Vacuolar-Type H+-ATPases Are Associated with the ... · Vacuolar-Type H+-ATPases Are Associated with the Endoplasmic Reticulum and Provacuoles of Root Tip Cells’ Eliot M. Herman,

Plant Physiol. (1994) 106: 1313-1324

Vacuolar-Type H+-ATPases Are Associated with the Endoplasmic Reticulum and Provacuoles of Root Tip Cells’

Eliot M. Herman, Xuhang Li, Robert T. Su, Paul Larsen, H e i 4 Hsu, and Heven Sze*

Department of Botany, University of Maryland, College Park, Maryland 20742 (X.L., P.L., H.S.); Plant Molecular Biology Laboratory (E.M.H.) and Florist and Nursery Crops Laboratory (H.-t.H.), United States Department of

Agriculture, Agricultural Research Service, Beltsville, Maryland 20705; and Division of Research Grants, National Institutes of Health, Bethesda, Maryland 20892 (R.T.S.)

To understand the origin of vacuolar H+-ATPases (V-ATPases) and their cellular functions, the subcellular location of V-H+- ATPases was examined immunologically in root cells of oat seedlings. A V-ATPase complex from oat roots consists of a large peripheral sector (V,) that includes the 70-kD (A) catalytic and the 60-kD (6) regulatory subunits. The soluble V1 complex, thought to be synthesized in the cytoplasm, i s assembled with the membrane integral sector (V.) at a yet undefined location. In mature cells, V- ATPase subunits A and B, detected in immunoblots with mqno- clonal antibodies (Mab) (7A5 and 2E7), were associated mainly with vacuolar membranes (20-22% sucrose) fractionated with an isopycnic sucrose gradient. However, in immature root tip cells, which lack large vacuoles, most of the V-ATPase was localized with the endoplasmic reticulum (ER) at 28 to 31% sucrose where a major ER-resident binding protein equilibrated. The peripheral subunits were also associated with membranes at 22% sucrose, at 31 to 34% sucrose (Golgi), and in plasma membranes at 38% sucrose. lmmunogold labeling of root tip cells with Mab 2E7 against subunit B showed gold particles decorating the ER as well as numerous small vesicles (0.1-0.3 pm diameter), presumably provacuoles. The immunological detection of the peripheral subunit B on the ER supports a model in which the V1 sector is assembled with the V,, on the ER. These results support the model in which the central vacuolar membrane originates ultimately from the ER. The presence of V-ATPases on several endomembranes indicates that this pump could participate in diverse functional roles.

In plants, acidification of the vacuolar compartment by the V-ATPase is essential to or involved in many diverse functions (Sze et al., 1992a). Depending on the tissue, the stage of development, and the signals received, these functions include osmoregulation, transport and storage of ions and metabolites, signal transduction, storage and turnover of proteins, and storage of secondary metabolites, defense proteins, and pigments (Boller and Wiemken, 1986; Martinoia, 1992). Vacuoles are dynamic, prominent organelles. Undif- ferentiated and immature plant cells often possess propor- tionately more cytoplasm that contains an extensive endo-

This work was supported in part by National Science Foundation grant DCB-90-06402 and by Maryland Agricultural Experiment Sta- tion (MAES) Hatch Project MD-J-151 to H.S. and by FY93 MAES competitive grant to E.M.H. and H.S. (contribution No. 8797, article NO. A-6585).

* Corresponding author; fax 1-301-314-9082.

membrane system and numerous small provacuoles. As cells mature, these small provacuoles merge to form a central vacuole that can occupy up to 80 to 90% of the intracellular space in differentiated cells.

V-ATPase from plants is a member of the V-type ATPases widely distributed among many eukaryotes (Nelson, 1992), including animal (Forgac, 1992; Gluck, 1992), fungal (Anraku et al., 1992; Bowman et al., 1992; Kane and Stevens, 1992), and plant cells (Sze et al., 1992a). The characteristic feature of this type of H+ pump is its sensitivity to nanomolar levels of bafilomycin, but not to vanadate or azide, which are inhibitors of the P- or F-type ATPases, respectively. In plants, the anion-sensitive V-ATPase is directly stimulated by 10 mM chloride and inhibited by 10 to 50 mM nitrate (Sze, 1985). The purified enzyme complex from several plants contains 7 to 10 different subunits. A peripheral sector, or Vl, is com- posed of 5 to 6 different subunits that are solubilized from the membrane by KI (Parry et al., 1989; Ward and Sze, 1992a). The VI complex includes three copies each of the nucleotide-binding catalytic (approximately 70 kD) and the regulatory (approximately 60 kD) subunits, also known as subunits A and B, respectively. The membrane integral sector, or V,, which forms the proton-conducting pathway, is made up of six copies of the 16-kD proteolipid together with 1 to 3 other subunits (Sze et al., 1992a).

In addition to elucidating the complex structure of V- ATPase, biochemical and molecular studies have provided evidence that plants contain a family of V-type ATPases. The first clue came from the diversity and variations in the subunit composition of the purified enzyme. For example, V-ATPase purified from red beet and barley contained a prominent 100- kD integral subunit (Parry et al., 1989; DuPont and Morrisey, 1992); however, this subunit was absent from a purified and

Abbreviations: BiP, a major ER-resident binding protein; FBS, fetal bovine serum; GERL, Golgi-ER-lysosome system; Mab, monoclonal antibody(ies); PM, plasma membrane; subunit A, the approximately 70-kD catalytic subunit of the V-ATPase; subunit 8, the approximately 60-kD regulatory subunit of the V-ATPase; TBST, Tris- buffered saline with Tween; TPCK, N-tosyl-L-phenylalanine chlo- romethyl ketone; V-ATPase, vacuolar H+-pumping adenosine triphosphatase; VI, the peripheral complex of the V-ATPase, which consists of subunits A and B plus three or four other subunits; VM23 or TIP, major tonoplast intrinsic protein of approximately 23 to 25 kD; V,, the membrane integral complex of the V-ATPase.

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1314 Herman et al. ' Plant Physiol. Vol. 106, 1994

transport-competent ATPase from oat (Avena sativa L.) roots (Ward and Sze, 1992b). Molecular studies clearly show that multiple genes encode the major subunits: subunit A (Starke et al., 1991), subunit B (Berkelman et al., 1994), and the 16- kD proteolipid (Lai et al., 1991). These results support the idea that there are several isoforms or subtypes of V-ATPases; however, it is not clear how the subtypes differ from one another in their cellular function and regulation. One working model is that the subtypes are spatially, temporally, or developmentally regulated, and that there may be organelle- specific isoforms (Lai et al., 1991; Gogarten et al., 1992).

Very little is known about the subcellular distribution of the V-ATPase from plants. Vacuoles purified from Kalanchoe leaves (Smith et al., 1984) or red beet roots (Bennett et al., 1984) possess an anion-sensitive ATPase activity. This find- ing led to the conclusion that the anion-sensitive ATPase was associated with the vacuolar membrane (Bennett et al., 1984; Mandala and Taiz, 1985). Consequently, this pump is often referred to as a vacuolar membrane marker. However, fractionation of microsomes showed that the V-ATPase is broadly distributed in linear Suc or dextran gradients. Several laboratories have suggested that the pump is associated with the ER and Golgi, based on co-migration of V-ATPase activity with ER (Churchill et al., 1983; Hager and Biber, 1984) and Golgi marker enzymes (Chanson and Taiz, 1985; Ali and Akazawa, 1986). Furthermore, immunoblotting with V-ATP- ase antibodies suggested that this enzyme is also found on purified clathrin-coated vesicles (Depta et al., 1991). These results would indicate that the V-ATPases have a broader distribution and physiological role than was previously thought. The most direct approach to verify the subcellular distribution of membrane proteins is by immunogold EM. In one study using polyclonal antibodies to subunit A, the V- ATPase was located on the Golgi, the vacuole, and, surpris- ingly, the PM of corn coleoptiles (Hurley and Taiz, 1989).

Although V-ATPases may be located on several endomembranes in plants, as in animals (Marquez-Sterling et al., 1991), it is not clear where this complex is synthesized and assembled. The current model based on work with yeast mutants defective in one V-ATPase subunit would suggest that the V, and VI complex are independently synthesized (Kane, 1992; Kane and Stevens, 1992). Like other multisubunit membrane complexes (Hurtley and Helenius, 1989), the V, complex is thought to be synthesized and assembled in the ER. However, evidence indicates that the VI subunits are independently synthesized and assembled in the cytosol. The VI is then assembled with the V, sector at a yet undefined location, resulting in a functional H+-ATPase in the vacuolar membrane. For example, yeast mutants in which the gene encoding the 16-kD proteolipid was disrupted were still capable of synthesizing the subunits of the VI; however, the VI remained in the cytosol and did not associate with the membrane (Kane and Stevens, 1992). Alternatively, if a gene encoding subunit A or B of the VI complex was disrupted, the major V, subunits (95 and 16 kD) were synthesized and associated to the membrane. Thus, an intact V, complex is required before V, can be attached to the membrane.

To understand better the cellular roles of V-ATPases and as first step in the study of the biogenesis of this proton pump, we have examined the subcellular distribution of

vacuolar ATPases in the roots of oat seedlings. Monocot roots serve as an ideal system in which to study the dev4opment and formation of vacuoles. Undifferentiated and meristematic cells are located at the root tip, and differentiated cells, like the epidermis, cortex, and procambium tissues, appear a few millimeters behind the tip. Furthermore, there is substantial background information about oat root, which has heen used extensively for studying PM and V-ATPases after membrane fractionation (Hodges and Leonard, 1974). Usin,; a Mab against subunit B for immunological assays, we show that the peripheral subunit B is located on the ER and ER fractions, as well as on Golgi-derived vesicles and provacuoles. The results support a model in which the V-ATPase is sy ithesized and assembled at the ER and in which the vacuolar membrane originates from the ER. The presence of V-ATPases on several endomembranes suggests that these H+ punips could play diverse roles in the growth and developmení of plant cells.

MATERIALS AND METHODS

Plant Material

Oat (Avena sativa L. var Ogle) seeds were germinated in the dark over an aerated solution of 0.5 mM Caso4. Roots or root tips were harvested after 4 d of growth.

Fractionation of Subcellular Membranes

Membrane vesicles were prepared using differential and linear density gradient centrifugation according to the pro- cedures of Ward and Sze (1992a) and Hager et al. (1991) with some modifications. Mature root sections (approximately 0.5 g fresh weight) or root tips (approximaíely 0.1 g fresh weight) were homogenized with a mortar and pestle in 50 mM Hepes-bis-tris propane, pH 7.4, 250 mM sorbitol, 6 mM EGTA, 1 mM DTT, 0.1 mM PMSF, and 0.05 mM TPCK at a medium-to-fresh tissue ratio of 6 or 20 mL/g fcr mature roots or root tips, respectively. Mature roots refer to approximately 4-cm sections excised 1 cm from the tip, and root tips were 1- to 2-mm tip sections. The homogenate was then strained through four layers of cheesecloth. The debris and mitochondria were removed by centrifugation at 13,OOOg for 15 min. The supematant was layered onto a 17 to 45% continuous Suc gradient (12 mL) over a I-mL 45% Suc cushion. The SUC gradient solution contained 25 m14 Hepes- bis-tris propane, 2 mM EGTA, 1 mM DTT, 0.1 mM PMSF, and 0.05 mM TPCK, pH 7.4. After centrifugation at 110,OOOg for 16 h (Beckman SW28, at maximum radius), 0.7-mL fractions were collected and diluted to less than 10% Suc in gradient solution. Proteins in each fraction were directly prwipitated with 20% TCA (final concentration) and washed with cold acetone. Protein concentration was determined using Bio- Rad protein assay solution.

SDS-PACE, Ag Stain, and lmmunoblot

Protein in each fraction was precipitated with 213% TCA (final concentration), washed with 100% acetone, and then solubilized in 60 pL of sample buffer containing 62.5 mM

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Vacuolar H+-ATPase Associated with the ER and Provacuoles 1315

Tris-HC1, pH 6.8, 10% (v/v) glycerol, 8 M urea, 5% 2- mercaptoethanol, and 0.002% (w/v) bromphenol blue. An equal volume fraction (typically one-fifth of 60 FL containing 2-4 r g of protein) was loaded on each lane. The proteins were separated on an 11% acrylamide gel, 15 X 20 cm, at 7 mA per gel ovemight at 15OC. One gel was silver stained for proteins.

After electrophoresis, gels were soaked in 25 II~M Tris, pH 8.3, 192 r m Gly, and 20% methanol for at least 10 min. Proteins were blotted onto Immobilon-P (Millipore, Bedford, MA) at constant voltage of 100 V for 2 h at 4OC using a Bio- Rad blotting apparatus. The Immobilon-P was incubated in TPBS (PBS with 0.1% Tween-20) containing 5% dry milk and 1% protease-free BSA (Sigma) for 1 h, and washed three times with TPBS for 5 min. The membrane was then incubated with Mab or polyclonal antibodies diluted with TPBS containing 1% BSA for 1 h and washed as above. The membrane was probed with either goat anti-mouse IgG (Sigma) or goat anti-rabbit IgG (Calbiochem, San Diego, CA) conjugated to alkaline phosphatase, and color was developed with 5-bromo-4-chloro-3-indoyl phosphate and nitroblue tet- razolium (Sigma). Antibodies used in these experiments include Mab 2E7 and 7A5 against the 60- and 70-kD subunits of the V-ATPase (Ward et al., 1992), and polyclonal antibodies anti-holo-V-ATPase (Ward and Sze, 1992a), anti-BiP (M. Chrispeels, San Diego, CA), anti-plasma membrane H+-ATP- ase (R.T. Leonard, Riverside, CA), and anti-VM23 (M. Mae- shima, Sapporo, Japan).

Mab and Ascites Fluid

Mab against the two major peripheral subunits of the oat V-ATPase were generated initially in mice as described by Ward et al. (1992). Hybridoma supematants of Mab 2E7 and 7A5 (both IgGJ at dilutions of 1:lOO to 1:200 were used in westem blots to detect subunits B and A, respectively. We have used Mab 7A5, instead of Mab 7D2, to detect subunit A because 7A5 reacts with all plants tested, whereas 7D2 is oat specific (Ward, 1991).

Because immunolocalization studies using the hybridoma supematants showed very weak labeling, we generated ascites fluid. The hybridoma cell lines 2E7 and 7A5 were grown in RPMI medium containing 20% fetal calf serum. Cells (lo6) were then washed with serum-free RPMI and introduced peritoneally into pristine-primed BALB/c mice (Hsu and Law- son, 1985). Two to 4 weeks after transplantation of the hybridoma cells, ascites fluid was collected and further purified with a protein G-Sepharose column (Harlow and Lane, 1988). The reactivity of each fraction with membrane-bound ATPase was tested by westem blotting. Fractions containing an antibody titer of 1:10,000 were pooled and stored at -2OOC in the presence of 0.1% Na azide. The labeling pattern of 7A5 was less specific, so 2E7 alone was used for immunolocalization studies.

Immunofluorescent Staining of V-ATPase

Immunofluorescent labeling of oat roots was performed using the method described by Baskin et al. (1992). Root tips, 2 to 3 mm in length, were cut from 4-d-old seedlings and

transferred to Eppendorf tubes containing 4% paraformal- dehyde in 15 m Hepes buffer, pH 7.2, with 15 II~M KC1. After fixation for 1 h, roots were rinsed thoroughly with 15 m KCl in 15 m Hepes, pH 7.2. After dehydration stepwise in ethanol, roots were embedded in methacrylate (Ladd Research Industries, Inc., Burlington, VT) (Baskin et al., 1992).

Sections approximately 2 pm thick were placed on polyly- sine (Sigma) coated slides in preparation for immunofluorescent labeling. All slides were camed through the following staining regimen. Slides with tissue sections were placed in acetone for 15 min to remove the embedding plastic. After rehydrating with PBS (pH 7.2) for 15 min, the slides were put in two changes of 0.1% Na borohydride in PBS for 10 to 15 min to remove residual fixative and then rinsed with PBS. Sections were blocked with 1% BSA in PBS for 10 to 15 min and then blotted to remove the BSA. Sections were then incubated with purified ascites fluid of 2E7 (1:l ddution) (30 pL/section) ovemight at 4OC in a humid chamber. To test for autofluorescence and unspecific staining of secondary antibody, control sections were treated with BSA/PBS. After three rinses with PBS, all the slides were blotted and then incubated with 30 pL of secondary antibody, sheep anti- mouse IgG conjugated with Texas Red (1:lO dilution in 1% BSA/PBS) (Jackson Immunoresearch Laboratories, Inc., West Grove, PA). All sections except the autofluorescence control were rinsed three times in PBS and stained for 30 min with 30 pL of Hoechst 33258 (5 pg/mL in PBS, Molecular Probes, Eugene, OR), which binds DNA. After rinsing with PBS, all the sections were mounted in Mowiol 4-88 (Calbiochem) with a coverglass and sealed with nail polish.

Sections were viewed with a Zeiss IM 35 microscope adapted for incident light fluorescent microscopy using a Zeiss l O O X achromat (numerical aperture = 1.3) objective lens. Sections were illuminated for fluorescence with a 75-W xenon arc lamp. An IR interference filter was placed in the optical path to narrow the excitation bandwidth. Irradiation with UV/violet light (approximately 365 nm) to excite the Hoechst dye and at 530 to 585 nm to excite the Texas Red was produced from standard filter packages (Carl Zeiss, New York, NY). Images were photographed with an Olympus OM-2N camera using T-Max 3200 film and the film was developed with T-Max developer (Eastman Kodak, Roches- ter, NY).

lmmunolocalization with the Electron Microscope

Immunocytochemical analysis was performed as described by Herman and Melroy (1990). Specifically, oat root tips (apical 2 mm) of 4-d-old oat seedlings were fixed in 4% formaldehyde, 2% glutaraldehyde, 0.1 M phosphate buffer (pH 7.4) at 7OC. Parallel aldehyde-fixed roots were postfixed in 1% OsO, for 2 h at room temperature to provide samples for ultrastructural analysis. The cells and tissues were dehy- drated in a graded ethanol series and embedded in hard- grade L.R. White resin. Ultrathin sections mounted on nickel grids were blocked with 10% FBS in TBST (10 m Tris-C1, pH 7.4, 0.15 M NaC1, 0.05% Tween-20). The grids were incubated in either purified IgG 2E7 (1:5 dilution) or control monoclonal IgG diluted in FBS/TBST. The 2E7 Mab was purified from the ascites fluid by affinity chromatography on



1316 Herman et al. Plant Physiol. Vol. 106, 1994

protein G-Sepharose (Harlow and Lane, 1988). The controlMab IgG consisted of an antibody directed at a thiol proteaseexpressed in maturing soybean seeds that was also purifiedby protein G-Sepharose chromatography (Kalinski et al.,1992). The grids were then washed in TEST and indirectlylabeled with anti-mouse IgG coupled to 10 nM colloidal gold(Ted Pella, Inc., Redding, CA) diluted 1:2 in FBS/TBST for10 min at room temperature. The grids were washed in TESTand distilled water and then stained in 5% uranyl acetate for30 min. For conventional EM, tissues were fixed in osmium,sectioned, and stained with 5% uranyl acetate for 30 min andlead citrate (33 mg/mL) for 10 min. The grids were examinedand photographed with Hitachi H300 and H500 electronmicroscopes.

RESULTS

Distribution of V-ATPase in Membranes Separated on aLinear Sue Gradient

To determine the subcellular distribution of V-ATPases,membranes from 4-d-old seedling roots were separated witha linear isopycnic Sue gradient, and Mab were used to detectV-ATPase subunits in western blots. Previously, we haveshown that Mab 2E7 and 7A5 specifically react with theperipheral subunits of approximately 60 and 70 kD, in eithernative membranes or in the purified enzyme from oat roots(Ward et al., 1992). To minimize aggregation of membranevesicles to one another, approximately 0.1 or 0.5 g fresh

weight of roots was homogenized and the post-mitochondrialsupernatant was directly separated with a Sue gradient (12mL) containing 2 HIM EGTA (Hager and Biber, 1984). Thepolypeptide profile indicated that the subcellular membraneshad been separated during gradient fractionation (see below).

In mature roots (without tip) that contain cells with fullydifferentiated vacuoles, subunits A and B of the V-ATPasewere associated with several endomembranes at Sue concen-trations ranging from 20 to 32% (Fig. IB). The highest levelswere detected in membranes at 20 to 22% Sue, where maturevacuolar membranes equilibrate (e.g. Bennett et al., 1984).Significant levels of the peripheral subunits were also de-tected at 28 to 31% Sue, where ER membranes equilibrated.Using anti-BiP, we show that BiP, a 78-kD ER lumen chap-erone, was predominantly associated with membranes of 28to 31% Sue (Fig. 1A). Interestingly, we have frequentlyobserved low levels of V-ATPase subunits at 38 to 41% Sue,where PMs equilibrate, as detected by the PM H+-ATPase of100 kD (Fig. 1A). In general, the V-ATPase distributed inparallel with the H+-PPase (not shown) and VM23 (Fig. 1C).VM23, or TIP, is a major integral protein of approximately23 kD (Johnson et al., 1989; Maeshima, 1992) that mediateswater transport across vacuolar membranes (Maurel et al.,1993). Two immunoreactive polypeprides (Fig. 1C) may beindicative of two isoforms of VM23 (Maeshima, 1992) asobserved for TIP in legume seeds (Johnson et al., 1989).

As a first step in determining the origin of V-ATPases, weexamined the distribution of the V-ATPase in immature,

MATURE ROOTS70 44 38 35 33 31 29 27 22 17 11

SUCROSE 45 41 36 34 32 30 28 25 20 14 10

PM-ATPase —

BiP —

70 kD —60 kD — B

VM23 —

10 12 14FRACTION NO.

16 18 20

Figure 1. Peripheral subunits A and B of V-ATPases isolated from mature oat roots are located on vacuolar membranesand other endomembranes. The post-mitochondrial supernatant from approximately 0.5 g fresh weight of mature rootsections was separated with a 17 to 45% Sue gradient (12 ml). After centrifugation and fractionation (0.7 ml each), eachfraction was diluted to 10% Sue and the proteins were precipitated with TCA and washed in acetone. Equal-volumefractions (one-fifth), or approximately 2 to 6 jig of protein, were analyzed by SDS-PACE (11% acrylamide). Results arefrom one experiment representative of two. A, Markers of the PM and ER equilibrate at 38 to 41% and 28 to 30% Sue,respectively. Gels were blotted to Immobilon P. Western blots were probed with a mixture of polyclonal anti-PM-ATPase(1:2000 dilution) and anti-BiP (1:30). B, Subunit A (70 kD) and subunit B (60 kD) of V-ATPase are associated mainly withvacuolar membranes (20-22%) and ER (28-31%). Western blots were probed with a mixture of Mab 2E7 (1:100 dilution)to B and Mab 7A5 (1:50) to A. C, VM 23 co-migrates with V-ATPase and is associated with the vacuolar membrane andER. Western blot was probed with polyclonal anti-VM23 (1:3000 dilution). www.plantphysiol.orgon February 18, 2020 - Published by Downloaded from




A SILVER45 41 35 33 31 29 26 22 17 13 9SUCROSE

kD220 —

105 —

75 —

46 —

27 —

19 —

16 —

B

43 37 34 32 30 27 24 19 14 11 9

g 10 12 14 16 18 20 22

FRACTION NO.ROOT TIPS

" 41 35 33 31 29 26 22 17 13 9SUCROSE 43 37 34 32 30 27 24 19 14 11 9

PM-ATPasc— - - . « . _ . - _ _ _ _BiP— — _ _ — _ w * . _ — - _

icant levels were detected in membranes at 22% Sue (maturevacuoles) and at 31 to 34% Sue, where Golgi marker IDPasedistributed (Hodges and Leonard, 1974). These results indi-cate that peripheral subunits of V-ATPase were associatedprimarily with the ER of root tips.

The distribution of the major peripheral subunits, A andB, reflected the presence of a fully assembled holoenzyme.Immunostaining with polyclonal antibodies to the purifiedenzyme (Ward and Sze, 1992a) confirmed that the majorsubunits of the V-ATPase, including the 95-, 70-, 60-, 48-,36-, and 16-kD polypeptides, were associated with mem-branes equilibrating at 23 to 35% Sue (Fig. 3A). These resultsresemble the broad distribution of vanadate-insensirive ornitrate-inhibited ATPase activity (24-32% Sue) previously

Immunoblot, anti-ATPase (Root tips)

SUCROSE 1*1

44 41 36 34 30 26 23 2O 16 13 12kDa kDa

95-

70-60-

- 105

- 75

70 kl> —

60 kD —10 12 I-IFRACTION NO.

20

Figure 2. Subunits A and B of V-ATPases were distributed in severalendomembranes, especially the ER of oat root tips. The post-mitochondrial supernatant from 0.1 g fresh weight of root tips (2mm) was separated with a 17 to 45% Sue gradient (12 ml). Fractionsof 0.7 ml each were precipitated with TCA, washed with acetone,and solubilized in sample buffer. Equal-volume fractions (one-fifth,containing 2-4 ,ug of protein) were then separated by SDS-PACE(11% acrylamide). Results are from one experiment representativeof two. A, Silver-stained gel shows separation of membrane poly-peptides. B, Top, ER and PM markers peak at 27 and 41% Sue,respectively, but the PM-ATPase is also associated with the ER ofroot tips. Western blot was probed with a mixture of polyclonalantibodies to the PM ATPase (1:2000) and ER BiP (1:30). Bottom,Both subunits A and B of V-ATPases are abundant in the ER as wellas other endomembranes. Western blot was probed with a mixtureof Mab2E7 (1:100 dilution) and Mab 7A5 (1:50).

differentiating cells that lack large vacuoles. On a freshweight basis, root tips were highly enriched in V-ATPases by5- to 10-fold relative to mature root sections. Therefore, onlyapproximately 0.1 g fresh weight of root dps 2 mm in lengthwere homogenized, and the post-mitochondrial supernatantwas separated on a Sue gradient. The major peripheral sub-units of 70 and 60 kD were predominantly associated withER at 27 to 30% Sue (Fig. 2B, lower panel); however, signif-

19-

16-

- 19

- 16

2 4 6 8 W 12 14 16 18 20 22

FRACTION NO.

BPM

FractionER

Fraction

Sucrose It] 44 41 36 34 30 26 23 20 16 13

60-

Fraction No. 2 4 6 8 10 12 14 16 18 20

Figure 3. Distribution and diversity of V-ATPase holoenzyme inmembranes from oat root tips. Membranes were fractionated witha Sue gradient and separated by SDS-PAGE as in Figure 2. A,Western blot was probed with polyclonal antibodies to the V-ATPase holoenzyme (1:300 dilution). Blot shows the presence of aputative 95-kD subunit and the diversity of the approximately 36-to 38-kD subunits between vacuolar membranes at 20 to 22% Sueand endomembranes at 24 to 26% and at 28 to 32% Sue. Thepolypeptide of approximately 65 kD is not part of the purified V-ATPase complex (Ward and Sze, 1992a). Fractions 17 to 22 con-tained solubilized proteins that were dissociated from the mem-brane. B, Fractions from the same experiment were western blottedand probed with Mab 2E7. Subunit B appeared in the PM fraction(38-41% Sue) as well as other endomembranes. www.plantphysiol.orgon February 18, 2020 - Published by Downloaded from




Figure 4. Immunofluorescent staining by 2E7, a Mab to subunit Bof V-ATPases, of perinuclear regions in root tip cells of 4-d-old oatseedlings. Root tips were fixed, embedded in methacrylate, sec-tioned, and stained with mouse ascites 2E7 and anti-mouse IgCconjugated to Texas Red. A, Bright aggregates of Texas Red fluo-rescence appear around the nuclei and in the cytoplasm of corticalcells. B, Location of nuclei is detected by Hoechst 33258 fluores-cence in the same cells as in A. C, Autofluorescence and nonspecificbinding of anti-mouse IgG conjugated to Texas Red is relativelylow. Control sections were treated as above, except the primaryantibody 2E7 was omitted. All the figures were photographed,exposed, developed, and printed at the same time under identicalconditions. Bar = 10 jim.

observed in oat roots and in corn coleoptiles (Churchill et al.,1983; Hager and Biber, 1984). Based on co-migration withenzyme markers of the ER and Golgi, it has been suggestedthat the V-ATPase could be associated with the tonoplast, aswell as the ER and Golgi membranes (Churchill et al., 1983;Hager and Biber, 1984). Furthermore, the immunoblot withMab 2E7 (Fig. 3B) showed V-ATPase association with thePM fraction (38-41% Sue). Interestingly, V-ATPases associ-ated with various endomembranes showed some diversity intheir subunit composition, especially in the size of a 36-to38-kD polypeptide (Fig. 3A).

Immunolocalization of V-ATPase by Light and EM

We verified the subcellular distribution of the V-ATPaseby immunocytochemical localization of V-ATPases in roottip cells. Initial attempts to use 2E7 tissue culture supernatantproved to be unsuccessful in immunoelectron microscopydue to the low concentration of antibody present in thesepreparations. So we generated ascites fluid of 2E7 and 7A5to subunit B and A, respectively, and purified the specificIgG by affinity chromatography with immobilized protein G.Purified ascites fluid 2E7 and 7A5 reacted specifically withsubunits B and A, respectively, in western blots at 1:5000dilutions. However, in preliminary tests of immunoelectronmicroscopy, 2E7 labeled endomembrane compartments moreeffectively than 7A5 and was therefore used for all subse-quent studies.

Immunofluorescent Labeling of Root Tip Cells

Cellular levels of the V-ATPase were visualized in semi-thick longitudinal sections of root tips that were labeled withmouse ascites 2E7 followed by anti-mouse IgG conjugated toTexas Red. Small, brightly fluorescent aggregates were ob-served residing in the perinuclear region of the cells in thecortex (Fig. 4A) and protoderm. The labeled structures ap-peared to be morphologically similar to small vacuoles ob-served in cells of root tips (Fig. 5). The large central nucleus

PROCAMBUM CORTEX

Figure 5. Light micrograph showing a longitudinal section of theroot tip of 4-d-old oat seedling. Roots were fixed in glutaraldehyde,embedded in Epon, sectioned, and stained with toluidine blue O.Dark material outside the protoderm (PD) is the mucilagenous layer.Bar = 5 Mm-




in the same section could be observed by fluorescence fromHoechst 33258 dye, which binds DNA (Fig. 4B). Controlsections exhibited little autofluorescence (Fig. 4C). Little orno labeling with 2E7 was detected in root sections takenapproximately 1 cm above the tip (not shown), a regioncontaining mature differentiated cells with large centralvacuoles.

Immunogold EM

Immunofluorescence microscopy had insufficient resolu-tion to identify the subcellular structures that contain V-ATPase, so additional immunocytochemical assays were un-dertaken at the EM level. The distribution of endomembraneV-ATPases may parallel the ontogeny of vacuoles, so weinitially analyzed root tip cells after aldehyde and OsO4fixation by conventional EM. At low magnification, the un-differentiated cells of the root had a dense cytoplasm andcontained numerous small vacuoles (approximately 1 /tmdiameter) as indicated by light microscopy (Fig. 5). At highermagnification as seen with EM, cells contained extensivelyelaborated ER and abundant Golgi dictyosomes and werefilled with numerous cytoplasmic vesicles (Figs. 6 and 7). TheGolgi consisted of eight or so closely appressed cisternae thatexhibit polar differences in electron density from cis to trans

(trans being the most electron dense) (Fig. 6). Electron-trans-lucent vesicles of 0.1 to 0.3 nm diameter were associated withthe trans face of the Golgi, apparently resulting from buddingat this face (Fig. 6). We suggest that these Golgi-derivedstructures are putative provacuoles. In some instances therewas apparent direct continuity between the putative prova-cuoles and tubular elements radiating from the Golgi appa-ratus (Fig. 6).

The fusion of small vacuoles is widely believed to mediatethe formation of the large central vacuole. Previous investi-gations have suggested that vacuoles form as a result offusion by extended trans Golgi cisternae or branched ERtubules that sequester and displace the cytoplasm (Marty etal., 1980; Amelunxen and Heinze, 1984). Examination of thetrans Golgi region and adjacent cytoplasm indicated thatvesicles or membrane tubules aggregated and apparentlyfused to form extended structures (Fig. 6). Vesicles orientedin single file appeared to be in the process of fusion (Fig. 6).Numerous small vesicles were frequently seen surroundingan enlarging vacuole of 1 to 2 nm in diameter (Fig. 7). Themembrane between the adjacent provacuoles appeared to beinternalized within the vacuole as a result of fusion. Themembrane within the vacuole seemed to be partially de-graded (Fig. 7, Iv).

Immunolocalization of the peripheral 60-kD subunit with

Figure 6. Interrelationship between provacuoles (Pv) associated with the Colgi apparatus (G) and the aggregation andfusion of the provacuoles. Provacuoles exhibit direct continuity with the medial and trans cisternae of the Colgi apparatus(arrowheads). Provacuoles immediately adjacent to the Colgi apparatus are oriented in a single-file array and appear tobe in the process of fusing. Also shown is an elongated, flattened membrane that is continuous with the provacuolemembranes (arrows). Bar = 0.5 ^m.




Figure 7. Association of provacuoles (Pv) withvacuoles (V). Vacuolar enlargement appears toresult from the fusion of provacuoles that com-pletely surround the vacuole shown. The proc-ess of provacuole fusion to the vacuole appearsto result in the sequestration of membraneswithin the vacuole. Iv, Intravacuolar material.

ER

8b0.5pm

Figure 8. Immunogold localization of peripheral subunit B of the V-ATPase in the ER. In a, the gold particles aredistributed throughout the segments of the ER. In contrast, b shows an example where the gold particles are bound toonly part of the continuous segments of ER, indicating that there may be spatial differences in V-ATPase content withinthe ER. Small vacuoles (V) are also labeled with gold particles. www.plantphysiol.orgon February 18, 2020 - Published by Downloaded from



Vacuolar H^-ATPase Associated with the ER and Provacuoles 1321

purified Mab 2E7 in root tip cells resulted in gold particlelabeling of small vesicles, provacuoles, and ER (Figs. 8 and9). Control Mab IgGi directed against a thiol protease p34(Kalinski et al., 1992) did not label oat root cells (not shown).The ER was identified by its characteristic cisternal structureand associated ribosomes (Fig. 8). Both the ER and vesiclelabel appeared to be associated with the membrane. The ERappeared to exhibit spatial variation of gold label on contig-uous ER cisternae, suggesting that there might be regionalspecialization of the ER with respect to vacuole formation(Fig. 8). Other immunocytochemical assays demonstratedlabeling of small vesicles and various sizes of provacuolesand vacuoles (Fig. 9). The smallest vesicles (0.1-0.2 /tmdiameter) appeared to be similar in size and morphology tothe putative provacuoles that were associated with the Golgi(Fig. 6). The relative labeling density was low, probablybecause the Mab 2E7 recognizes a single epitope that mightnot always be correctly oriented. Additional gold label wasseen inside the vacuoles (Fig. 9). This could be due to V-ATPases on membranes that were internalized during fusionof the provacuoles and subsequently degraded (Fig. 7). Thisis consistent with the proposed mechanism of vacuole en-largement by fusion of provacuoles and internalization/deg-radarion of excess tonoplast as seen in reformation of coty-

Figure 9. Immunogold localization of the peripheral subunit B ofthe V-ATPase on small vacuoles (V) and provacuoles (Pv). Note goldparticles labeling a membranous structure within the vacuole(arrow).

ledon vacuoles from protein bodies (Melroy and Herman,1991). The immunocytochemical evidence for subunit B onER, endomembrane compartments, and provacuoles is con-sistent with the results obtained from biochemical fractiona-tion (Figs. 1-3).

DISCUSSION

To understand the relationship between the ontogeny ofplant vacuoles and the origin of the V-ATPase, we have useda specific Mab, 2E7, to determine the subcellular location ofV-ATPases in root tip cells. Previous ultrasrrucrural studieshave indicated that vacuoles are ultimately derived from theER. According to one model, small vacuoles are thought tooriginate from a tubular membrane network at the trans faceof the Golgi system called GERL (Marty et al., 1980). Al-though the term "GERL" is no longer in use, enclosure of thecytoplasm by this membrane network, followed by lateralexpansion and fusion of the membrane tubules, seals off theenclosed cytoplasm from the cell cytoplasm. The sequesteredcytoplasm is then digested by autophagy to form a smallvacuole. The subsequent fusion of many small vacuolesresults in the formation of a large central vacuole. Anothermodel suggests that smooth ER tubules form a circle at thesite to be occupied by the tonoplast (Amelunxen and Heinze,1984; Hilling and Amelunxen, 1985). The tubules fuse intorelatively flat sacks that dilate to form small vacuoles. Con-tinued fusion of small vacuoles with nearby ER tubules andsubsequent dilation results in the formation of large vacuoles.The cytoplasm enclosed by the smooth ER ring is graduallydisplaced and excess membrane can be contained within thevacuole. According to this model, the entire smooth ERbecomes the tonoplast. Our immunological results (Figs. 6-9) are in general consistent with either of these two models;however, the distinction between smooth ER and the GERLnetwork cannot be made.

V-ATPases are associated with purified mature vacuoles(Smith et al., 1984; Sze, 1985); however, it is not clear wherethis pump is synthesized and assembled. There are severalpossibilities. V-ATPases could be assembled at the maturevacuole, provacuoles, trans Golgi-derived vesicles, or even atthe ER. Using both cell fractionation and immunolocalizationtechniques, we have shown that V-ATPases are associatedwith the ER in immature cells of the root tip (Figs. 2 and 8).In these cells, higher levels of V-ATPase subunits were as-sociated with the ER fraction and Golgi-derived vesicles thanwith the mature vacuoles (Fig. 2). Conversely, in maturedifferentiated cells, the V-ATPases were mostly located onthe mature vacuolar membrane (Fig. IB). These results sup-port the idea that vacuoles are initially derived from the ERmembrane (Ameluxen and Heinze, 1984; Hortensteiner etal., 1992) and that the V-ATPase originated at the ER.

Conventional EM suggests that vacuole precursors (i.e.provacuoles) could originate from the trans Golgi network(Fig. 6). Much of the V-ATPases were associated with smallvesicles (0.1-0.3 pm) that may be derived from the Golgi(Fig. 9). Immunogold labeling on the Golgi dictyosomes wasnot observed in oat roots. However, the same 2E7 antibodydid label the Golgi from sycamore suspension-cultured cellsthat were prepared by the rapid freeze/freeze substitution www.plantphysiol.orgon February 18, 2020 - Published by Downloaded from



1322 Herman et ai. Plant Physiol. Vol. 106, 1994

method (G.F. Zhang and A. Staehelin, personal communi- cation). The V-ATPase was recently shown to be associated with purified Golgi membranes from maize roots (Oberbeck et al., 1994). The Golgi apparatus has been shown to mediate the deposition of soluble proteins in the vacuole; however, there is far less information on the role of Golgi in mediating the traffic of tonoplast proteins. The Golgi trafficking inhibitors brefeldin A or monensin impede the progression of newly synthesized soluble proteins to the vacuole, but not the vacuolar membrane-bound proteins (Gomez and Chris- peels, 1993). Thus, soluble and membrane-bound proteins use different mechanisms of transport to reach the vacuole. The tonoplast integral protein aTIP has been localized in the Golgi (Melroy and Herman, 1991), suggesting that some tonoplast proteins do progress through the Golgi. Taken together, these results support the idea that Golgi membranes possess vacuolar-type proton pumps. Some of the pumps may be in transit to the vacuole, whereas others may be resident pumps that acidify Golgi compartments.

Mature vacuoles are formed as a result of fusion of many small vacuoles; however, the events leading to the recogni- tion, docking, and subsequent fusion are not understood. As two vesicles fuse, the membranes that divide the two compartments appear to be intemalized and degraded to form one compartment (Fig. 7). This intemalization and degrada- tion may be necessary for the reduction of membrane surface area to volume ratio as the vacuole enlarges. We suggest that V-ATPase subunits sometimes observed inside an enlarging vacuole could result from the fusion of two or more vesicles (Fig. 9). This interpretation is consistent with the results with negatively stained vesicles in which the peripheral knobs of V-ATPases of one vesicle are observed facing the knobs of a neighboring vesicle (Ward and Sze, 1992a). This could ex- plain the inaccessibility of approximately 50% of V-ATPase peripheral subunits to KI washing of native membranes (Rea et al., 1987). Because the fusion of biological membranes can be facilitated by Ca2+, an acidic vesicle interior, ion move- ment, and osmotic swelling due to water uptake (Wilshut and Hoekstra, 1984; Lucy and Ahkong, 1986), these results imply that endomembrane V-ATPases could play a role in membrane fusion and therefore vacuole ontogeny. This idea is supported by the observation that small GTP-binding proteins (probably involved in vesicle trafficking and fusion) associate with pancreatic microsomes when the intravesicular pH is acidified by a V-ATPase (Zeuzem et al., 1992).

Interestingly, low levels of V-ATPase subunits were con- sistently detected on plasma membrane fractions separated with a Suc gradient. PM-associated V-ATPase was relatively more prominent in root tip cells (Figs. 2 and 3B) and could be enhanced in roots grown in alkaline medium (X. Li, data not shown). Although we do not understand the role of a PM-associated V-ATPase yet, it is possible that V-ATPases are associated with secretory vesicles of immature cells, which actively secrete new wall materials (such as pectins and hemicelluloses) as well as the mucilagenous layer that pro- tects the root tip (Fig. 5). We did not, however, detect V- ATPases on the PM by immunogold labeling, perhaps because the V-ATPases appeared on the PM transiently and therefore the antigen levels were relatively low.

Very little is known about the biosynthesis and assembly

of V-ATPases in plants or other eukaryotes. According to the model derived from studies in yeast (Kane and Stevens, 1992), the V, complex is synthesized and assembled at the rough ER. We have some evidence supporting this idea. When the cDNA encoding an oat 16-kD subunit is tran- scribed and translated in vitro, the major integral protein is inserted stably into microsomal membranes (Szi? et al., 1992b). In yeast, evidence shows that the V1 is syrthesized and assembled in the cytosol; however, it is unclear whether the VI is attached to the V, at the Golgi or the vacuole (Kane and Stevens, 1992). Our results showing subunit B on the ER (Figs. 2 and 8) would strongly indicate that the peripheral subunit B or the V1 complex is attached to the V, 0'1 the ER from oat roots. Thus, the fully assembled V-ATPase on the ER could (a) directly become part of the vacuole membrane during vacuole formation or (b) be sorted via the Golgi to specific endomembrane destinations or (c) both.

This model for the assembly of VI with V, at the ER is supported by recent experiments in both plants and animals using different approaches. Purified membrane fra ztions of the ER, Golgi apparatus, and clathrin-coated vesicles from maize roots reacted positively with antibodies ta the V- ATPase on western blots. Because both peripheral (70, 57, and 44 kD) and integral (16 kD) subunits were present, these results suggest that the V-ATPase was already asseinbled at the ER (Oberbeck et al., 1994). In pulse-chase experiments of bovine kidney epithelial cells, Myers and Forgac (1 393) bovine showed that the VI complex was associated wi'h membranes at 15OC or in the presence of brefeldin A. Since brefeldin A disrupts the Golgi and perturbs ER-Golgi traffic and 15OC temperatures stop traffic between the ER arid Golgi, their results would suggest that the VI is assembled with the V, in the ER.

These results raise several significant questions: (a) Is the ER-bound V-ATPase functionally active? According to membrane fractionation studies, the V-ATPase associaíed with smooth ER and low-density endomembranes in oat or com seedlings is a functional proton pump. Membranes equilibrating between 24 and 28% Suc show both vanadate- sensitive and nitrate-sensitive ATPase activity (ChL rchill et al., 1983) and are active in proton pumping (DuPont et al., 1982; Hager and Biber, 1984) or in ApH-driven Ca transport (Schumaker and Sze, 1985). (b) Are all the endomvmbrane V-ATPases destined for the mature vacuole or are they sorted to specific destinations? We do not know yet, but we suspect that there is active sorting to maintain compartment diversity. Endomembranes are dynamic and can participate in diverse functions, like cell plate formation (fusion of Golgi and ER vesicles), provacuole fusion to form central vacuoles, and secretion of wall components during cell wall sjgnthesis. Because of the diverse roles attributed to endomembranes, it is very likely that compartments are functionally distinct. If so, there might be organelle- or compartment-spec ific isoforms of V-ATPases that could differ in their subunit composition and be independently regulated (Gogarten et al., 1992). A provocative indication of subunit diversity is observed among subunits of 36 to 38 kD between membranes of densities corresponding to 20 to 23% and 28 to 32% Suc (Fig. 3A).

In summary, the plant V-ATPases are located not only on




mature vacuoles but also on many endomembrane compartments of rapidly proliferating, differentiating cells. In oat root tips, V-ATPases are associated with the ER, Golgi-derived vesicles, provacuoles, tonoplast, and possibly secretory vesicles and the PM. These results suggest that the V-ATPase complex originates at the ER and is then sorted to other endomembranes. Therefore, the V-ATPase is not a reliable marker for the vacuole in immature cells. However, the V- ATPase appears to be a better marker of the tonoplast in mature cells, where there is little ER and Golgi, and the V- ATPase is associated mainly with the mature vacuolar membrane (Smith et al., 1984). The broad distibution of V- ATPases in plant cells, as in other eukaryote cells (Marquez- Sterling et al., 1991; Nolta et al., 1993), suggests that this pump is involved in organelle biogenesis and in diverse functions of the endomembrane system, including synthesis, folding and assembly of proteins, cytosolic Ca regulation, osmoregulation, vesicle trafficking, and membrane fusion. Future studies to identify the various isoforms of V-ATPases and to immunolocalize isoform-specific V-ATPases are one approach to developing an understanding of the roles and significance of this endomembrane proton pump.

ACKNOWLEDCMENTS

We thank M. Chrispeels (University of Califomia, San Diego), R.T. Leonard (University of Califomia, Riverside), and M. Maeshima (Hokkaido University, Sapporo, Japan) for providing antibodies to BiP, PM-ATPase, and VM23, respectively. We also thank Stephen Wolniak for his advice about immunofluorescence microscopy and Todd Cooke (University of Maryland, College Park) for stimulating discussions regarding plant structure and function.

Received May 26, 1994; accepted July 14, 1994. Copyright Clearance Center: 0032-0889/94/106/1313/12.

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