Localization of phosphatidylcholine synthesizing enzymes in etiolated bean seedlings (Phaseolus vulgaris L.)

Plant Science Letters, 21 ( 1 9 8 1 ) 3 8 9 - - 3 9 6

389

© Elsevier/North-HollandScientific Publishers Ltd.

LOCALIZATION OF PHOSPHATIDYLCHOLINE SYNTHESIZING ENZYMES IN ETIOLATED BEAN SEEDLINGS ( P H A S E O L U S V U L G A R I S L.)

K. HOCK and E. HARTMANN* Institut fiir AUgemeine Botanik der Universita't Mainz, Saarstrasse 21, 6500 Mainz (F.R.G.)

(Received October 13th, 1980) (Revision received January 8th, 1981) (Accepted January 9th, 1981)

SUMMARY

The enzymes of the CDP-choline pathway of lecithin biosynthesis are present in etiolated hypocotyl hooks of bean seedlings (Phaseolus vulgaris L.). The subcellular distribution of the enzymes was determined by sucrose density gradient centrifugation. The enzyme choline kinase (EC 2.7.1.32) proved to be soluble protein while phosphorylcholine-cytidyl transferase (EC 2.7.7.15) and phosphorylcholine
INTRODUCTION Many investigations have shown that the nucleotide pathway, which was first demonstrated in animal tissues [1]. is at least one important route for lecithin synthesis. Biosynthesis involves three steps: (1) the phosphorylation of choline to phosphorylcholine by choline kinase (EC 2.7.1.32), (2) the reaction between phosphorylcholine and CTP to form CDP-choline, catalyzed by phosphorylcholine-cytidyl transferase (EC 2.7.7.15) and (3) the formation of lecithin from CDP-choline and diglyceride by the enzyme CDP-choline:diglyceride phosphorylcholine transferase (EC 2.7.8.2 .). In plant cells the nucleotide pathway was reported the first time by Morrd et al. [ 2]. Intensive studies have also been made on the cells of castor bean endosperm [3,4]. Besides these investigations the three enzymes of the *To whom correspondence should be sent.

390

CDP-choline pathway have also been reported from other plant sources [5--9]. The hypocotyl hook was chosen because it is very sensitive to light; a fast growth reaction is observed after illumination. One effect of light is to change phospholipid metabolism [ 10] and mediate choline kinase activity [ 11]. This study presents data which characterize the compartmentation of phosphollpid synthesizing enzymes in the etiolated hypocotyl hook region and provides further information about changes in lipid metabolism which follows the light stimulus. MATERIALS AND METHODS Seeds of bush beans (Phaseolus vulgaris L., cv. St. Andreas I. Ernte, grfinhtUsig) were grown on sterile moist vermiculite in plastic boxes covered with glass plates in absolute darkness at 20 -+ I°C. Six-day-old seedlings were cut below the hypocotyl hook and the elbow region of the hypocotyl was frozen in liquid nitrogen immediately. A quantity (3 g) of the frozen hypocotyl hook preparation was chopped with razor blades into 5 ml of grinding medium in a mortar on ice. The grinding medium [12] contained 0.15 M tricine (pH 7.5), 10 mM KC1, 1 mM MgC12, 1 mM EDTA-Na2 (pH 7.5) and 13% (w/w) sucrose. The crude homogenate was filtered through two layers of nylon cloth (mesh 55 urn) and centrifuged for 10 rain at 200 × g to remove cell debris. All procedures were carried out at 0--4°C. The separation of organelles was achieved by centrifuging 5 ml samples of the precentrifuged homogenate onto sucrose gradients. The gradients were made in 1 × 3 in. cellulose nitrate tubes and contained the following solutions (from the bottom to the top of the tube): Vol. (ml)

Sucrose conc. % (w/w)

2 6 3 3 6 6

60 60--50.5 (linear gradient) 50.5 48 48--32 (linear gradient) 32

All sucrose solutions contained 1 mM EDTA-Na2 (pH 7.5). The sucrose concentration was measured with a 'Leitz' refractometer. The samples were centrifuged in a Beckman rotor SW 25.1 on a Beckman L 50 ultracentrifuge at 2°C for 6 h at 20 000 rev/min. After centrifugation 1.2-ml fractions were collected using an ISCO density gradient fractionator, model 640. A TP:cholinephosphotransferase (EC 2.7.1.32) was assayed as described

391 by Butt and Brody [13]. The incubation medium contained 0.1 M 2-amino2-methyi-l,3-propandiol-buffer (pH 9.0), 10 mM MgC12, 10 mM ATP, 10 mM [methyl-~4C] choline choride (spec. act., 1 m C i / m m o l ) , 1.0 mM ouabain, 20 mM phosphoenolpyruvate and 81 U/ml pyruvate kinase. An ATP-regenerating system was used in this assay, because ADP proved to be a choline kinase inhibitor [ 13]. Fresh medium was prepared prior to use. The assay was carried out with 15 pl of the incubation medium and a 5-pl aliquot of the gradient fractions at 37°C for 30 rain. The reaction product phosphorylcholine was separated from choline with sodium tetraphenylborate in a mixture of acetonitrile-heptanone-3 (1 : 5) [14]. The aqueous phase was washed twice with acetonitrile-heptanone-3, placed in a scintillation vial with 10 ml scintillation cocktail KL 402 (Zinsser) and counted in a Berthold BF 8000 scintillation counter. The dpm values were automatically calculated from the ESCR-program. Phosphorylcholine-cytidyl transferase (EC 2. 7. 7.15). This enzyme was assayed according to the method of Montague and Ray [9]. The incubation medium contained 0.33 pCi phosphoryl-[~4C] methylcholine (spec. act., 52 mCi/mmol), 2 mM CTP, 10 mM MgC12, 100 mM Tris-maleate-buffer (pH 6.4) and 0.4 ml of the gradient fraction in a final volume of 0.5 ml. The assay was carried out at 28°C for 1 h. The separation of the reaction product from the incubation medium was made as described by Montague and Ray [ 9] with no change except that the radioactivity was counted with a Berthold LB 6210 U gas flow counter. CDP-choline:diglyceride phosphorylcholine transferase (EC 2.7.8.2). This enzyme was assayed by the method of Weiss et al. [15] modified by Lord et al. [3]. The assay mixture contained, in a final volume of 0.5 ml, 20 #mol Tris--HC1 (pH 7.0), 10 pmol MgC12 or alternatively 5 pmol MnC12, 20 pmol dithiothreitol, 5 pmol dipalmitin (added as an emulsion in 50/~1 0.3% (w/v) Tween 20), 0.1 pCi CDP-methyl[ ~4C] choline (spec. act., 55 mCi/mmol) and 0.1 ml gradient fraction. The dipalmitin had been sonicated in 0.3% (w/v) Tween 20 before additon to the enzyme preparation. The incubation was carried out at 28°C for 60 min. NADH:cytochrome c reductase (EC 1.6.99.3) was assayed spectrophotometrically at 550 nm as described by Donaldson et al. [16]. Cytochrome c oxidase (EC 1.11.1.5) was assayed using the procedure of Tolbert et al. [17] at 550 nm with cytochrome c which was reduced with NaS204. Triosephosphate isomerase (EC 5. 3.1.1) was assayed after Beisenherz [ 18]. NADP:triosephosphate dehydrogenase (EC 1.2.1.13) was assayed after the method of Heber et al. [19].

Malate dehydrogenase (EC 1.1.1.37)and lactate dehydrogenase (EC 1.1.1.27) were assayed after Bergmeyer [20].. Protein was determined by the method of Lowry et al. [21] using bovine serum albumin (Sigma fraction V) as standard. All radiochemicais were purchased from Amersham Buchler.

392 RRSULTS

The separation of various organelles from hypocotyl hook homogenates by density gradient centrifugation is shown in Fig. 1. Two peaks of NADPH: cytochrome c reductase are present; one peak at a density of 1 10 g cmo3 l

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Fig. 1. Equilibrium eucroee den&y grandient eeperation of organeilee from bean hypocotyl hook homogenates. (1) cytochrome c oxidwe activity; (2) NADH:cytochrome c reductase activity; (3) lactate dehydrogenue activity; (4) malate dehydrogenrue activity; (6) trioeephoephate dehydrogenese a&My; (0) trioeephorghate bomeraee activity. In end uucroae concentration (A,B) ahaorhan& of protein b&own IWa continous line (-) (w/w) as dotted line (- - - - -).

393

represents the microsomat enzyme, and the other at a density of I • 20 g cm -3 is contributed by the mitochondrial enzymes. Cytochrome c oxidase shows one major peak at the density of I • 20 g cm -3 which gives strong evidence for the localization of the mitochondria in these fractions. Triosephosphate isomerase locates the position of the proplastids in the gradient. q

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Fig. 2. Distribution of phosphatidylcholine synthesizing enzymes. (1) ATP:choline phosphotransferase; (2) phosphorylcholine-cytidyl transferase; (3) CDP-choline :diglyceride phosphoryleholine transferase; o o, e n z y m e activity with 10 mM Mg =÷-concentration; v, e n z y m e activity with 5 mM Mn=+-concentration. In (A) absorbance trace of protein is shown as a continuous line ( ) and sucrose concentration ( w / w ) as dotted line ( . . . . . ).

394 The comparison of the profiles of cytochrome c oxidase and triosephosphate isomerase shows that a separation between mitochondria and proplastids could not be achieved with sucrose density gradient centrifugation. Lactate dehydrogenase activity occurred in the first five fractions of the gradients, which represented the cytoplasmic phase of the homogenate. The distribution of the enzyme ATP:cholinephosphotransferase (EC 2.7.1.32) among the gradient fractions is shown in Fig. 2. The activity of the enzyme is found in the first six fractions of the density gradient. This gives strong evidence that the enzyme is exclusively localized in the cytoplasm of the bean hypocotyl hooks, without binding to any membranes. The enzyme phosphorylcholine-cytidyl transferase (EC 2.7.7.15), which catalyzes the reaction from phosphorylcholine with CTP to CDP-choline, seems to be localized in two different compartments of the cell. The distribution of the enzyme across the gradient is shown in Fig. 2. One peak of activity occurred in the gradient fractions which represent the microsomal membranes (fraction 6--10) and another peak of activity was found in the fractions 12 and 13, which are considered to be mitochondria or proplastids. No activity of the enzyme was detected at the top of the gradient. This indicates that the enzyme is a membrane bound enzyme, which is localized in the microsomes and in the mitochondria or proplastids. CDP-choline:diglyceride phosphorylcholine transferase. (EC 2.7.8.2), which catalyzes the last reaction in the nucleotide pathway leading to phosphatidylcholine, seems to be exclusively localized in the microsomal fractions as seen in Fig. 2. After centrifugation for 6 h in the sucrose density gradient the activity of the enzyme was found as a sharp peak in the gradient fractions 6--9 (density I • 10 g cm -3). No activity was detected in the soluble fractions of the gradient or in the mitochondria or proplastids. Also after differential pelleting all activity of the enzyme was found in the 100 000-g pellet with only very small traces ( < 1 % ) o f the activity in the 10 000-g pellet. The enzyme proved to be SH-group dependent because its activity could be strongly inhibited by p-chloromercuribenzoic acid and iodoacetic acid. Dithiothreitol was therefore added to the medium as a SH-group protecting agent. The two profiles in the Fig. 2 show the influence of different divalent cations on the activity of CDP-choline:diglyceride phosphorylcholine transferase. We tested Mg 2÷ and Mn 2÷ and found that both ions stimulated the incorporation of CDP-choline in phosphatidylcholine. Manganese (5-mM) is as effective as 10 mM magnesium. DISCUSSION

The data reported in this paper provide evidence that the CDP-choline pathway is at least one general way leading to phosphatidylcholine, which is one of the major phospholipids in biological membranes. Besides the nucleotide pathway there is also a methylation pathway from phosphatidylethanolamine using S-adenosyl-methionine as methylgroup donor. Although

395

this methylation has been reported in different plant species [22--27] other evidence indicated that the nucleotide pathway is the major way to phosphatidylcholine in plants. In general, the endoplasmic reticulum or the microsomal fractions, depending on the methods of separation, seem to be the sites of phospholipid synthesis in animal and plant cells. The enzyme choline phosphotransferase which has been characterized by Hartmann and Schleicher [11] in Phaseolus vulgaris hypocotyl hooks and by Tanaka et al. [28] in other plants, seemed to be localized only in the cytoplasm of the hypocotyl h o o k cells. This result is in agreement with the report of Morr~ et al. [2], who first demonstrated the nucleotide pathway in plants. Lord et al. [3] came to the same result in castor bean endosperm. Comparing our data with numerous investigations on castor bean endosperm [2,3,12,29,30,31] there are some differences in the distribution of the enzymes phosphorylcholinecytidyl transferase and CDP-choline:diglyceride phosphorylcholine transferase. Whereas in castor bean endosperm both enzymes were exclusively localized in microsomal fractions, we found in bean hypototyl hooks the enzyme phosphorylcholine-cytidyl transferase in microsomes and in mitochondria or proplastids. Morr~ et al. [2] reported that phosphorylcholinecytidyl transferase was distributed among all cell fractions in onion stem, but the highest specific activity was found in the Golgi apparatus. They suggested that there were different enzymes in each fraction which showed differences in pH-optima and requirement for divalent cations. The result, that the enzyme CDP-choline:diglyceride phosphorylcholine transferase is only localized in the microsomal fractions of our preparation is in agreement with data reported from other plants [2,3,6,9]. We found also that the enzyme required divalent cations either Mg2÷ or Mn 2÷ as Devor and Mudd [6] did with their investigation on spinach leaves. The enzyme of bean hypocotyl hooks could be inhibited like the enzyme of spinach leaves by sulfhydryl reagents. The result, that the enzyme phosphorylcholine-cytidyl transferase was localized in both microsomal and mitochondrial fractions whereas the enzyme CDP~holine:diglyceride phosphorylcholine transferase was only found in the microsomes may be interesting for the pathways of lecithin synthesis in this plant organ. The explanation that the mitochondrial fractions were contaminated by microsomal membranes is not acceptable because no CDP~holine:diglyceride phosphorylcholine transferase activity could be detected. In lipid exchange experiments [32] a cross-contamination of about 10% between microsomal and mitochondrial fractions is found, but this value is n o t high enough to explain the enzyme data presented in this paper. Taking into account that other reports showed a separation of the microsomes into ER and dictyosomal membranes [9] it could be possible that the Golgi dicytosomes usually having a higher density than ER might contaminate the mitochondrial-proplastid fractions. By assuming such a situation it is further possible to conclude that the microsomal fractions

396 are n o t h o m o g e n e o u s a n d t h a t t h e e n z y m e s are s e p a r a t e d in d i f f e r e n t m e m b r a n e s y s t e m s . T o a n s w e r this q u e s t i o n i n v e s t i g a t i o n s e m p l o y i n g o t h e r e n z y m e m a r k e r s a n d m o r e s o p h i s t i c a t e d s e p a r a t i o n t e c h n i q u e s h a v e t o be c a r r i e d o u t in f u t u r e e x p e r i m e n t s . ACKNOWLEDGEMENTS

This w o r k was supported by the Deutsche Forschungsgemeinschaft (Ha 865/9). W e wish to thank Dr. N. Grimsley (Leeds) for reading and correcting the manuscript. REFERENCES

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