Planta (1984)160:348-356

P l a n t a 9 Springer-Verlag 1984

Subcellular localization of H+-ATPase from pumpkin hypoeotyls (Cucurbita maxima L.) by membrane fractionation G/inther F.E. Scherer Botanisches Institut der Universit/it, Venusbergweg 22, D-5300 Bonn 1, Federal Republic of Germany

Abstract. A new method of preparing sealed vesicles from membrane fractions of pumpkin hypocotyls in ethanolamine-containing buffers was used to investigate the subcellular localization of H +ATPase measured as nigericin-stimulated ATPase. In a fluorescence-quench assay, the H + pump was directly demonstrated. The H + pump was substrate-specific for M g . A T P and 0.1 mM diethylstilbestrol completely prevented the development of a A pH. The presence of unspecific phosphatase hampered the detection of nigericin-stimulated ATPase. Unspecific phosphatases could be demonstrated by comparing the broad substrate specificity of the hydrolytic activities of the fractions with the clear preference for Mg. ATP as the substrate for the proton pump. Inhibitor studies showed that neither orthovanadate nor molybdate are absolutely specific for ATPase or acid phosphatase, respectively. Diethylstilbestrol seemed to be a specific inhibitor of ATPase activity in fractions containing nigericin-stimulated ATPase, but it stimulated acid phosphatase which tended to obscure its effect on ATPase activity. Nigericin-stimulated ATPase had its optimum at pH 6.0 and the nigericin effect was K+-dependent. The combination of valinomycin and carbonylcyanide m-chlorophenylhydrazone had a similar effect to nigericin, but singly these ionophores were much less stimulatory. After prolonged centrifugation on linear sucrose gradients, nigericin-stimulated ATPase correlated in dense fractions with plasma membrane markers but a part of it remained at the interphase. This lessdense part of the nigericin-stimulated ATPase could be derived from tonoplast vesicles because c~-mannosidase, an enzyme of the vacuolar sap, remained in the upper part of the gradient. NigericinAbbreviations: CCCP=carbonylcyanide m - chlorophenylhydrazone i DES - diethylstilbestrol

stimulated ATPase did not correlate with the mitochondrial marker, cytochrome c oxidase, whereas azide inhibition of ATPase activity did. Key words: Cucurbita - ATPase (protonated) Plasma membrane (H+-ATPase) - Vacuole (H § ATPase).

Introduction Many important physiological phenomena in plants appear to be closely linked to ATP-dependent H + transport, e.g. cell extension growth and its hormonal regulation (Cleland 1975), aminoacid transport (Kinraide and Etherton 1982), vacuolar transport (Komor etal. 1980; Doll and Hauer 1981), and root gravitropism (Behrens et al. 1982). Therefore, methods have been developed to study proton transport in vitro (Sze 1980; Hager et al. 1980; Hager and Helmle 1981 ; Dupont et al. 1982; Mandala etal. 1982; Stout and Cleland 1982). The method of membrane isolation used here is based on the inhibition of phospholipases during the isolation (Scherer and Morr~ 1978a, b) and it is shown that such membrane vesicles are suitable for studying proton transport. Here specifically, the subcellular localization of H +ATPase was investigated by using the nigericinstimulated part of ATPase activity as a parameter for proton transport (Sze 1980) because studies of this kind have been few and the results and their interpretation has been controversial (Dupont et al. 1982; Mandala etal. 1982; Scherer 1982; Rasi-Caldogno et al. 1982). It was found that in sucrose gradients, nigericin-stimulated ATPase correlated in part with marker enzymes for the

G.F.E. Scherer: Subcellular localization of H §

plasma membrane and equilibrated in part at low density where tonoplast vesicles may sediment. Material and methods Plant material. Pumpkin seeds (Gelber Zentner; Chrysant, Bonn, FRG) were surface-sterilized and grown for 4 d at 30 ~ C on moist cotton in the dark. Hypocotyl hooks about 1 cm long were harvested. Chemicals. Chemicals were of the highest grade commercially available. Nupercaine was a gift from Hoffmann-La Roche (Basel, Switzerland) and nigericin from Ciba-Geigy (Wehr, FRG). Membrane preparation. Membranes for ATPase and markerenzyme tests were prepared as described by Scherer (1981). After separation of the postmitochondrial membrane homogehate from soluble proteins by Sepharose 2B-CL chromatography and overnight centrifugation on a linear sucrose gradient, the fractions were chromatographed on small prepacked Sephadex G25 columns (Pharmacia, Uppsala, Sweden) in 5 m M 2-(Nmorpholino)ethanesulfonic acid-piperidine, pH 6.0, containing 10 mM KC1. Calculations of the activities of ATPase and marker enzymes were based on protein content prior to Sephadex chromatography. Marker-enzyme tests were done with the modifications as described previously by Scherer (1982). In particular, the following methods have been used: glucan synthetase I and II (Jesaitis et al. 1977); NADH-cytochrome c reductase (antimycin A-insensitive) and cytochrome c oxidase (Jesaitis et al. 1977); uridine 5'-diphosphate glucose-sterol-fl-D-glucosyltransferase (Quantin et al. 1980); acid phosphatase and cr mannosidase (Boller and Kende 1979). Carotenoids were determined spectrophotometrically in the total lipid extract (Bligh and Dyer 1959) and protein according to Lowry et al. (1951). Assay of ATPase. The activity of ATPase was assayed for 20-30 min at 30 ~ C in a total volume of 0.25 ml of 15 m M piperidine pH 6.0 (or other pH values as indicated) with 3 mM ATP (piperidine salt), 3 m M MgCI> and 0.3 M sucrose. Basal ATPase activity was measured in the presence of 10 mM KCI and KCl-stimulated ATPase activity was determined as the difference of activities in the presence of 50 mM KC1 and 10 mM KC1. The ATPase activity at pH 8.0 was tested in 3 m M Mes and 12 m M 2-(N-morpholino)propanesulfonic acid (Mops)-piperidine (instead of 15 mM Mes-piperidine) correspondingly. Where indicated with standard deviation bars, experiments were done in triplicate. All other di- or triphophates were assayed at 3 m M substrate concentrations in duplicate. Ionophores and diethylstilbestrol (DES) were added from ethanolic stock solutions up to 0.4% ethanol in the assay, ionophorestimulated ATPase activity was determined as the difference between ATPase activities in the presence and absence of the ionophore. Release of inorganic phosphate was determined as described (Scherer and Morr~ 1978 a). Fluorescence-quench assay for H + transport. For fluorescencequench assays, membranes were prepared with a modified procedure. The tissue was ground with mortar and pestle in 8% ethanolamine (v/v), 2 0 m M ethylenediaminetetraacetic acid (EDTA), 0.4 M sodium glycerol-l-phosphate (grade III, Sigma, Munich, FRG), 2 mM dithioerythritol and 0.15 mM nupercaine or tetracaine titrated to pH 6.5 with HC1. The Sepharose 2B-CI chromatography was omitted and the postmitochondrial supernatant was loaded onto a step gradient of 4 ml 1.0 M, 6 ml 0.5 M and 6 ml 0.3 M sucrose in the homogenization buffer (large buckets, SW 27 rotor; Beckmann, D/isseldorf,

349 FRG). The gradients were spun for 30 min at 25,000 rpm and the middle fraction was used for H § transport assays. This fraction was plasma membrane-enriched and contained 20-30% of the H+-pumping activity whereas the lowermost fraction contained up to two thirds of the mitochondria but not more than 5% of the H § transport activity (data not shown). Membranes were chromatographed on small prepacked Sephadex G25 columns in 10 m M Mes-arginine pH 6.5, 0.3 M sucrose and 50 mM KC1. Proton-pumping activity was assayed in 2 ml of the same buffer in the presence of 15 IxM quinacrine, 3 mM MgSO 4 and 3 mM ATP in an Eppendorf (Hamburg, FRG) fluorimeter with 313 +366 nm as the excitation beam, and the emission above 420 nm was recorded (Schuldiner et al. 1972). Conditions for fluorimetry had been optimized.

Results

The presence of sealed vesicles containing an H § ATPase was demonstrated by monitoring H § transport directly. The proton transport into the GTP

,oo o quench

C~CP

f

[ D

5 rain

~

A

CCCP

Fig. 1. Proton transport in heavy microsomes from pumpkin hypocotyls. The fraction at the 0.3 M/0.5 M sucrose interphase from a step gradient (density >_1.125 g c m - 3) was used to assay proton transport as the fluorescence quench of quinacrine. The effect of three nucleosides on H + pumping activity is shown Table 1. Substrate specificity of the proton pump for di- and

trinucleotides in the fluorescence-quench assay using quinacrine. A plasma-membrane-enriched fraction from a step gradient was used (p >-1.125 g c m - 3). Units were measured as the fluorescence increase by uncoupling with NH4CI after the steady state had been reached. Substrate concentrations were 3 mM each and 3 mM MgSO 4 Nucleotide

Fluorescence increase (%)

Adenosine 5'-triphosphate ( + Mg z+) (ATP) Adenosine 5'-triphosphate ( - Mg 2 +) Gnanosine 5'-triphosphate (GTP) lnosine 5"-triphosphate (ITP) Cytosine 5'-triphosphate (CTP) Uridine 5'-triphosphate (UTP) Adenosine 5'-diphosphate (ADP) Guanosine 5'-diphosphate (GDP) Inosine 5'-diphosphate (IDP) Cytosine 5'-diphosphate (CDP) Uridine 5'-diphosphate (UDP)

100 0 11 19 2 6 0 0 1

0 0

350

G.F.E. Scherer: Subcellular localization of H +-ATPase

~----...

r--

a2s.M

i0opNl

z""

No- vanadate 10 % fluorescence quench

N

~

]

S

60bin

DES l

10 mM

NH4CI

10 mN

Fig. 2. Inhibition by DES of proton transport in light microsomes from pumpkin hypocotyls. The top fraction of a step gradient was used for the experiment (density > 1.10 g cm 3). The effect of increasing concentrations of DES on the fluorescence quench is shown

0.6-

9

(3

0.6- b

ATP

NH40

Fig. 3. Inhibition by orthovanadate of proton transport in heavy microsomes. The fraction at the 0.3 M/0.5 M sucrose interphase from a step gradient (density>l.125 g cm -3) was used to assay proton transport as the fluorescence quench of quinacrine. The effect of increasing concentrations of sodium orthovanadate on the fluorescence quench is shown

o UTP CTP

0.4

0.4-

~

@

@ 0.2-

0-04

"-. 0 2

"0

o

0

i

i

C



1.0>.

x\

[

i

ADP ZxGDP 1:3 IDP 9

-1.20~'~E ~

10I d

?/!

I

[

UDP DP

+

:a.

8 -100

~o.

>

~6 aS.

-

mo 0.5-

\

>"

~

2 0.5

-50

om o_

o_

0

i

5 I'0 ~raction number

; fraction

membrane vesicles was assayed in step-gradient fractions as the fluorescence quench of quinacrine. Figure 1 shows the recorder tracing of such an experiment with a plasma membrane-enriched fraction. Usually, after 30-40 rain a steady state was reached which lasted at least an additional 30-60 min. Ammonium chloride, carbonylcyanide m-chlorophenylhydrazone (CCCP), and nigericin ( + KC1) reverted the fluorescence quench, thus indicating that protons were transported. The uncoupling effect of all three uncouplers was similar. A correction for fluorescence quench by CCCP alone had to be made so that for most experiments ammonium chloride was used. Proton transport

lb number

Fig. 4a-d. Phosphatase activities with diand triphosphates in membrane fractions of a linear sucrose gradient. Phosphatase activities were tested in the presence of 50 mM KC1 and 3 mM MgC12 at pH 6.0, similar to ATPase activity. a e--e b o--o

ATP; A--~ UTP; o--o

GTP; m--~ CTP.

ITP.

e e - - e ADP; zx--zx GDP; ra--c3 IDP; • • density. d o--o UDP; o--o CDP; +--+ glycerol-l-phosphate. All measurements were done in duplicate

+

was strictly Mg z+-dependent and was very specific for ATP (Table J ; Fig. i). Other di- and trinucleotides had little effect. In a microsomal fraction containing both plasma membrane and tonoplast vesicles, DES effectively blocked the proton transport at 67 pM (Fig. 2), a concentration which had little influence on ATPase activity. With orthovanadate in a plasma membrane-enriched fraction, strong inhibition of H + transport at an inhibitor concentration of 100 gM was obtained (Fig. 3). Orthovanadate sensitivity is characteristic for plant plasma membrane proton transport (Coccuci et al. 1980; Goffeau and Slayman 1981). The presence of H +-ATPase can also be tested

G.F.E. Scherer: Subcellular localization of H+-ATPase

351 9 control &lO0/JM DES

06f

0

Z~ /"z~

10,. mo,yb-/ \ date

_

0'.

~_ o.4-

,s0

M

]

~

,,

-

o

o o4

\

A e-

c

0.2-

0.1-

0.1 mM DES i

i

9 control o 50/JN vanadate

i

b

9 100',uM

0.6-

25. 5_

~

0.1 0.2-

r

I

----I



0.5m M Molybdate I

1

-1.20

o=- 0.6-

~--1.15 E o

04

-~ 0.4c ._> "5 0.2-

-1.10

\

"~ ~z

x 0.5 mM Vanadate 0

\

i

1

[

1

5

10

fraction

r

5

T

10

fraction number

I

C x-x

(3b-

Subcellular localization of H(+)-ATPase from pumpkin hypocotyls (Cucurbita maxima L.) by membrane fractionation.

A new method of preparing sealed vesicles from membrane fractions of pumpkin hypocotyls in ethanolamine-containing buffers was used to investigate the...
869KB Sizes 0 Downloads 0 Views