Corn Coleoptiles - NCBI

Plant Physiol. (1985) 78, 232-240 0032-0889/85/78/0232/09/$01 .00/0

Evidence for an ATP-Dependent Proton Pump on the Golgi of Corn Coleoptiles' Received for publication November 5, 1984 and in revised form December 20, 1984

ALAIN CHANSON2 AND LINCOLN TAIZ*

Department of Biology, Thimann Laboratories, University of California, Santa Cruz, California 95064 ABSTRACT inferred that the PM and tonoplast ATP-dependent proton pumps represent two distinct classes of H+-ATPases based on a Corn (Zea mays L. cv Trojan T929) coleoptile membranes were number of criteria, including sensitivity to inhibitors, stimulation fractimted on scrose density gr Nts, ad ATP-dependent proton by ions, and pH optima (21). Little is known about possible pumping activity was loalzed by the techiques of ll4cmethylami transport ATPases on other endomembranes in plant cells. A uptake and q e florescee q ig. Two pealks of proton putative secretory vesicle ATPase with properties similar to the pumping activity were detected: a light peak (1.07 grams/cubic cenime- plasma membrane ATPase (e.g. vanadate sensitivity) was identer) corresponding to the previously characterized tonoplast-type Ht- tified in a membrane vesicle preparation from suspension culATPase, and a second peak (1.13 grams/cubic centimeter) which coin- tured oat cells (2). Unfortunately, the purity of the preparation cided with the Golgi markers, latent UDPase, and glUcan synthase I. was not established using marker enzymes, and it is not clear The second peak was lighter than that of the plasma membrae marker, whether the activity was associated with secretory vesicles or uridine diphosphoglucosesterol glucosyltransferase (1.16 grams/cubic contaminating plasma membranes. No vanadate-sensitive ATPcenimeter) ndwas not inhibited by vanadate, an inhibitor of the plasma ase was found to be associated with the secretory vesicles of pea membrane ATPase. The activity was also better correlated with the Golgi stem homogenates (22). Nevertheless, an ATP-driven proton cisternae marker, glucan synthase I, than with latent UDPase, a secretory pump has been reported to be present on the Golgi of rat and vesicle marker, but a secretory vesicle location cannot be ruled out. The mouse liver (1, 8, 25). In a previous paper, we reported the tonophast-type and Golg proton pumps were similar in several respects, occurrence of a KCl-stimulated Mg2+-ATPase which appeared including a pH optimum at 7.2, stimulation by chbride, inhibition by to be specifically associated with the Golgi of corn coleoptiles diethylstilbestrol ad N,N'-dicyclohexylcarbodlimide (DCCD), inei- (5). The Golgi ATPase overlapped with the plasma membrane tivity to oligomycin and azide, and nucleotide specificity for Mge-ATP. ATPase on sucrose density gradients, but could be resolved as a However, the Golgi H' pump was much less sensitive to nitrate and separate peak by assaying the activity at pH 7.5 instead of 6.5. iodide, and more sensitive to the anion channel blockers, 4-acetamido- The two major objectives of the present study were: (a) to 4'-isothiocyano-2,2'-stilbene suUfonic acid (SITS) and 4,4'-diisothiocy- determine whether the Golgi ATPase of corn coleoptiles funcano-2,2'-stilbene disulfonic acid (DIDS) than the tonoplast-type Hl- tions as a proton pump; and (b) to compare the properties of the pump. The Golgi pump, but not the tonoplast-type pump, was stimulated Golgi proton pump with those of the previously characterized by valinomycin in the prsce of KO. It is concluded that the Golgi of tonoplast-type proton pump (10, 1 1). Because of the lack of an corn coleoptiles contains a KC-stimulated H+-ATPase which can acidify independent marker for the tonoplast, the term 'tonoplast-type' the interior of Goli cisternse ad associated vesicles. is used throughout this paper (21). However, recent studies with intact vacuoles isolated from corn coleoptiles indicate that the tonoplast-type H+-ATPase purified on sucrose gradients has the same density and properties as the H+-ATPase associated with purified vacuolar membranes (Mandala and Taiz, unpublished data). In recent years, evidence has accumulated that the PM3 and MATERIAILS AND METHODS tonoplast of plant cells contain H+-ATPases which carry out the electrogenic transport of protons. Such proton pumps apparently Plant Material. Corn (Zea mays L. cv Trojan T929, Pfizerserve as primary transport mechanisms, driving the transfer of DeKalb) seeds were soaked 6 to 8 h in distilled H20 and sown other solutes across their respective membranes (21). It has been in trays with moist vermiculite. Seedlings were grown in the dark at 20°C with 2 h of dim red light daily to inhibit mesocotyl 'Supported by grant PCM-8301995 from the National Science Foun- growth. After 5 to 6 d, coleoptiles (-3 cm) were harvested, dation. debladed, and collected on ice under room lights. Homogeniza2 Recipient of a postdoctoral fellowship from the Swiss National tion and subsequent treatments were performed at 0 to 4°C. Foundation. Present address: Institut de Biologie et de Physiologie VeHomogenization. Coleoptiles were homogenized and a 1,OOOg getales, Universite de Lausanne, Batiment de Biologie, 1015 Lausanne, supernatant (lKS) was prepared as previously described (5). Switzerland. Briefly, coleoptiles (13 g) were chopped by hand (10 min) with 3Abbreviations: PM, plasma membrane; GS I, glucan synthase I; razor blades in 6 ml of homogenization medium (250 mm IDPase, inosine diphosphatase; MeA, methylamine; UDPase, uridine sucrose, 2 mM EDTA, 1 mM DTT, 0.1% BSA, 50 mm Tris-Mes diphosphatase; UDPG-ST, uridine diphosphoglucose-sterolglucosyl- [pH 7.8], except for Figures 1 and 2, in which BSA was omitted). transferase; BTP, bis-tris propane; DES, diethylstilbestrol; KIDA, potas- The tissue was then ground very lightly with a mortar and pestle sium iminodiacetate; DIDS, 4,4'-diisothiocyano-2,2'-stilbene disulfonic and strained through nylon. The remaining tissue was lightly acid; SITS, 4-acetamido-4'-isothiocyano-2,2'-stilbene sulfonic acid; reground in an additional 6 ml of homogenization buffer. The DCCD, N,N'-dicyclohexylcarbodiimide. final homogenate was filtered through nylon and combined with 232

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GOLGI PROTON PUMP OF CORN the first homogenate. Unbroken cells, cell wall fragments, starch, and nuclei were removed by a 5-min centrifugation at lOOOg (Sorvall, SS-34 rotor) and the supernatant (1 KS) was collected. Linear Sucrose Gradients. The 1 KS was layered onto a linear gradient consisting of a 2-ml cushion of 45% sucrose (w/w), 20 ml of 15 to 45% sucrose (Fig. 1) or 10 to 40% sucrose (Fig. 4), and 1 ml of a 15 or 10% sucrose overlay. The gradient buffer included 20 mm KCI, 1 mM DTT, 0.5 mM EDTA, and 2.5 mM Tris-Mes (pH 7.5). The gradients were centrifuged at 80,000g for 3 h (Beckman L2-65B ultracentrifuge, SW 28 rotor) and fractionated into 16 fractions (1.5 ml). Nonlinear Sucrose Gradients. The 1 KS fraction was layered onto a step gradient consisting of a 3-ml cushion of 35% (w/w) sucrose and 5 ml each of 25, 18, and 10% sucrose (in 0.5 mm EDTA, 20 mm KCI, 1 mM DTT, 2.5 mM Tris-Mes, pH 7.5). The gradients were centrifuged 2 h at 80,000g, and the membranes at the different interfaces were collected with a Pasteur pipet. The pellet was discarded. Rate-Zonal Centrifugation. The lKS was layered onto a linear gradient (20 ml, 15 to 35% sucrose in 0.5 mm EDTA, 20 mM KC1, 1 mM DTT, 2.5 mM Tris-Mes, pH 7.5) with a 2-ml cushion of45% sucrose and a l-ml overlay of 15% sucrose. The gradients were centrifuged 25 min at 30,000g (SW 28 rotor) and fractionated into 16 fractions (1.5 ml). Dilution of the Fractions. For proton pumping experiments, the gradient fractions were diluted to 10% sucrose (w/w) with 0.5 mM EDTA, 1 mM DTT, 20 mM KCI, and 2.5 mm Tris-Mes (pH 7.5). The diluted fractions were assayed directly or frozen in liquid N2 and stored at -70°C for up to 2 weeks without loss of activity. Freezing prior to dilution resulted in a loss of activity. Enzyme Assays. NADPH-Cyt c reductase, Cyt c oxidase, latent UDPase, UDPG-ST, and GS I activities were determined as previously described (5). ATP hydrolyzing activity was determined by released Pi (5) after a 30-min incubation at 37°C. The reaction mixture contained 3 mm Tris-ATP (plus or minus 3 mM MgSO4), 250 mm sucrose, 50 mM LiCl, 2 ,uM gramicidin, I mM Na-molybdate (to inhibit nonspecific phosphatase), 1 mM NaN3 (to inhibit mitochondrial ATPase), and 25 mM Tris-Mes, pH 7.2. The activity is expressed as the Mg2+-stimulated activity. Methylamine Uptake. The uptake of MeA was determined by a Millipore filtration technique (1 1). At time zero, 20 Ml of test solution was added to 100 Ml of diluted membranes (in 10% sucrose). The final concentrations were: 3 mm Mg2+-ATP or Mg2+-ADP (control), 18 Mm MeA (0.77 MCi/ml/assay, 44 mCi/ mmol in ethanol), 20 mm KCI, 10% sucrose, and 12 mM TrisMes (pH 7.5). After a 5-min incubation at 30°C, the reaction mixture (110 Ml) was filtered on a prewetted O.45-Mm Millipore filter (HATF). The filter was immediately rinsed with 3 ml of cold buffer (20 mm KCI, 10% sucrose, 2.5 mm Tris-Mes, pH 7.5). The entire stop process took less than 15 s. Radioactivity was determined in a liquid scintillation counter. MeA uptake was calculated as the difference in activity between Mg2+-ATP and the Mg2+-ADP control. Quinacrine Fluorescence Quenching. Membrane vesicles (300400 Mg protein/experiment), the appropriate salts or inhibitors, and 10 uM quinacrine were added to an assay buffer of 25 mM BTP-Mes (pH indicated in text) to a final volume of 0.6 ml. Fluorescence was measured at room temperature with an Hitachi-Perkin Elmer fluorescence spectrophotometer. The excitation wavelength was 420 nm, and the emission wavelength was 495 nm. After temperature equilibration, the reaction was initiated by the addition of Mg2+-ATP. At the end of the experiment, the proton gradient was collapsed by the addition of 3 M1 of 1 mM monensin dissolved in ethanol (5 Mm, final monensin con-

centration). Other Assays. Sucrose concentrations were determined refractometrically. Protein was assayed by the Lowry method (cited in

Ref. 5) after a TCA precipitation, and using BSA as a standard. Chemicals. ATP (disodium salt), valinomycin, DIDS, SITS, and ouabain were purchased from Calbiochem. Sodium azide and I-amino-2-naphthal-4-sulfonic acid were purchased from Eastman Kodak Co.; sodium lauryl sulfate (SDS) was obtained from Bio-Rad; N,N'-dicyclohexylcarbodiimide was obtained from the Aldrich Chemical Co.; and sodium vanadate was from Fisher Scientific Co. All other chemicals were purchased from Sigma Chemical Co. or Mallinckrodt. RESULTS Localization of the Golgi ATPase Proton Pump. Two peaks of proton pumping activity (ATP-dependent MeA-uptake) were detected after centrifugation of the 1 KS fraction on a near isopycnic linear sucrose gradient: one near the top of the gradient at 1.07 g/cc, less dense than the ER marker, NADPH-Cyt c reductase, and a second at 1.13 g/cm3, between the Golgi marker, latent UDPase, and the PM marker, UDPG-ST (Fig. 1). The distribution of valinomycin-stimulated proton pumping is also shown. Valinomycin stimulated the proton pumping activity of the second peak, but did not affect the activity of the first peak. In the presence of 50 mm nitrate, the lighter peak was completely abolished while the denser peak was only inhibited by about 50% (Fig. 1). On the basis of its low density and marked nitrate sensitivity, we infer that the first peak represents tonoplast vesicles, although this conclusion remains tentative because of the lack of an independent marker for tonoplast membranes (21). The identity of the peak less sensitive to nitrate is not immediately apparent because of its position midway between latent UDPase and UDPG-ST. Figure 2 illustrates a rate-zonal sucrose gradient of the 1 KS 0

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