full inhibition of the ATPase activity (Kaestner et al., 1988). The 16-kDa proteolipid was cloned from oat (Lai et al., 1991), cotton (Hasenfratz et al., 1995), chromaffin granules (Mandel et al., 1988), Droosophila (Meagher et al., 1990), and yeast (Nelson and Nelson, 1989). The open reading frame of the cDNA clone of oat predicted a polypeptide of 165 amino acids with a molecular mass of 16,641 Da. Based on hydropathy plots, a molecule with four membrane-

98

KUNlHlRO KASAMO AND HIROYASU YAMANISHI

spanning domains (I-IV) was predicted (Lai et al., 1991). Domain IV might be a functionally conserved region because it showed 80%identity in nucleotide or amino acid sequences between the oat and the bovine proteolipids and contained a glutamate residue that is the putative DCCD-binding residue. Transmembrane domain IV, which contains a DCCD-binding glutamine residue, is the most conserved domain among the species (Hasenfratz et al., 1995), which is consistent with the role of this domain in proton translocation (Mandel et al., 1988; Noumi et al., 1991). DCCD is thought to inhibit H+-pumpingV-ATPase by reacting with carboxyl groups of glutamate or aspartate found in the hydrophobic environment of the membrane. The 16-kDa proteolipid in oat is encoded by a small multigene family; this was supported by Southern blot analysis of oat nuclear DNA digested with EcoR1, BarnH1, and Hind111 (Lai et al., 1991). In cotton, genes encoding the 16-kDa proteolipid are organized as small gene families (Hasenfratz et al., 1995).

D. Molecular Cloning of the 95- to 116-kDa Subunit Gene Initially, the 116-kDa subunit present in V-ATPase from clathrin-coated vesicles and chromaffin granules was not found in vacuolar H+ pumps that were partially purified from lower organisms; preparations from plants, fungi, and yeast were first thought to be composed of only three subunits of 70, 58, and 17-kDa, respectively. Recently, the subunit compositions of the vacuolar H+ pumps from yeast (Kane et al., 1989) and plants (Parry et al., 1989) have been reexamined, and these H+-ATPases were found to have polypeptide compositions that include a 100-kDa component and are also otherwise similar to that of the clathrin-coated vesicle H+ pump. Especially in plants, the red beet H+-ATPasecontains a 100-kDa proteolipid subunit, but this subunit is not present in oat H+-ATPase (Parry et al., 1989; Ward and Sze, 1992a). The 21-kDa subunit (VMA21 and 22) from yeast was reported to have a function of degradation of the 100-kDa (Hill and Stevens, 1995). The deletion of the 100-kDa subunit may be related to the 21-kDa subunit function. A 116-kDa subunit of the V-ATPase was purified and sequenced from clathrin-coated vesicles of bovine brain (Perlin et al., 1991;Peng et al., 1994) and yeast (Manolson et al., 1992).At least two isoforms (types I and 11) were identified in cDNA from bovine brain (Peng el al., 1994).Type I, containing the 18-base pair (bp) insert, was found in the brain, whereas the truncated (type 11) form was found in all tissues examined (brain, heart, kidney, liver, and spleen). The deletion site of the 18bp insert contains a predicted protease sensitivity motif (PEST site), suggesting that differences in the biological half-life of the two 116-kDa isoforms may exist. The cDNAs encoding the

FUNCTIONAL RECONSTITUTION OF PROTON-ATPASE

99

116-kDapolypeptide of the clathrin-coated vesicle and yeast consist of 96,267 and 95,600 Da, respectively, and 838 and 840 amino acids were deduced, respectively. The 116-kDa subunit consists of two fundamental domains: a hydrophilic amino-terminal half that is composed of >30% charged residues and a hydrophobic carboxyl-terminal half that contains at least six transmembrane regions (Perlin et al., 1991). The structural properties of the 116-kDa polypeptide exhibit the function of coupling ATP hydrolysis by cytoplasmic subunits to proton translocation by the intramembraneous components of the pump. The 100-kDa subunit covalently binds carbohydrate, which is most likely exposed on the luminal side of the membrane (Adachi et al., 1990). In the absence of the membrane-bound 95-kDa subunit, the peripheral bound subunits (69 and 60 kDa) are no longer correctly targeted to the vacuolar membrane (Manolson et al., 1992). Thus, this subunit, is also necessary for targeting of the enzymes to specific organelles.

E. H+-ATPasePeripheral Subunits of Eukaryotes and Prokaryotes Vacuolar Ht-ATPase belongs to a highly conserved family of proton pumps that provide most of the energy required for transport processes in the vacuolar system in eukaryotic cells. This type of enzyme functions in archaebacteria both in ATP-dependent proton pumping and ATP synthesis utilizing proton-motive force. A related enzyme was also found in eubacteria that was adapted for sodium pumping (Takase et af., 1994). All these enzymes share a common structure of distinct catalytic and membrane sectors, each containing multiple subunits. This general structure is similar to F-ATPases that function in ATP synthesis in eubacteria, chloroplasts, and mitochondria. The catalytic sectors of F- and V-ATPases contains at least five subunits. The subunits of the F-ATPase catalytic sector were named a (55 kDa), fi (50 kDa), y (31 kDa), 6 (19 kDa), and E (15 kDa) and show little variation (Futai et al., 1989). The subunits of the catalytic sector of eukaryotic V-ATPase were designated A (72 kDa, VMAl), B (57-kDa, VMA2), C (40 kDa, VMAS), D (34 kDa, VMAS), and E (33 kDa, VMA4) (Moriyama, 1989b). Mammalian cDNA encoding subunits A, B, C , and E were cloned and sequenced (Sudhof et al., 1989; Puopolo et al., 1992a; Nelson et al., 1990; Hirsch et af., 1988). In plants, the genes encoding A, B, and E (Dietz et al., 1995) were cloned and sequenced. Biochemical and genetic analyses have demonstrated that the yeast V-ATPase complex comprises at least 10 polypeptides ranging in molecular mass from 100 to 14 kDa. The genes encoding the 100-kDa (VPH1) (Manolson et af., 1992), 69-kDa (VMAUTFPl) (Shih et al., 1988; Hirata et al., 1990), 60-kDa (VMA2NAT2) (Nelson et al., 1989;Yamashiro

100

KUNlHlRO KASAMO AND HIROYASU YAMANISHI

et al., 1990),54-kDa (VMA13) (Ho et af.,1993b),42-kDa (VMAS) (Beltran et al., 1992; Ho et af., 1993a)) 36-kDa (VMA6) (Bauerle et al., 1993)) 32-kDa (VMA 8) (Nelson et al., 1995; Graham et al., 1995), 27-kDa (VMA4) (Foury, 1990), and 14-kDa (VMA7) subunits (Nelson et af., 1994; Graham et al., 1994),and two hydrophobic polypeptides of 17-kDa (VMA3) (Nelson and Nelson, 1989) and VMAll (Umemoto et al., 1991) have been cloned and sequenced (Table 11). In N. crassa, VMAl (E. J. Bowman et uf., 1988b), VMA2 (B. Bowman et af., 1988), and VMA4 (Bowman et al., 1995) were isolated and cloned. V. Functional Reconstitution of the H+ Pump V-ATPases are large multimeric components composed of a peripheral sector and a membrane integral sector as described in the previous section. TABLE II The Correspondence between Subunits of V-ATPase and the Gene Encoding Subunits

Subunits (yeast) (kDa) 100

Genes (yeast)

Subunits (bovaine chromaffin)

Subunits (F,-ATPase)

A

P

B

(Y

VPH1"

69 60

VMAl (TFPl)b VMA2 (VAT2)'

54

VMA13d

42

VMA5'

36

VMA6'

32 31(27)

VMA8R VMA4h

D E

14

VMA7'

F

17

VMA3' VMAllk

'Manolson et al. (1992).

Shih et al. (1988) and Hirata et al. (1990). Nelson et al. (1989) and Yamashiro et al. (1990) Ho et al. (1993b). Beltran et al. (1992) and H o et al. (1993a). fBauerle et al. (1993). g Nelson et al. (1995) and Graham et al. (1995). Foury (1990). ' Nelson et al. (1994) and Graham et al. (1994). 'Nelson and Nelson (1989). Umemoto et al. (1991).

C

Y E

FUNCTIONAL RECONSTITUTION OF PROTON-ATPASE

101

The peripheral sector could be removed from the membrane by chaotropic reagents (Parry et al., 1989;Ward et al., 1992) or cold treatment (MatsuuraEndo et al., 1992).The dissociation of the peripheral sector induced inactivation of the enzyme, and it could be reassembled again by removal of the reagents (Ward et al., 1992). This process is termed functional reassembly. On the other hand, incorporation of a holoenzyme, once it has been solubilized from biological membranes, into artificial phospholipid vesicles is termed reconstitution. In the following sections, we describe the significance of reconstitution of V-ATPase to research in this field, the general aspects of reconstitution methods, and the practical application to V-ATPase.

A. General Aspects of the Reconstitution Methods Since the pioneering work of reconstitution of the FIFo-ATPasefrom bovine heart mitochondria by Kagawa and Racker (1971) more than two decades ago, a large number of other ATPases have been studied in reconstitution systems. However, few of the V-ATPases from plant vacuoles have been reconstituted into proteoliposomes. Several reviews (Villalobo, 1990; Rigaud et al., 1995) described the general approach of the reconstitution with respect to different classes of membrane proteins. In these reviews, only one example for plant V-ATPase (Bennett and Spanswick, 1983) had been referred to out of more than 400 references (Villalobo, 1990). The reason is that the identities of the vacuolar membrane and V-ATPase have been established within this decade. Plant vacuolar membrane, namely the tonoplast, is a structurally and functionally complex asymmetrical barrier separating the cytoplasm and the vacuolar lumen. The semifluid lipidic bilayers are constructed with lipids and proteins, including V-ATPase and V-PPase, as the primary active transporter (Rea and Sanders, 1987),secondary transporter proteins that are powered by an H + gradient generated by the primary active transporter systems (Taiz, 1992), voltage-sensitive ion channel proteins (Hedrich and Schroeder, 1989), water channel proteins (Chrispeels and Agre, 1994), and permease-like proteins (Martinoia et al., 1991). Other Mg2+-ATP-dependent,Hf electrochemical potential difference-independent transporters different from V-ATPase have been reported in the tonoplast (Martinoia et al., 1993;Li et al., 1995;Hortensteiner et al., 1993; Salt and Rauser, 1995). Studies of tonoplast proteins in their native environment can be difficult to interpret because of restrictions arising from the complexity of the native membranes and interference from other membrane constituents or other reactions. Proteins that have only a vectorial function (transport) and no enzymic functions could be used to examine the transport functions solely by means of solubilization and functional reconstitution into artificial phospholipid vesicles. Only after functional reconstitution of a particular protein into artificial phospholipid vesi-

102

KUNlHlRO KASAMO AND HIROYASU YAMANISHI

cles can we show direct evidence for the protein function. In membrane research, phospholipid vesicles (proteoliposomes) incorporating purified membrane protein are therefore a powerful tool for elucidating both functional and structural aspects of these membrane-associated proteins. This situation is the same with respect to V-ATPase. Functional reconstitution of the purified V-ATPase into artificial phospholipid vesicles helps to improve our understanding not only of the mode of action of the enzyme but also of lipid-protein interaction. Reconstitution of the V-ATPase into liposomes makes an important contribution to the studies dealing with the mechanisms of Hf transport, the electrical properties of the transport mechanism, the iodsubstrate stoichiometric ratios, and the analysis of the coupling between transport systems. The general approach is outlined for the V-ATPase as follows: (i) preparation of purified tonoplast in large quantity; (ii) solubilization of the VATPase from the tonoplast; (iii) purification of the V-ATPase possessing high specific activity; (iv) reconstitution of the V-ATPase into proteoliposomes; and (v) artificial manipulation of lipids and/or protein components. 1. Preparation of Purified Tonoplast in Large Quantities The cell wall in plant cells represents a major obstacle in preparing a homogenate of the plant tissue to obtain the plant cell membrane. Two strategies for the homogenization of plant tissues have been developedone in which no attempt is made to digest the cell wall and the other in which the cell membrane and organelles are isolated from the protoplast. The tonoplast could be isolated from other intracellular membranes of a microsome fraction depending on its density. Several improved methods other than the conventional sucrose density gradient (Yoshida et al., 1986) have been used for the tonoplast, namely, the dextran T-70 step gradient centrifugation method (Kasamo et al., 1991; Mandala and Taiz, 1985; Randall and Sze, 1986) and the floating centrifugation method (Matsuura-Endo et al., 1990; Yamanishi and Kasamo, 1994). Other isolation methods based on the electrical properties of the tonoplast, such as a two-polymer phase partitioning method (Yoshida et al., 1986) and a free-flow electrophoresis method (Morre et al., 1991), are sometimes used after the density gradient methods to achieve greater purity. Protoplasts prepared by cell wall-degrading enzyme digestion are lysed under osmotic and pH shock conditions, releasing their large central vacuoles. Disruption of protoplasts to yield the tonoplast can be achieved by relatively gentle mechanical shear forces. The protoplast disruption methods are often used to obtain the tonoplast without loss of the integrity of native membranes and with high purity (Keller, 1988; Rentsch and Martinoia, 1991; Getz, 1987; Preisser and Komor, 1991). An appropriate method

FUNCTIONAL RECONSTITUTION OF PROTON-ATPASE

103

for preparing the tonoplast from plant tissues should be selected with regard to the objective of the experiment, yield, purity, and the time for isolation. 2. Solubilization of V-ATPase from the Tonoplast

It is very important to select detergents that are most effective for solubilizing the V-ATPases and to determine the conditions required to maintain the enzyme in active form because the membrane proteins often lose their activity during solubilization. In the standard procedure, the V-ATPases are first cosolubilized with phospholipids in an appropriate detergent in order to form an isotropic solution of lipid-protein-detergent and lipiddetergent micelles. Next, the detergent is removed, resulting in the progressive formation of proteoliposomes. Various detergents have been used to solubilize the V-ATPases, including Triton X-100 [poly(oxyethelene glycol], Triton X-114 [octylphenolpoly(ethylene glycol ether)n], CI2ES[dodecylpoly(ethyleneglycolether)8], ClzE9 [dodecylpoly(ethyleneglycolether)9], n-octylglucoside (1-0-n-octyl-P-D-glucopyranoside), cholic acid, sodium salt (sodium cholate), deoxycholic acid, 3-[(3-cholamidopropyl)dimethylammoniol-1-propane sulfonate (CHAPS), 3-[(3-~holamidopropyl)dirnethylammonio]-2-hydroxy-l-propanesulfonate (CHAPSO), Zwittergent 3-14 (N-tetradecyl-N,N-dimethyl-3-ammonio-l-propane sulfonate), and lysophosphatidylcholine (lyso PC). The physicochemical properties of various detergents and the effects on the V-ATPase of B. vulgaris were investigated (Christine et al., 1992). The physicochemical properties of detergents; critical micelle concentration (cmc), which is defined as the concentration at which the detergent monomer forms micellar aggregates; micellar size in relation to the aggregation number of the detergents in a micelle; and the hydrophilic-lipophilic balance (HLB), which relates to the amphiphilicity of the detergent, are important for solubilization and for subsequent detergent removal resulting in the formation of proteoliposomes. Detergents with high cmcs (cholate, deoxycholate, octylglucoside,CHAPS, and CHAPSO) form relatively small micelles and can be removed by dialysis or gel filtration. Detergents with high HLB, such as sodium dodecylsulfate, have denaturing action, and detergents with HLBs between 12.5 and 14.5 are suitable for the solubilization of V-ATPase. Detergents are classified into ionic and nonionic types. These ionic properties of detergents should be given consideration in selecting the proper detergent for solubilization of the V-ATPase and later purification procedures. Other precautions, for example, the proper ratio of protein and detergent, the selection of appropriate pH range for the enzyme activity, the addition of glycerol, sucrose, and/or phospholipids stabilizing the enzyme, and a combination of these solubilizing conditions, should be taken

104

KUNlHlRO KASAMO AND HIROYASU YAMANISHI

to prevent the inactivation of V-ATPase during solubilization. Two-step solubilization procedures using a combination of two detergents are sometimes adapted for effective purification and solubilization. In these cases, the first detergent is used to remove proteins other than V-ATPase, probably proteins loosely bound to the tonoplast, and then the V-ATPases are solubilized by the second detergent. Combinations such as deoxycholate and Zwittegent 3-14 (Bennett and Spanswick, 1983), deoxycholate and noctylglucoside (Mandala and Taiz, 1985; Kasamo et al., 1991), Triton X100 and lyso-PC (Matsuura-Endo et aL, 1990; Yamanishi and Kasamo 1992a), and Triton X-114 and n-octylglucoside (Warren et aL, 1992) were used.

3. Purification of V-ATPase Possessing High Specific Activity V-ATPases of plant tonoplast were solubilized and reconstituted into liposomes without any purification steps after solubilization (Bennett and Spanswick, 1983; Kasamo et al., 1991; Warren et al., 1992; Banuls et al., 1992). Although the V-ATPases were selectively incorporated in the proteoliposomes, the possibility of minor contamination by other tonoplast components cannot be completely eliminated in these cases; thus, it will be necessary to purify the V-ATPase before reconstitution into liposomes for the accurate examination of the properties of the ion-translocating features of the enzyme. The V-ATPase is a relatively large enzyme in its native state compared to the other tonoplast proteins; thus, it can be purified partially by glycerol density gradient centrifugation in the case of yeast (Uchida et al., 1985) or by sucrose density gradient centrifugation in the case of plants (Mandala and Taiz, 1985) after solubilizing it from the tonoplast. In some cases, ammonium sulfate fractionation is effective to purify and remove the detergent used to solubilize the membrane (Moriyama and Nelson, 1987a; Xie and Stone, 1986). However, several chromatographic purification steps are necessary to achieve further purification. Chromatography on a gel filtration column (Randall and Sze, 1986), an ion-exchange column (Matsuura-Endo et al., 1990; Yamanishi and Kasamo, 1992a), and a combination of the two (Parry et al., 1989;Ward and Sze, 1992a) were used to purify the V-ATPase from plants. A hydroxylapatite column was used to purify the V-ATPase from animal cells (Moriyama and Nelson, 1987a; Xie and Stone, 1986). These chromatographic procedures, except in the case of V-ATPase from mung bean (Matsuura-Endo et al., 1990; Yamanishi and Kasamo, 1992a), were carried out in the presence of phospholipids (0.03-0.05 mg/ml) to retain the enzyme activity; thus, the purified V-ATPase fractions contained exogenously added phospholipids. These V-ATPases with high purity are usable for subsequent experiments on reconstitution. The V-ATPase puri-

FUNCTIONAL RECONSTITUTION OF PROTON-ATPASE

105

fied in the absence of exogenously added phospholipids (Matsuura-Endo etal., 1990;Yamanishi and Kasamo, 1992a)will be better suited for determination of the effects of lipid composition on proton-pumping activity.

4. Reconstitution of V-ATPase into Liposomes The mechanisms of the formation of proteoliposomes have been reviewed by other authors (Rigaud et al., 1995). Here, we describe practical methods of detergent removal to reconstitute proteoliposomes and other methods relevant to reconstitution applied to the V-ATPase. Mechanical methods, such as sonication and/or freeze-thaw techniques, have also been used for the reconstitution of the V-ATPase from animal cells (Xie and Stone, 1986; Xie et al., 1986). However, detergent-mediated reconstitution methods are the main techniques with respect to plant V-ATPase because the mechanical methods can possibly cause unfavorable perturbation of the membrane protein structure due to subtle changes produced by sonication and/or the freeze-thaw process. Proteoliposomes are formed by removal of the detergent from lipidprotein-detergent micelles. There are many variations of removing detergent based on the physicochemical properties of detergents as described under Section V,A,2. Detergents with high cmcs (cholate, deoxycholate, n-octylglucoside, CHAPS, and CHAPSO) generally form small micelles and can be removed by gel-filtration techniques. Depending on the size of the micelles, one can use different gel-sized columns ranging from Sephadex G-25 (Kasamo et al., 1991; Yamanishi and Kasamo, 1992b) to Sephadex G-200 (Bennett and Spanswick, 1983). The most significant advantage of this technique is its rapidity compared to the dialysis technique (avoiding long periods of contact between detergents and proteins), which, however, becomes a disadvantage in terms of incomplete protein incorporation and also a broader size distribution of proteoliposomes than that obtained by dialysis. Detergents (such as Triton X-100) that have a low cmc and consequently form large micelles are not readily removed by gel chromatography, and even less removed by dialysis, but can be efficiently removed through adsorption on hydrophobic resins such as Bio-Beads SM2 (Ward and Sze, 1992b) or Amberlite XAD beads (Banuls et al., 1992). A resin-prepacked column (Ampure DT column) is also available for this purpose (Yamanishi and Kasamo, 1994).From the abundant literature, it appears that reconstitution from lipid-protein-detergent mixtures yields proteoliposomes of different sizes and compositions, depending on the nature of the detergent, the particular procedure used to remove it, the protein and lipid ratio, the ionic conditions, and the temperature when proteoliposomes are formed. It is important to examine such characteristics of the reconstitution systems thoroughly to achieve optimal reconstitution and avoid artifactual interpre-

106

KUNlHlRO KASAMO AND HIROYASU YAMANISHI

tation of the results. Another procedure for obtaining proteoliposomes from lipid-protein-detergent micellar solutions consists of diluting the reconstitution mixture. Dilution lowers the detergent concentration to below its cmc and proteoliposomes form spontaneously. Detergents with high cmcs, such as cholate or n-octylglucoside, have been used, and generally the dilution was followed by centrifugation of the diluted mixture containing proteoliposomes. The dilution technique failed in the case of V-ATPase from the tonoplast of mung bean hypocotyls. Spontaneous incorporation of V-ATPases into preformed sonicated liposomes has not yet been reported. Organic solvents have had limited use for solubilization of the V-ATPase because the exposure to organic solvents often denatures amphiphilic proteins. Toluene (Sun et d., 1987) or chloroform/methanol (Kaestner et d , 1988) are used to extract the highly hydrophobic Vo fraction that is deeply embedded into the tonoplast.

5. Artificial Manipulation of Lipids andor Protein Components Although only limited information is available on the purified plant VATPases reconstituted into artificial liposomes, the reconstituted proteoliposome systems are very useful for our direct understanding of their structure, function, and regulation. It was conclusively demonstrated that the structure of the V-ATPase composed of at least nine polypeptides was sufficient for coupled ATP hydrolysis and H+ translocation, and the direct regulation of V-ATPase by C1- concentration was also demonstrated (Ward and Sze, 1992b). The proton-pumping kinetics without other components of the tonoplast have been studied (Warren et al., 1992). Artificial changes in the lipid compositions of proteoliposomes clarify the effects of lipids on the protonpumping activity of the enzyme (Warren et aZ., 1992; Yamanishi and Kasamo, 1994). A coreconstitution system like that for bacteriorhodopsin and FoF1-ATPaseshas not yet been established with respect to the V-ATPase and other tonoplast proteins such as antiporters or ion channels. Our understanding of ion translocation across the vacuolar membranes will progress enormously provided that a coreconstitution system can be established.

6. Practical Approach of Reconstitution of V-ATPases into Liposomes

Preparation of tonoplast vesicles of high purity and in a large quantity is important for the subsequent purification of V-ATPase and its reconstitution into liposomes. A floating centrifugation method developed by Matsu-

FUNCTIONAL RECONSTITUTION OF PROTON-ATPASE

107

ura-Endo er al. (1990), in which the tonoplast vesicles suspended in 0.3 M sucrose solution migrate to the interface portion between 0.3 M sucrose and 0.25 M sorbitol during centrifugation for 40 min, is a rapid and effective procedure for purification of the tonoplast. The lumen of the tonoplast vesicles from mung bean hypocotyls isolated by the floating centrifugation method was acidified by adding Mg2+-ATP, as measured by fluorescence quenching of quinacrine. Proton pumping was inhibited completely by 50 mM KN03,lO p M NEM, or 100 p M DCCD, but V 0 4 had no inhibitory effect. The proton gradient formed across the membrane was collapsed by nigericin, which catalyzes the electroneutral exchange of protons for potassium (Pressman, 1976), and the nonspecific ionophore gramicidin, or FCCP and CCCP, which are known to be uncouplers. Bafilomycin A l was less effective in collapsing the proton gradient once it had formed. O n the other hand, the addition of bafilomycin before proton pumping was begun by adding MgS04 strongly inhibited Mg’+-ATPase-dependent proton pumping at a concentration at the nanomolar level. The concentration of bafilomycin at half-maximal inhibition was about 0.5 nM. These facts indicate that the tonoplast vesicles are useable as the starting material for the purification of the V-ATPase. Two-step solubilizing methods sometimes are quite effective for purification of the V-ATPase. Tonoplast vesicles of Kulanchoe were suspended in buffer containing 0.62% Triton X-114, and the Triton-insoluble material was washed again in the same buffer. This procedure removed almost all proteins other than V-ATPase, which was then solubilized with n-octylglucoside (Warren et al., 1992). These authors stressed that the concentration of Triton X-114 and the conditions of solubilization by noctylglucoside were of critical importance. Similar to this case, a two-step solubilizing system with Triton X-100 and lyso-PC was used for mung bean tonoplast (Matsuura-Endo et al. 1990; Yamanishi and Kasamo, 1992a). The solubilized mung bean V-ATPase with lyso-PC was purified on a Mono Q ion-exchange column. Many phospholipids in the tonoplast were removed by treatment with Triton X-100. Subsequent purification by Mono Q ion-exchange FPLC removed almost all phospholipids from the ATPase. Lyso-PC was used to solubilize the VATPase passing through the Mono Q ion-exchange column, whereas the V-ATPase was retained and later eluted by NaC1. If the molecular mass of ATPase is taken to be 650 kDa (Ward and Sze, 1992a), one molecule of phospholipid or less per molecule of ATPase was detectable in the purified ATPase fraction. Sterols and glycolipids were still associated to some extent with the purified ATPase [lo-15 sterol molecules and 25-30 glycolipid molecules per ATPase molecule (Yamanishi and Kasamo, 19931. Lipids located at the lipid-protein interface are distinguished from bulk lipids of the membrane bilayer and are referred to as “boundary layer

108

KUNlHlRO KASAMO AND HIROYASU YAMANISHI

lipids” or “lipid annulus” because they may be able to influence membrane protein activity (Quinn and Williams, 1990). The boundary layer lipids or lipid annulus are thought to surround a protein with a single layer (Kasamo and Sakakibara, 1995). Warren et al. (1975) reported that 30 phospholipid molecules in the annulus surrounding the Ca2’-ATPase of the sarcoplasmic reticulum were necessary to activate the enzyme. Only the Vo portion of V-type ATPase, whose molecular mass is about 250 kDa (Zhang et al., 1992), is embedded in the membrane. Assuming a cylindrical shape, with its radius proportional to the square root of the molecular mass of a protein, the calculated boundary lipids of V-type ATPase are about 1.5 times those in the sarcoplasmic reticulum CaZt-ATPase,whose molecular mass is about 110 kDa. The purified and delipidated tonoplast H+-ATPase from mung bean hypocotyls still retained 10-15 sterol molecules and 25-30 glycolipid molecules per ATPase molecule. Thus, 35-45 sterol and glycolipid molecules is a reasonable number for the amount of boundary lipid molecules of the enzyme. However, essentially no ATPase activity was detectable without the addition of exogenous phospholipid. Asolectin at a concentration of 0.005-0.01% was necessary to achieve maximal ATPase activity. The activity of membrane-bound enzymes depends to a great extent on the physical state of the lipid constituents of the membrane, which in turn is affected by temperature. Thus, for example, phospholipid classes and the degree of unsaturation of fatty acids and/or sterol content all influence such activities. Certain membrane-bound enzymes have been shown to require a liquid-crystalline phospholipid environment (Warren et al., 1975; George et al., 1989) and phospholipids with a specific acyl chain length (Montecucco et al., 1982; Kasamo, 1990; Yamanishi and Kasamo, 1993). Kasamo et al. (1991), as well as Bennett and Spanswick (1983), successfully reconstituted a proton pump by incorporating H+-ATPase,which had been solubilized from a tonoplast-enriched membrane fraction, in liposomes. The resultant proteoliposomes may have contained considerable amounts of intrinsic membrane components. Subsequently, purified Ht-ATPases from two plant sources were functionally incorporated in liposomes (Warren et al., 1992;Ward and Sze, 1992b).These ATPases had been solubilized and/or purified in the presence of exogenously added phospholipids, and thus preparations may have contained small amounts of native phospholipids. To examine the effects of lipids on proton-pumping activity, purified H+-ATPase must first be freed of lipids and then maximally activated by exogenously added phospholipids. H+-ATPase purified by the method described previously should be ideal for such examinations because it is free from phospholipids other than the boundary layer and has little activity in the absence of exogenously added phospholipids. A simple and rapid method for the functional reconstitution of a proton pump by incorporating

FUNCTIONAL RECONSTITUTION OF PROTON-ATPASE

109

delipidated and purified H+-ATPase in liposomes has been developed by modifying the earlier methods (Kasamo et al. 1991). Mainly two methods have been used to remove detergent from lipidprotein-detergent miccelles to form proteoliposomes with respect to the plant V-ATPase. One is the gel-filtration method (Kasamo et al., 1991; Yamanishi and Kasamo, 1992b; Warren et al., 1992), and the other is the resin absorption method (Ward and Sze, 1992a; Yamanishi and Kasamo, 1994).The proteoliposomes prepared by the resin absorption method (Yamanishi and Kasamo, 1994) are likely to be more stable than those prepared by the gel-filtration method (Yamanishi and Kasamo, 1992b) because the proteoliposomes prepared by the gel-filtration method may lose their proton-pumping activity due to the freeze-thaw treatment. Reconstituted proteoliposomes of V-ATPase of mung bean hypocotyls required the proper content of cholesterol for stable proton pumping (Yamanishi and Kasamo, 1994). Proteoliposomes prepared from cholesterol and asolectin at a ratio of 45 : 55 (w/w) and at a ratio of lipid to protein of 200: 1 (w/w) generated the largest pH gradient, as determined by the ATPgenerated quenching of quinacrine fluorescence. In the presence of cholesterol, the pH gradient formed across the membranes of proteoliposomes and the average diameter (168 nm) of proteoliposomes increased about 2fold. The initial rate of proton pumping decreased to 20% of that observed with proteoliposomes prepared from asolectin alone. The addition of cerebroside to asolectin at a ratio of 5:95 (w/w) caused a 1.6-fold increase in the maximum pH gradient without any significant change in the initial rate of proton pumping or the average diameter of proteoliposomes, but the maximum pH gradient decreased greatly at ratios above 20: 80 (w/w). After the addition of cerebroside to the reconstitution mixture for preparation of proteoliposomes, the maximum ApH increased with increasing amounts of cerebroside, being greatest at 5%, but the maximum ApH decreased with more than 5% (w/w) cerebroside. The latter effect may have been due to the formation of clusters of cerebroside in the phospholipid bilayer. Model membranes composed of phosphatidylcholine and cerebroside exist as mixed fluid crystals at concentrations of cerebroside from 1 to 5 mol%, whereas clusters form above 20 mol% at physiologically relevant temperatures such as 37°C (Curatolo, 1986). The maximum pH gradient was transient and decreased spontaneously when asolectin alone was used to prepare proteoliposomes or when cerebroside and asolectin were used together. The disappearance of the proton gradient once it had formed and/or leakage of protons were suppressed by cholesterol at ratios above 30 : 70 (w/w). It was clear, therefore, that cholesterol and asolectin at ratios of 30 :70 to 45 : 55 (w/w) formed larger and more stable proteoliposomes than did asolectin alone (Fig. 2). Valinomycin induced the quenching of quinacrine fluorescence in the absence of Mg2+-ATPwhen K'-loaded proteoliposomes

110

KUNlHlRO KASAMO AND HIROYASU YAMANISHI

3 m M MgSO4

7

4 a

b

Asolectln

+5% Cerebroside

+30% Cholesterol

1 mln

A

5 pM Nigericln

FIG. 2 Effects of cerebroside and cholesterol on the characteristics of Htpumping across membranes of proteoliposomes with V-ATPase (Y amanishi and Kasamo, 1994).

that had been prepared from asolectin alone were diluted in K+-free reaction mixture. Thus, leakage of protons into the proteoliposomes was driven by a K+/valinomycin-generated membrane potential (Fig. 3). Valinomycin did not induce leakage of protons in the case of proteoliposomes prepared from cholesterol and asolectin at a ratio of 30 :70 (w/w), an indication that proteoliposomes that contained 30% cholesterol were less leaky to protons than proteoliposomes without cholesterol. The addition of cholesterol at 30% or more of the total lipid content completely suppressed the spontaneous decrease in pH (Fig. 2c). As noted by Yeagle (1985), cholesterol might reduce the permeability of phospholipid bilayers to cations. Proteoliposomes containing 30% cholesterol were less leaky to protons than those prepared from asolectin alone (Fig. 3). The proton gradient that formed across the membranes of proteoliposomes prepared from cholesterol and

111

FUNCTIONAL RECONSTITUTION OF PROTON-ATPASE

+Cholesterol

-Cholesterol (-ATPI

(-ATPI

Valinomycin c

+

Valinomycin

.) ”

w

“‘w?

FIG. 3 Effects of cholesterol on the permeability to protons of proteoliposomes (Yamanishi and Kasamo, 1994).

asolectin at a ratio of 30:70 (w/w) was more stable than the gradient across membranes of proteoliposomes prepared from asolectin alone (Fig. 2a and 2c). Proteoliposomes prepared from cholesterol and asolectin (30 :70, w/w) were therefore used to compare the characteristics of proton pumping with those of the native tonoplast. The effects of various inhibitors on proton pumping across membranes of native tonoplast vesicles and proteoliposomes reconstituted with the HC-ATPasewere studied. Proton-pumping across membranes of native tonoplast vesicles and proteoliposomes reconstituted with the enzyme was inhibited by NO3-,NEM, and DCCD, all of which inhibit ATP-hydrolyzing and proton-pumping activities of the V-ATPases. V04, which inhibits plasma membrane ATPases, failed to inhibit the proton pumping in either system. A more specific inhibitor of V-ATPases is bafilomycin Al (E. J. Bowman et af., 1988a). Bafilomycin Al at 1 nM inhibited proton pumping by about 50%. The proton gradient across membranes of native tonoplast vesicles and reconstituted proteoliposomes was collapsed with the addition of various ionophores and uncouplers, such as nigericin, gramicidin, FCCP (Fig. 4). Cholesterol at certain relative levels appeared to reduce the maximum ApH (Fig. 2). Cholesterol maximally decreased the maximum ApH at 10% (w/w) but gradually increased it beyond 30% (w/w). This behavior was also observed by Xie et af. (1986), although the maximum ApH for proteoliposomes that contained 15-20% (w/w) cholesterol was the lowest level, and the ApH for proteoliposomes that contained 25-27% (w/w) cholesterol was

112

KUNlHlRO KASAMO AND HIROYASU YAMANISHI

the highest level. Changes in the physical state of the lipid bilayer may explain the complex effects of cholesterol. A mixture of cholesterol and phosphatidylcholine may exist as two immiscible solid phases below 20 mol% cholesterol and may be a eutectic mixture above 20 mol% cholesterol (Mabrey et af., 1978). Twenty mol% cholesterol corresponds to 10% by weight, if the molecular weight of cholesterol is assumed to be about half that of asolectin. Thus, the critical ratios of cholesterol to total lipid for the lowest and highest values of pH would appear to reflect the different physics of mixtures of cholesterol and phospholipid. The lipid composition with a mixture of phosphatidylcholine, phosphatidylserine, and cholesterol, in the ratio 27 : 53 :20, was found to be close to the optimum for both the ATP-hydrolytic and proton-pumping activities of the enzyeme (Warren et af., 1992). Similar stimulation by lipids was observed with the chromaffin granule V-ATPase (Perez-Castineira and Apps, 1990). The V-ATPase purified from the tonoplast of mung bean hypocotyls could also be functionally reconstituted with a mixture of phosphatidylcholine, phosphatidylserine, and cholesterol in the ratio 40 :40 :20. Phosphatidylethanolamine could not be substituted for phosphatidylcholine or phosphatidylserine (H. Yamanishi, unpublished data). The V-ATPase of chromaffin granules can be substantially delipidated by ammonium sulfate precipitation, and the resulting delipidated enzyme could be fully reactivated by soybean or extracted chromaffin granule phospholipids (Buckland et af., 1981).Reconstitution with dipalmitoylphosphatidylcholineor dimyristoylphosphatidylcholine resulted in the generation of an ATPase, which showed two activation energies with a change in slope at a temperature almost identical to the gel-to-liquid crystalline phase transition temperature as measured by fluorescence polarization of the probe diphenylhexatriene, suggesting that the ATPase activity could be regulated by the viscosity of the membrane (Buckland et af., 1981). The lipid requirement for reconstitution was also examined with plasma membrane ATPase (Kasamo and Yamanishi, 1991). Both H' pumping and the hydrolysis of ATP by the plasma membrane ATPase are strongly affected by the polar head group and compositon of the fatty acyl chain of the phospholipids used to prepare liposomes for reconstitution of the ATPase. Kasamo et af. (1992) reported that the proportion of unsaturated fatty acyl chain in the total phospholipids of the plasma membrane and tonoplast from chilling-insensitivecultured rice cells was much higher than that from chilling-sensitive cells. This fact suggests a higher fluidity of membranes in chilling-insensitivecells than in chilling-sensitiveones. Suspension-cultured cells of tomato that originated from high altitudes in the Andes showed a great tolerance to chilling and the proton-pumping activity acclimated to a lower temperature range after being precultured at a low temperature (DuPont and Mudd, 1985). Molecular mechanisms that cause the difference between proton-pumping activity of the tonoplast from chilling-sensitive

113

FUNCTIONAL RECONSTITUTION OF PROTON-ATPASE

and chilling-insensitive plants are still unclear. Differences in the H+ -ATPase molecule itself, in the physical state of lipid bilayer, or in the interaction of the enzyme with the membrane may be responsible for the chilling sensitivity. Reconstitution of a proton pump in proteoliposomes prepared with H+-ATPasefrom chilling-sensitiveplants and tonoplast lipids from chilling-insensitive plants, and vice versa, will clarify the molecular mechanisms concerning chilling sensitivity (data not shown). The method developed in the current study will provide a useful tool to gain more insight into the effects of the lipid bilayer on the proton-pumping activity of the tonoplast. To date, no proteins that transport solutes, such as ions, amino acids, or sugars, have been purified other than H+-ATPase and H+-PPase,and their identity has not been ascertained because they have no enzymic activity after solubilization. If we are able to incorporate one of these proteins into reconstituted proteoliposomes with H+-ATPase,it will be possible to transport a particular solute by energizing the proteoliposomes with ATP. As a result, the function and identity of the protein will be ascertained. The presented method will also provide a useful tool for such future studies. The V-ATPase purified from mung bean is inhibited by free fatty acids, especially polyunsaturated ones (Fig. 5). Unsaturated free fatty acids also inhibit Na+, K+-ATPase (Swarts et al., 1990) and dissipate proton electrochemical gradients in pea stem microsomes (Macri et al., 1991). The role of free fatty acids in biological membranes has not clarified. The reconstitution method developed for the V-ATPase is applicable to various fields of membrane biology such as those mentioned previously.

VI. Future Prospects V-ATPase consists of at least 9 or 10 subunits, which were grouped into and 16 or 17 kDa. 67- to 37- and five groups; 115-95,67-73,62-55,45-28, 2 p M Vaiinomycin 3mM MgS04

1

5nM Bafilomycin A 1 50mM KN03

A

inhibitor

“L g

1 min

A

10pM Nigericin

FIG. 4 Reconstitution of V-ATF’ase into lipid mixture: PC/PS/cholesterol = 4 : 4: 2.

114

KUNlHlRO KASAMO AND HIROYASU YAMANISHI

FIG. 5 Effects of fatty acids on the activity of V-ATPase from mung bean hypocotyls (H. Yamanishi and K. Kasamo, unpublished data).

62- to 55-kDa subunits have been classified as peripheral and 115- to 95and 16- or 17-kDa subunits are integral. Subunit groups 45-28 kDa contain peripheral and integral groups and relate to assembly and activity. The 40to 42-kDa subunit is mainly peripheral and the 32-kDa subunit is an integral sector. Subunits 67-73, 62-55 and 16 or 17 kDa are common to all VATPases. All the bovine chromaffin granule and coated vesicle and yeast H+-ATPaseshave subunits of about 100-115 kDa (Arai et af., 1988;Forgac, 1989; Kane et al., 1989), whereas the bovine Golgi membrane enzyme lacks a subunit of this molecular mass (Moriyama and Nelson, 1989a). In plants, the 100-kDa subunit was also found in the purified enzyme fraction (Parry et al., 1989), as well as chromaffin granules, coated vesicles, and yeast. The cloning of the 100-kDa subunit of plants will be successful in the future. The function of the 100-kDa subunit remains unknown. The 100-kDa subunit can be bound to carbohydrate and exposed on the outside of the cell (Adachi et al., 1990), suggesting that this subunit may possibly function in signal transduction. Subfamilies of each subunit (57,100, and 16 kDa) were isolated and cloned (Berkelman et af., 1994; Peng et af., 1994; Hasenfratz et al., 1995). The significance of a small multigene family encoding the

FUNCTIONAL RECONSTITUTION OF PROTON-ATPASE

115

subunit is not yet clear; however, the V-ATPase genes represented by the cDNA clones of the 16-kDa subunit are differentially expressed in mature and expanding tissue (Hasenfratz et ul., 1995). As shown in Table 11, the genes encoding from 100 to 14 kDa of the V-ATPase from yeast could be systematically described. A slight correspondence was observed between the genes encoding yeast and plant V-ATPases. VMA1, VMA2, and VMA3 are 70-68, 57-60, and 16 or 17 kDa, respectively. The 0 , p, y, and E subunits of FIFo-ATPase are equivalent to VMA2, VMA1, VMA8, VMA7, respectively. The A, B, C, D, and E subunits of bovine chromaffin granules are equivalent to VMAl, VMA2, VMA5, VMA8, and VMA4, respectively. In the future, the correspondence of these genes between yeast, Neurosporu, animal, and plants will be more clear.

VII. Concluding Remarks The V-ATPase actively transports protons across the vacuolar membranes using the free energy liberated by the hydrolysis of ATP to generate an inside acid pH difference and an inside positive electrical potential difference that provides the driving force for the secondary transport of numerous ions and metabolites. The V-ATPase has an apparent functional mass of 400-600 kDa and comprises at least 9 or 10 different subunits, of which the catalytic 67-73 kDa, the neucleotide-binding 55-62 kDa, proteolipids 95-115 and 16 or 17 kDa,and 44-29 kDa subunits required for activity and assembly are universal components. In plants, the 70- to 68-, 55- to 60-, 44-, 42-, 36-, and 29-kDa subunits are peripheral and the loo-, 16-, and 32-kDa subunits are integral. The 100-kDa subunit is present in barley and red beet but is not present in oat and mung bean. Molecular cloning of the genes encoding the 69-, 57-, 31-, and 16-kDa subunits in plants has been successful; however, it has been unsuccessful for the 100-kDa subunit. Multigene families encoding the 57- and 16-kDa subunits are present in cotton. A multigene family of 100-kDa will be found in plants. Subunits 57 and 70-kDa are found to be necessary for Hf-ATPase activity by reconstitution of animal Hf-ATPase, and the 16- and 100-kDa subunits are also needed to induced the proton pumping. Reconstitution of the V-ATPase complex and the recombinant subunit of Hf-ATPase from clathrin-coated vesicles has been successful. In plants, reconstitution of the V-ATPase complex from mung bean, corn, citrus, and crassulacean plants has been reported; however, reconstitution of the recombinant subunit of Hf-ATPase has not been successful.

116

KUNlHlRO KASAMO AND HIROYASU YAMANISHI

References Adachi, I., Puopolo, K., Sterling, N. M., Arai, H., and Forgac, M. (1990). Dissocition, crosslinking, and glycosylation of the coated vesicles proton pump. J. Biol. Chem. 265,967-973. Amalou, Z., Bibrat, R., Brugidou, C., Triuslot, P., and d'Auzac, J. (1992). Evidence for an amiloride-inhibited Mg2+M+antiporter in lutoid (vacuolar) vesicles from latex of Hevea brasiliensis. Plant Physiol. 100, 255-260. Amzel, L. M., and Pedersen, P. L. (1983). Proton ATPases: Structure and mechanism. Annu. Rev. Biochem. 52,801-824. Arai, H., Berne, H., and Forgac, M. (1987a). Inhibition of the coated vesicle proton pump and labeling of a 17,000-dalton polypeptide by N,N'-dicyclohexylcarbodiimide.J. Biol. Chem. 262,11006-11011. Arai, H., Berne, M., Terres, G., Terres, H., Puopolo, K., and Forgac, M. (1987b). Subunit composition and ATP site labeling of the coated vesicle proton-translocating adenosinetriphosphatase. Biochemistry 26, 6632-6638. Arai, H., Terres, G., Pink, S., and Forgac, M. (1988). Inhibition of the coated vesicles proton pump and labeling of a 17000-dalton polypeptide by N,N'-dicyclohoxylcarbodiimide. J. Biol. Chem. 263,8796-8802. Askerlund, P. (1996). Modulation an intracellular calmodulin-stimulated Ca2+-pumpingATPase in cauliflower by trypsin: The use of calcium green-5N to measure Ca2+transport in membrane vesicles. Plant Physiol. 110, 913-922. Banuls, J., Ratajczak, R., and LUttge, U. (1992). Solubilization and functional reconstitution of the tonoplast H+-ATPase from Citrus in liposomes. J. Plant Physiol. 144, 74-79 Barkla, B. J., Zingarelli, L., Blumwald, E., and Smith, J. A. C. (1995). Tonoplast Na+/H+ antiport activity and its energization by the vacuolar Ht-ATPase in the halophytic plant Mesembryanthemum crystallinum L. Plant Physiol. 109, 549-556. Bassham, D. C., and Raikhel, N. V. (1996). Transport proteins in the plasma membrane and the secretory system. Trends Plant Sci. 1, 15-20. Bauerle, C., Ho, M. N., Lindorfer, M. A., and Stevens, T. H. (1993). The Succharomyces cerevisiae VMA6 gene encodes the 36-kDa subunit of the vacuolar Ht-ATPase membrane sector. J. Biol. Chem. 268, 12749-12757. Beltran, C., Kopecky, J., Pan, Y.-C. E., Nelson, H., and Nelson, N. (1992). Cloning and mutational analysis of the gene encoding subunit c of yeast vacuolar H+-ATPase. J. Biol. Chem. 267,774-779. Bennett, A. B., and Spanswick, R. M. (1983). Solubilization and reconstitution of an anionsensitive H+-ATPase from corn roots. J. Membr. Biol. 75, 21-31. Berkelman, T., Houtchens, K. A., and DuPont, F. M. (1994). Two cDNA clones encoding isoforms of the B subunit of the vacuolar ATPase from barley roots. Plant Physiol. 104, 287-288. Blackford, S . , Rea, P. A., and Sanders, D. (1990). Voltage sensitivity of H+/Ca2+antiport in higher plant tonoplast suggests a role in vacuolar calucium accumulation. J. Biol. Chem. 265, 9617-9620. Boller, T., and Wiemken, A. (1986). Dynamics of vacuolar compartmention. Annu. Rev. Plant Physiol. 37, 137-164. Bowman, B., Allen, R., Wechser, M. A., and Bowman, E. J. (1988). Isolation of gene encoding the Neurospora vacuolar ATPase. Analysis of Vma-2 encoding the 57-kDa polypeptide and comparison to Vma-1.J. Biol. Chem. 263,14002-14007. Bowman, B. J., and Bowman, E. J. (1986). H+-ATPasefrom mitochondria, plasma membranes and vacuoles of fungal cells. J. Membr. Biol. 94, 83-97. Bowman, B. J., Dschida, J., Harris, T., and Bowmane, E. J. (1989). The vacuolar ATPase of Neurospora crassa contains an F1-like structure. J. Biol. Chem. 264, 15606-15612.

FUNCTIONAL RECONSTITUTION OF PROTON-ATPASE

117

Bowman, E., Steinhardt, A., and Bowman, B. J. (1995). Isolation of the vma4 gene encoding the 24-kDa subunit of the Neurospora crassa vacuolar ATPase. Biochim. Biophys. Acta l237,95-98. Bowman, E. J. (1983). Comparison of the vacuolar membrane ATPase of Neurospora crassa with the mitochondorial and plasma membrane ATPases. J. Biol. Chem. 258,15238-15244. Bowman, E. J., Mandala, S., Taiz, L., and Bowman, B. J. (1986). Structural studies of the vacuolar membrane ATPase from Neurospora crassa and comparison with the tonoplast membrane ATPase from Zea mays. Proc. Natl. Acad. Sci. USA 83,48-52. Bowman, E. J., Siebers, A., and Altendorf, K. (1988a). Bafilomycins: A class of inhibitors of membrane ATPases from microorganisms, animal cells, and plant cells. Proc. Natl. Acad. Sci. USA 85,7972-7976. Bowman, E. J., Tenney, K., and Bowman, B. J. (1988b). Isolation of genes encoding the Neurospora vacuolar ATPase. Analysis of Vma-1 encoding the 67-kDa subunit reveals homology to other ATPases. J. Biol. Chem. 263, 13994-14001. Brernberger, C., and Luttge, U. (1992). Dynamics of tonoplast proton pumps and other transport proteins of Mesembryanthemum crystallinurn L. during the induction of Crassulacean acid metabolism. Planta 188, 575-580. Bremberger, C., Haschke, H.-P., and Luttge, U. (1988). Separation and puification of the tonoplast ATPase and pyrophosphatase from plants with constitutive and inducible Crassulacean acid metabolism. Planta 175, 465-479. Buckland, R. M., Radda, G. K., and Wakefield, L. M. (1981). The role of phospholipids in the modulation of enzyme activities in the chromaffin granule membrane. Biochim. Biophys. Acta 643, 363-375. Carystinos, G. D., MacDonald, H. R., Monroy, A. F., Dhindsa, R. S., and Poole, R. J. (1995). Vacuolar H+-translocatingpyrophosphatase is induced by anoxia or chilling in seedlings of rice. Plant Physiol. 108, 641-649. Chanson, A,, and Taiz, L. (1985). Evidence for an ATP-dependent proton pump on the golgi of corn coleoptiles. Plant Physiol. 78, 232-240. Chrispeels, M. J., and Agre, P. (1994). Aquapoins: Water channel proteins of plants and animal cells. Trends Biochem. Sci. 19, 421-425. Christine, M. E., Ford, R. C.,and Holzenburg, A. (1992). Detergent sensitivity of the tonoplast H+-ATPase and its purification from Beta vulgaris. Biochim. Biophys. Acta 1136,319-326. Cidon, S., and Nelson, N. (1983). A novel ATPase in the chromaffin granule membrane. J. Biol. Chem. 258,2892-2898. Cidon, S . , and Nelson, N. (1986). Purification of N-ethylmaleimide-sensitive ATPase from chromaffin granule membranes. J. Biol. Chem. 261,9222-9227. Crider, B. P., Xie, X.-S., and Stone, D. K. (1994). Bafilomycin inhibits proton flow through the H' channel of vacuolar proton pumps. J. Biol. Chem. 269,17379-17381. Curatolo, W. (1986). The interactions of 1-palmitoyl-2-oleyl-phosphatidylcholine and bovine brain cerebroside. Biochim. Biophys. Acta 861, 373-376. Darley, C. P., Davis, J. M., and Sanders, D. (1995). Chill-induced changes in the activity and abundance of the vacuolar proton-pumping pyrophosphatase from mung bean hypocotyls. Plant Physiol. 109,659-665. Dean, G . E., Fishkes, H., Nelson, P. J., and Rudnick, G. (1984). The hydrogen ion-pumping adenosinetriphosphatase of platelet dense granule membrane. Differences from FIFO-and phosphoenzyme-type ATPases. J. Biol. Chem. 259,9569-9574. Depta, H., Hostein, S. E. H., Robinso, D. G., Lutzelschwab, M., and Michalke, W. (1991). Membrane markers in highly purified clathrin-coated vasicles from Cucurbita hypocotyls. Planta 183,434-442. Dietz, K.-J., Rudloff, S., Ageorges, A., Eckerskorn, C., Fischer, K., and Arbinger, B. (1995). Subunit E of the vacuolar H+-ATPase of Hordeum vulgare L.: cDNA cloning, expression and immunological analysis. Plant J. 8, 521-529.

118

KUNlHlRO KASAMO AND HIROYASU YAMANISHI

Dschida, W. J., and Bowman, B. J. (1992). Structure of the vacuolar ATPase from Neurospora crassa as determined by electron microscopy. J. Biol. Chem. 267, 18783-18789. DuPont, F. M., and Morrissey, P. J. (1992). Subunit composition and Ca2+-ATPase activity of the vacuolar ATPase from barley roots. Arch. Biochem. Biophys. 294, 341-346. DuPont, F. M., and Mudd, J. B. (1985). Acclimation to low temperature by microsomal membranes from tomato cell cultures. Plant Physiol. 77,74-78. Eisenberg, D., Schwarz, E. K., Komaromy, M., and Wull, R. (1984). Analysis of membrane and surface protein sequences with the hydrophobic moment plot. J. Mol. Biol. 179,125-142. Fichmann, J., Taiz, L., Gallagher, S., Leonard, R. T., Depta, H., and Robinson, D. G. (1989). Immnological comparison of the coated vesicle H--ATPases of plants and animals. Protoplasma 153,117-125. Forgac, M. (1989). Structure and function of vacuolar class of ATP-driven proton pumps. Physiol. Rev. 69,765-796. Forgac, M., and Cantley, L.(1984). Characterization of the ATP-dependent proton pump of clathrincoated vesicles. J. Biol. Chem. 259, 8101-8105. Foury, F. (1990). The 31-kDa polypeptide is an essential subunit of the vacuolar ATPase in Saccharomyces cerevisiae. J. Biol. Chem. 265, 18554-18560. Futai, M., Noumi, T., and Maeda, M. (1989). ATP synthase (H+-ATPase): Results by combine biochemical and molecular biological approaches. Annu. Rev. Biochem. 58, 111-136. George, R., Lewis, R. N. A. H., Mahajan, S., and McElhaney, R. N. (1989). Studies on the purified, lipid-reconstituted (Na+ + Mg2+)-ATPasefrom Acholeplasma laidlawii B membranes: Dependence of enzyme activity on lipid head group and hydrocarbon chain structure. J. Biol. Chem. 264, 11598-11604. Getz, H. P. (1987). Accumulation of sucrose in vacuoles released from isolated beet root protoplasts. Plant Physiol. Biochem. 25, 573-579. Getz, H. P., and Klein, M. (1995a). The vacuolar ATPase of red beet storage tissue: Electron microscopic demonstration of the “head-and-stalk” structure. Bot. Acfa 108, 14-23. Getz, H. P., and Klein, M. (1995b) Characteristics of sucrose transport and sucrose-induced H+ transport on the tonoplast of red beet (Beta vulgaris L.) storage tissue. Plant Physiol. 107,459-467. Gluck, S . , and Caldwell, J. (1987). Immuno affinity purification and characterization of vacuolar Ht-ATPase from bovine kidney. J. Biol. Chem. 262, 15780-15789. Gogarten, J. P., Fichmann, J., Morgan, L., DeLapp, K., Styles, P., Taiz, S. L., and Taiz, L. (1992). The use of antisense mRNA to inhibit the tonoplast H+-ATPase of carrot. Plant Cell 4, 851-864. Gomez, L., and Chrispeels, M. J. (1993). Tonoplast and soluble vacuolar proteins are targeted by different mechanisms. Plant Cell 5, 1113-1124. Graham, L. A., Hill, K. J., and Stevens, T. H. (1994). VMA 7 encodes a novel 14-kDa subunit of the Saccharomyces cerevisiae vacuolar Hi-ATPase complex. J. Biol. Chem. 269,25974-25977. Graham, L. A., Hill, K. J., and Stevens, T. H. (1995). VMA8 encodes a 32-kDa V1 subunit of the Saccharomyces cerevicea vacuolar H+-ATPase required for function and assembly of the enzyme complex. J. Biol. Chem. 270,15037-15044. Greutert, H., and Keller, F. (1993). Further evidence for stachyose and sucrose/H+ antiporter on the tonoplast of Japanese artichoke (Stachys sieboldii) tubers. Plant Physiol. 101,13171322. Hager, A., Berthold, W., Biber, W., Edel, H.-G., Lanz, C. H., and Schiebel, G. (1986). Primary and secondary energized ion translocating systems on membranes of plant cells. Ber. Deutsh. Bot. Ges. 99, 281-295. Hanada, H., Moriyama, Y., Maeda, M., and Futai, M. (1990). Kinetic studies of chromaffin granule H+-ATPase and effects of bafilomycin A. Biochem. Biophys. Res. Commun. 170, 873-878.

FUNCTIONAL RECONSTITUTION OF PROTON-ATPASE

119

Hasenfratz, M.-P., Tsou, C.-L., and Wilkins, T. A. (1995). Expression of two related vacuolar H+-ATPase 16-kilodalton proteolipid genes is differentially regulated in a tissue-specific manner. Plant Physiol. 108, 1395-1404. Hedrich, R., and Schroeder, J. I. (1989). The physiology of ion channels and electrogenic pumps in higher plants. Annu. Rev. Plant Physiol. 40, 539-569 Herman, E. M., Li, X., Su, R. T., Larsen, P., Hsu, H. T., and Sze, H. (1994). Vacuolar-type H+-ATPases are associated with the endoplasmic reticulum and provacuoles of root tip cells. Plant Physiol. 106, 1313-1324. Hill, K. J., and Stevens, T. H. (1995). VMA22p is a novel endoplasmic reticulum-associated protein required for assembly of the yeast vacuolar H+-ATPase complex. J. Biol. Chem. 270,22329-22336. Hirata, R., Oshumi, Y., Nakano, A., Kawasaki, H., Suzuki, K., and Anraku, Y. (1990). Molecular structure of a gene, VMA1, encoding the catalytic subunit of H+-translocatingadenosine triphosphatase from vacuolar membranes of Saccharomyces cerevisiae. J. Biol. Chem. 265, 6726-6733. Hirata, R., Ho, M. N., Umemoto, N., Ohya, Y., Stevens, T. H., and Anraku, Y. (1993). VMA12 is essential for assembly of the vacuolar H'-ATPase subunits onto the vacuolar membrane in Saccharomyces cerevisiae. J. Biol. Chem. 268, 961-967. Hirsch, S., Straws, A., Masood, K., Cee, S., Sukhatme, V., and Gluck, S. (1988). Isolation and sequence of a cDNA clone encoding the 31-kDa subunit of bovine kidney vacuolar Hi-ATPase. Proc. Natl. Acad. Sci. USA. 85, 3004-3008. Ho, M. N., Hill, K. J., Lindorfer, M. A., and Stevens, T. H. (1993a). VMA13 encodes a 54-kDa vacuolar H+-ATPase subunit required for activity but not assembly of the enzyme complex in Saccharomyces cerevisiae. J. Biol. Chem. 268,221-227. Ho, M. N., Hirata, R., Umemoto, N., Ohya, Y., Takatsuki, A., Stevens, T. H., and Anraku, Y. (1993b). VMA13 encodes a 54-kDa vacuolar H+-ATPase subunit required for activity but not assembly of the enzyme complex in Saccharomyces cerevisiae. J. Biol. Chem. 268, 18286-18292. Hortensteiner, S., Vogt, E., Hagenbuch, B., Meier, P. J., Amrheim, N., and Martinoia, E. (1993). Direct energization of bile acid transport into plant vacuoles. J. B i d . Chem. 268, 18446-18449. Kaestner, K. H.. Randall, S. K., and Sze, H.(1988). N,N'-dicyclohexylcrbodiimide-binding proteolipid of the vacuolar Hi-ATPase from oat roots. J. B i d . Chem. 263, 1282-1287. Kagawa, Y., and Racker, E. (1971). Partial resolution of the enzymes catalyzing oxidative phosphorylation: XXV. Reconstitution of vesicles catalyzing 32Pi-adenosine triphosphate exchange. J. Biol. Chem. 246,5477-5487. Kane, P. M., Yamashiro, C. T., and Stevens, T. H.(1989). Biochemical characterization of the yeast vacuolar H+-ATPase. J. Biol. Chem. 264, 19236-19244. Kasai, M., Yamamoto, Y., Maeshima, M., and Matsumoto, H. (1993). Effects of in vivo treatment with abscisic acid andlor cytokinin on activities of vacuolar H' pumps of tonoplast-enriched membrane vesicles prepared from barley roots. Plant Cell Physiol. 34, 1107-1115. Kasamo, K. (1990). Mechanism for the activation of plasma membrane H+-ATPase from rice (Oryza sativa L.) culture cells by molecular species of a phospholipid. Plant Physiol. 93, 1049-1052. Kasamo, K., and Sakakibara, Y. (1995). The plasma membrane H+-ATPase from higher plants: Functional reconstitution into liposomes and its regulation by phospholipids. Plant Sci. 111, 117-131. Kasamo, K., Yamanishi, H., Kagita, F., and Saji, H. (1991). Reconstitution of tonoplast H'ATPase from mung bean (Vigna radiata L.) hypocotyls in liposomes. Plant Cell Physiol. 32, 643-651.

120

KUNlHlRO KASAMO AND HIROYASU YAMANISHI

Kasamo, K., Kagita, F., Yamanishi, H., and Sakaki, T. (1992). Low temperature-induced changes in the thermotropic properties and fatty acid composition of the plasma membrane and tonoplast of cultured rice (Oryza sativa L.) cells. Plant Cell P h y s d 33, 609-616. Keller, F. (1988). A large-scale isolation of vacuoles from protoplasts of mature carrot tap roots. J. Plant Physiol. 132, 199-203. Lai, S., Randall, S. K., and Sze, H. (1988). Peripheral and integral subunits of the tonoplast H+-ATPase from oat roots. J. Biol. Chem. 263,16731-16737. Lai, S., Watson, J. C., Hansen, J. N., and Sze, H. (1991). Molecular cloning and sequencing of cDNAs encoding the proteolipid subunit of the vacuolar H+-ATPasefrom a higher plant. J. Biol. Chem. 266,16078-16084. Li, Z . S., Zhao, Y., and Rea, P. A. (1995). Magnesium adenosine 5’4riphosphate-energized transport of glutathione-s-conjugates by plant vacuolar membrane vesicles. Plant Physiol. 107, 1257-1268. Low,P. S., and Chandra, S. (1994). Endocytosis in plants. Annu. Rev. Plant Physiol. Planf Mol. Biol. 45, 609-631. Mabrey, S., Mateo, P. L., and Sturtevant, J. M. (1978). High-sensitivity scanning calorimetric study of mixtures of cholesterol with dimyristoyl- and dipalmitoyl-phosphatidylcholines. Biochemistry 17, 2464-2468. Macri, F., Vianell, A., Braidot, E., and Zancani, M. (1991). Free fatty acids dissipate proton electrochemical gradients in pea stem microsomes and submitochondrial particle. Biochim. Biophys. Acta 1058,249-255. Macri, F., Zancani, M., Petrussa, E., Dell’Atone, P., and Vianello, A. (1995). Pyrophosphate and Ht-pyrophosphatase maintain the vacuolar proton gradient in metabolic inhibitortreated Acer pseudoplatanus cells. Biochim. Biophys. Acta 1229, 323-328. Maeshima, M. (1990). Development of vacuolar membranes during elongation of cells in mung bean hypocotyls. Plant Cell Physiol. 31, 311-317. Maeshima, M. (1992). Characterization of the major integral protein of vacuolar membrane. Plant Physiol. 98, 1248-1254. Maeshima, M., and Yoshida, S. (1989). Puriification and properties of vacuolar membrane proton-translocating inorganic pyrophosphatase from mung bean. J. Biol. Chem. 264, 20068-20073. Maeshima, M., Nakanishi, Y., Matsuura-Endo, C., and Tanaka, Y. (1996). Proton pumps of the vacuolar membrane in growing cells. J. Plant Res. 109, 119-125. Mandala, S., and Taiz, L. (1985). Partial purification of a tonoplast ATPase from corn coleoptiles. Plant Physiol. 78, 327-333. Mandala, S., and Taiz, L. (1986). Characterization of the subunit structure of the maize tonoplast. Immunological and inhibitor binding studies. J. Biol. Chem. 261, 12850-12855. Mandel, M., Moriyama, Y., Hulmes, J. D., Pan, k’.-C., Nelson, H., and Nelson, N. (1988). cDNA sequence encoding the 16-kDa proteolipid of chromaffin granules implies gene duplication in the evolution of Hi-ATPase. Proc. Nutl. Acad. Sci. USA 85, 5521-5524. Manolson, M. F., Rea, P. A., and Poole, R. J. (1985). Identification of 3-0-(4-benzoxyl)benzoyladenosine 5’triphosphate and N,N’-dicycloheximide-bindingsubunits of a higher plant H+-translocating tonoplast ATPase. J. Biol. Chem. 260, 12273-12279. Manolson, M. F., Quellette, B. F., Filion, M., and Poole, R. J. (1988). cDNA sequence and homologies of the “57-kDa” nucleotide-binding subunit of the vacuolar ATPase from Arabidopsis. J. Biol. Chem. 263, 17987-17994. Manolson, M. F., Profeau, D., Preston, R. A., Stebnit, A., Roberts, B. T., Hoyt, M. A., Preuss, D., Mulholland, J., Botstein, D., and Jones, E. W. (1992). The VPHl gene encodes a 95-kDa integral membrane polypeptide required for in vivo assembly and activity of the yeast vacuolar Hi-ATPase. J. Biol. Chem. 267, 14294-14303. Martinoia, E., Thume, M., Vogt, E., Rentsch, D., and Dietz, K-J. (1991). Transport of arginine and aspartic acid into isolated barley mesophyll vacuoles. Plant Physiol. 97, 644-650.

FUNCTIONAL RECONSTITUTION OF PROTON-ATPASE

121

Martinoia, E., Grill, E., Tommasini, R., Kreuz, K., and Amrheim, N. (1993). ATP-dependent glutathione-S-conjugate ‘exprot’ pump in the vacuolar membrane of plants. Nature 364, 247-249. Martiny-Baron, G., Manolson, M. F., Poole, R. J., Hecker, D., and Scherer, G. F. E. (1992). Proton transport and phosphorylation of tonoplast polypeptides from zucchini are stimulated by the phospholipid platelet-activating factor. Plant Physiol. 99, 1635-1641. Marty, F., Branton, D., and Leigh, R. A. (1980). Plant vacuoles. In “The Biochemistry of Plants: A Comprehensive Treaties, Vol. 1” (N. E. Tobert, Ed.), pp. 625-658. Macmillan, New York. Matile, P. (1978). Biochemistry and function of vacuoles. Annu. Rev. Plant Physiol. 29,193-213. Matsuura-Endo, C., Maeshima, M., and Yoshida, S. (1990). Subunit composition of vacuolar membrane H+-ATPase from mung bean. Eur. J. Biochem. 187,745-751. Matsuura-Endo, C., Maeshima, M., and Yoshida, S. (1992). . . Mechanism of the decline in vacuolar Hi-ATPase activity in mung bean hypocotyls during chilling. Plant Physiol. 100,718-722. Meagher, L., Mclean, P., and Finbow, M. E. (1990). Sequence of a cDNA from Drosophila coding for the 16-kDa proteolipid component of the vacuolar H’-ATPase. Nucleic Acids Res. 18, 6712. Miller, A. J., and Smith, S. J. (1992). The mechanism of nitrate transport across the tonoplast of barley root cells. Planta 187, 554-557. Mimura, T., Dietz, K-J., Kaiser, W., Schramm, M. J., Kaiser, G., and Heber, H. (1990). Phosphate transport across biomembranes and cytosolic phosphate homeostasis in barley leaves. Planta 180, 139-146. Montecucco, C., Smith, G. A., Dabbeni-Sala, F., Johannsson, A., Galante, Y. M., and Bisson, R. (1982). Bilayer thickness and enzymatic activity in the mitochondria1 cytochrome c oxidase and ATPase complex. FEBS Lett. 144, 145-148. Moriyama, Y., and Nelson, N. (1987a). The purified ATPase from chromaffin granule membranes is an anion-dependent proton pump. J. Biol. Chem. 262, 9175-9180. Moriyama, Y., and Nelson, N. (1987b). Nucleotide binding sites and chemical modification of the chromaffin granule proton ATPase. J. Biol. Chem. 262, 14723-14729. Moriyama, Y., and Nelson, N. (1989a). H+-translocating ATPase in golgi apparatus: Characterization as vacuolar H+-ATPase and its subunit structures. J. Biol. Chem. 264,18445-18450. Moriyama, Y., and Nelson, N. (1989b). Cold inactivation of vacuolar proton-ATPases. J. Biol. Chem. 264,3577-3582. Moriyama, Y., Takano, T., and Ohkuma, S. (1984). Proton translocating ATPase in lysosomal membrane ghosts. Evidence that alkaline Mg2+-ATPaseacts as a proton pump. J. Biochem. (Tokyo) 94,995-1007. Morre, D. J., Liedtke, C., Brightman, A. O., and Scherer, F. E. (1991). Head and stalk structures of soybean vacuolar membranes. Planta 184, 343-349. Nakamura, Y.,Kasamo, K., Shimosato, N., Sakata, M., and Ohta, E. (1992). Stimulation of the extrusion of protons and H+-ATPase activities with the decline in pyrophosphatase activity of the tonoplast in intact mung bean roots under high-NaCI stress and its relation to external Ca2+ions.Plant Cell Physiol. 33, 139-149. Narasimhan, M. L., Binzel, M. L., Perez-Prat, E., Chen, Z., Nelson, D. E., Singh, N. K., Bressan, R. A,, and Hasegawa, P. M. (1991). NaCl regulation of tonoplast ATPase 70 kilodalton subunit mRNA in tobacco cells. Plant Physiol. 97, 562-568. Nelson, H., and Nelson, N. (1989). The progenitor of ATP synthesis was closely related to the current vacuolar Hi-ATPase. FEBS Lett. 247, 147-153. Nelson, H., and Nelson, N. (1990). Disruption of genes encoding subunits of yeast vacuolar H+-ATPase causes conditional lethality. Proc. Natl. Acad. Sci. USA 87, 3503-3507. Nelson, H., Mandiyan, S., and Nelson, N. (1989). A conserved gene encoding the 57-kDa subunit of the yeast vacuolar H+-ATPase.J. Biol. Chem. 264, 177551778,

122

KUNlHlRO KASAMO AND HIROYASU YAMANISHI

Nelson, H., Mandiyan, S., Noumi, T., Moriyama, Y.,Miedel, M. C., and Nelson, N. (1990). Molecular cloning of cDNA encoding the C subunit of H+-ATPasefrom bovine chromaffin granules. J. Biol. Chem. 265,20390-20393. Nelson, H., Mandiyan, S., and Nelson, N. (1994). The Sacchuromyces cerevisiae VMA7 gene encodes a 14-kDa subunit of the vacuolar Ht-ATPase catalytic sector. J. Biol. Chem. 269, 24150-24155. Nelson, H., Mandiyan, S., and Nelson, N. (1995). A bovine cDNA and yeast gene (VMA8) encoding the subunit D of the vacuolar H+-ATPase.Proc. Natl. Acud. Sci. USA 92,497-501. Noumi, T., Beltran, C., Nelson, H., and Nelson, N. (1991). Mutational analysis of yeast vacuolar Ht-ATPase. Proc. Nutl. Acad. Sci. USA 188, 1938-1942. Njus, D., Kelley, P. M., and Harnadek, G . J. (1986). Bioenergetics of secretory vesicles. Biochem. Biophys. Acta 853,237-265. Ohya, Y., Umemoto, N., Tanida, I., Ohta, A., Iida, H., and Anraku, Y. (1991). Calciumsensitive CIS mutants of Saccharomyces cerevisiae showing a Pet- phenotype are ascribable to defects of vacuolar membrane H+-ATPase activity. J. Biol. Chem. 266, 13971-13977. Okazaki, Y., Tazawa, M., Moriyama, Y., and Iwasaki, N. (1992). Bafilomycin inhibits vacuolar pH regulation in a fresh water charophyte, Charu corullina. Bot. Acta 105, 421-426. O’Neill, S. D., and Spanswick, R. M. (1984). Characterization of native and reconstituted plasma membrane Ht-ATPase from the plasma membrane of Beta valguries. J. Membr. Biol. 79, 245-256. Orr, W., White, T. C., Iu, B., Robert, L., and Singh, J. (1995). Characterization of a lowtemperature-induced cDNA from winter Brassicu napus encoding the 70-kDa subunit of tonoplast ATPase. Plant Mol. Biol. 28, 943-948. Parry, R. V., Turner, J. C., and Rea, P. A. (1989). High purity preparations of higher plant vacuolar Ht-ATPase reveal additional subunits. Revised subunit composition. J. Biol. Chem. 264,20025-20032. Peng, S.-B., Crider, B. P., Xie, X.-S., and Stone, D. K. (1994). Alternative mRNA splicing generates tissue-specific isoforms of 116-KDa polypeptide of vacuolar proton pump. J. Biol. Chem. 269,17262-17266. Perez-Castineira, J. R., and Apps, D. K. (1990). Vacuolar H+-ATPase of adrenal secretory granules: Rapid partial purification and reconstitution into proteoliposomes. Biochem. J. 271, 127-131. Perlin, M. S., Fried, V. A., Stone, D. K., Xie, X.-S., and Sudhof, T. C. (1991). Structure of the 116-kDa polypeptide of the clathrin-coated vesiclelsynaptic vesicle proton pump. J. Biol. Chem. 266,3877-3881. Pfeiffer, W., and Hager, A. (1993). A Ca2+-ATPaseand a Mg*+/H+-antiporterare present on tonoplast membranes from roots of Zea mays L. Planta 191, 377-385. Preisser, J., and Komor, E. (1991). Sucrose uptake into vacuole of sugarcane suspension cells. Planta 186, 109-114. Preisser, J., Sprugel, H., and Komor, E. (1992). Solute distribution between vacuole and cytosol of sugarcane suspension cells: Sucrose is not accumulated in the vacuole. Planta 186,203-211. Pressman, B. C. (1976). Biological applications of ionophores. Annu. Rev. Biochem. 45, 501-530. Puopolo, K., Kumamoto, C., Adachi, I., and Forgac, M. (1991). A single gene encodes the catalytic “A” subunit of the Bovine vacuolar H+-ATPase.J. Biol. Chem. 266,24564-24572. Puopolo, K., Kumamoto, C., Adachi, I., Magner, R., and Forgac, M. (1992a). Differential expression of the “B” subunit of the vacuolar H+-ATPasein Bovine tissues. J. Biol. Chem. 267,3696-3706. Puopolo, K., Sczekan, M., Magner, R., and Forgac, M. (1992b). The 40-kDa subunit enhances but is not required for activity of the coated vesicle proton pump. J. Biol. Chem. 267,51715176.

FUNCTIONAL RECONSTITUTION OF PROTON-ATPASE

123

Quinn, P. J., and Williams, W. P. (1990).Structure and dynamics of plant membranes. I n “Method in Plant Biochemistry,” Vol. 4, pp. 297-340. Academic Press, New York. Randall, S., and Sze, H. (1987).Probing the catalytic subunit of the tonoplast H+-ATPase from oat roots. Binding of 7-chloro-4-nitrobenzo-2-oxa-1,3,-diazole to the 72 kilodalton polypeptide. J. Biol. Chem. 262, 7135-7141. Randall, S.K., and Sze, H. (1986).Properties of the partially purified tonoplast H+-pumping ATPase from oat roots. J. Biol. Chem. 261, 1364-1371. Rausch, T., Butcher, D. N., and Taiz, L. (1987).Active glucose transport and proton pumping in tonoplast membrane of Zea mays L. coleoptiles are inhibited by anti-Ht-ATPase antibodies. Plant Physiol. 85,996-999. Rautiala, T. J., Koskinen, A. P., and Vaananen, H. K. (1993).Purification of vacuolar ATPase with bafilomycin C1 affinity chromatography. Biochem. Biophys. Res. Commun. 194,50-56. Rea, P.A., and Poole, R. J. (1993). Vacuolar Ht-translocating pyrophosphatase. Annu. Rev. Plant Physiol. Plant Mol. Biol. 44, 157-180. Rea, P. A., and Sanders, D.(1987).Tonoplast energization: Two H’ pumps, one membrane. Physiol. Plant. 71, 131-141. Rea, P. A., Griffith, C. J., Manolson, M. F., and Sanders, D.(1987a).Irreversible inhibition of H+-ATPase of higher plant tonoplast by chaotropic anions: Evidence for peripheral location of nucleotide-binding subunits. Biochim. Biophys. Acta 904, 1-12. Rea, P. A., Griffith, C. J., and Sanders, D. (1987b). Purification of the N,N’-dicyclohexiimide binding proteolipid of a higher plant tonoplast H+-ATPase.J. Biol. Chem. 262,14745-14752. Rentsch, D.,and Martinoia, E. (1991). Citrate transport into barley mesophyll vacuoleComparison with malate-uptake activity. Planta 184, 532-537. Reuveni, M., Bennett, A. B., Bressan, R. A., and Hasegawa, P. M. (1990). Enhanced H + transport capacity and ATP hydrolysis activity of the tonoplast H+-ATPase after NaCl adaptation. Plant Physiol. 94, 524-530. Rigaud, J-L., Pitard, B., and Levy, D. (1995). Reconstitution of membrane proteins into liposomes: Application to energy-transducing membrane proteins. Biochim. Biophys. Acta

1231, 223-246.

Rothman, J. H., Yamashiro, C. T., Raymond, C. K., Kane, P. M., and Stevens, T. H. (1989). Acidification of the lysosome-like vacuole and the vacuolar H+-ATPase are deficient in two yeast mutants that fail to sort vacuolar proteins. J. Cell Biol. 109,93-100. Salt, D.E.,and Rauser, W.E. (1995). MgATP-dependent transport of phytochelatins across the tonoplast of oat roots. Plant Physiol. 107,1293-1301. Salt, D. E., and Wagner, G. J. (1993). Cadmium transport across tonoplast of vesicles from oat roots: Evidence for a Cd2+/H+antiport activity. J. Biol. Chem. 268, 12297-12302. Scherer, G. F. E., Martiny-Baron, G., and Stoffel, B. (1988).A new set of regulatory molecules in plants: A plant phospholipid similar to platelet-activating factor stimulates protein kinase and proton-translocating ATF’ase in membrane vesicles. Planta 175, 241-253. Schumaker, K. S.,and Sze, H. (1990).Solubilization and reconstitution of the oat root vacuolar Ht/Ca2+ exchanger. Plant Physiol. 92,340-345. Shih, C.-K., Wagner, R., Feinstein, S., Kanik-Ennulat, C., and Neff, N. (1988). A dominant tritluoperazine resistance gene from Saccharomyces cerevisiae has homology with FoFlATP synthase and confers calcium-sensitive growth. Mol. Cell. Biol. 8, 3094-3103. SUdhof, T.C., Fried, V. A., Stone, D. K., Tohnston P. A., and Xie, X.-S. (1989). Human endomembrane Ht pump strongly resembles the ATP-synthetase of Archaebacteria. Proc. Natl. Acad. Sci. USA 86, 6067-6071. Sun, S-Z., Xie, X-S., and Stone, D.K. (1987).Isolation and reconstitution of the dicyclohexylcarbodiimide-sensitive proton pore of the clathrin-coated vesicle proton translocating complex. J. Biol. Chem. 262, 14790-14794. Suzuki, K., and Kasamo, K. (1993).Effects of aging on the ATP- and pyrophosphate-dependent pumping of protons across the tonoplast isolated from pumpkin cotyledons. Plant Cell Physiol. 34,613-619.

124

KUNlHlRO KASAMO AND HIROYASU YAMANISHI

Swarts, H. G. P., Schuurmans-Stekhoven, F. M. A. H., and De Pont, J. J. H. H. M. (1990). Binding of unsaturated fatty acids to Na+,K+-ATPaseleading to inhibition and inactivation. Biochim. Biophys. Acta 1024, 32-40. Sze, H. (1985). Ht-translocating ATPases: Advances using membrane vesicles. Annu. Rev. Plant Physiol. 36, 175-208. Sze, H., Ward, J. M., and Lai, S . (1992a). Vacuolar H+-translocating ATPase from plants: Structure, function and isoforms. J. Bioenerg. Biomembr. 24, 371-381. Sze, H., Ward, J. M., Lai, S.,and Pereda, I. (1992b).Vacuolar-type H+-translocatingATPasesin plant endomembranes: Subunit organization and multigene fami1y.J. Exp. Biol. 172,123-135. Taiz, L. (1992). The plant vacuole. J. Exp. Biol. 172, 113-122. Takase, K., Kakinuma, S., Yamato, I., Konishi, K., Igarashi, K., and Kakinuma, Y. (1994). Sequencing and characterization of the ntp gene cluster for vacuolar-type Na+-translocating ATPase of Enterococcus hirae. J. Biol. Chem. 269, 11037-11044. Tognoli, L. (1985). Partial purification and characterization of an anion-activated ATPase from radish microsomes. Eur. J. Biochem. 146,581-588. Uchida, E., Ohsumi, Y., and Anraku, Y. (1985). Purification and properties of H+-translocating, Mg2+-adenosinetriphosphatasefrom vacuolar membranes of Saccharomyces cerevisiae. J. Biol. Chem. 260, 1090-1095. Umemoto, N., Yshihisa, T., Hirata, R., and Anraku, Y. (1990). Roles of the VMA3 gene product, subunits c of the vacuolar membrane ATPase on vacuolar acidification and protein transport. J. Biol. Chem. 265, 18447-18453. Umemoto, N., Ohya, Y., and Anraku, Y. (1991). VMA11, a novel gene that encodes a putative proteolipid, is indispensable for expression of yeast vacuolar membrane H+-ATPaseactivity. J. Biol. Chem. 266, 24526-24532. Villalobo, A. (1990). Reconstitution of ion-motive transport ATPases in artificial lipid membrane. Biochim. Biophys. Acta 1017, 1-48. Wan, C.-Y., and Wilkins, T. A. (1994). Isolation of multiple cDNAs encoding the vacuolar H+-ATPasesubunit B from developing cotton (Gossypium hirsutum L.) ovules. Plant Physiol. 106, 393-394. Wang, Y., and Sze, H. (1985). Similarities and differences between the tonoplast type and mitochondria1 H+-ATPase of oat roots. J. Biol. Chem. 260, 10434-10443. Ward, J. M., and Sze, H. (1992a). Subunit composition and organization of the vacuolar H+ATPase from oat roots. Plant Physiol. 99, 170-179. Ward, J. M., and Sze, H. (1992b). Proton transport activity of the purified vacuolar Ht-ATPase from oats. Plant Physiol. 99, 925-931. Ward, J. M., Reinders, A., Hsu, H-T., and Sze, H. (1992). Dissociation and reassembly of the vacuolar Ht-ATPase complex from oat roots. Plant Physiol. 99, 161-169. Warren, G. B., Houslay, M. D., Metcalfe, J. C., and Birdsall, N. J. M. (1975). Cholesterol is excluded from the phospholipid annulus surrounding an active calcium transport protein. Nature 255,684-687. Warren, M., Smith, J. A., and App, D. K. (1992). Rapid purification and reconstitution of plant vacuolar ATPase using Triton X-114 fractionation: Subunit composition and substrate kinetics of the H'-ATPase from the tonoplast of Klanchoe daigremontiana. Biochim. Biophys. Acta 1106, 117-125. Werner, G., Hagenmaier, H., Drautz, H., Baumgartner, A., and Zahner, H. (1984). Metabolic products of microorganisms. 224. Bafilomycins, a new group of macrolide antibiotics. Production, isolation, chemical structure and biological activity. J. Antibiotics 37, 110-117. Wilkins, T. A. (1993). Vacuolar H+-ATPase69-kilodalton catalytic subunit cDNA from developing cotton (Grossypium hirsutum) ovules. Plant Physiol. 102, 679-680. Wink, M. (1993). The plant vacuole: A multifunctional compartment. J. Exp. Bot. 44,231-246. Xie, X.-S., and Stone, D. K. (1986). Isolation and reconstitution of the clathrin-coated vesicle proton translocating complex. J. Biol. Chem. 261, 2492-2495.

FUNCTIONAL RECONSTITUTION OF PROTON-ATPASE

125

Xie, X.-S., and Stone, D. K. (1988). Partial resolution and reconstitution of the subunits of the clathrin-coated vesicle proton ATPase responsible for Ca2+-activated ATP hydrolysis. J. Biol. Chem. 263, 9859-9867. Xie, X.-S., Tsai, S.-J., and Stone, D. K. (1986). Lipid requirements for reconstitution of the proton-translocating complex of clathrin-coated vesicles. Proc. Natl. Acad. Sci. USA 83,8913-8917. Yamanishi, H., and Kasamo, K. (1992a). Binding of 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole to an essential cysteine residue(s) in the tonoplast Ht-ATPase from mung bean (Vigna radiata L.) hypocotyls. Plant Physiol. 99, 652-658. Yamanishi, H., and Kasamo, K. (1992b). Reconstitution of purified tonoplast H+-ATPase from mung bean (Vigna radiata L.) hyplcotyls in liposomes. In “Plant Cell Wall as Biopolymers with Physical Functions” (Y. Masuda, Ed.), pp. 350-352, Keihanna, Osaka, Japan. Yamanishi, H., and Kasamo, K. (1993). Modulation of the activity of purified tonoplast H+ATPase from mung bean (Vigna radiata L.) hypocotyls by various lipids. Plant Cell Physiol. 34,411-419. Yamanish, H., and Kasamo, K. (1994). Effect of cerebrocide and cholesterol on the reconstitution of tonoplast Ht-ATPase purified from mung bean (Vignu radiata L.) hypocotyls in liposomes. Plant Cell Physiol. 35, 655-663. Yamashiro, C. T., Kano, P. M., Wolczyk, D. F., Preston, R. A., and Stevens, T. H. (1990). Role of vacuolar acidification in protein sorting and zymogen activation: A genetic analysis of the yeast vacuolar proton translocating ATPase. Mol. Cell. Biol. 10,3737-3749. Yaver, D. S., Nelson, H., Nelson, N., and Klinosky, D. J. (1993). Vacuolar ATPase mutants accumulate precursor proteins in a pre-vacuolar compartment. J. Biol. Chem. 268, 1056410572. Yeagle, P. L. (1985). Cholesterol and the cell membrane. Biochim. Biophys. Acta 822,267-287. Yoshida, S . (1994). Low tempertature-induced cytoplasmic acidosis in cultured mung bean (Vigna radiata [L.] Wilczek) cells. Plant Physiol. 104, 1131-1138. Yoshida, S., Kawata, T., Uemura, M., and Niki, T. (1986). Isolation and characterization of tonoplast from chilling-sensitive etiolated seedling of Vigna radiata L. Plant Physiol. 80, 161-166. Young, G. P.-H., Qiao, J. Z., and Awqati, A. L. Q. (1988). Purification and reconstitution of the proton-translocating ATPase of golgi enriched membranes. Proc. Natl. Acad. Sci. USA 85, 9590-9594. Zeiger, E. (1983). The biology fo stomata guard cells. Annu. Rev. Plant Physiol. 34,441-475. Zhang, J., Myers, M., and Forgac, M. (1992). Characterization of the Vo domain of the coated vesicles (H+)-ATPase.J. Biol. Chern. 267, 9773-9778. Zhang, J., Feng, Y., and Forgac, M. (1994). Proton conduction and bafilomycin binding by the Vo domain of the coated vesicle V-ATPase. J. Biol. Chem. 269,23518-23523. Zimniak, L., Dittrich, P., Gogarten, J. P., Kibak, H., and Taiz, L. (1988). The cDNA sequence of the 69-kDa subunit of the carrot vacuolar Ht-ATPase. Homology to the @chain of FoF1ATPase. J. Biol. Chem. 263, 9102-9112.