Planta (1989) 178:19 24

Planta

9 Springer-Verlag 1989

Evidence for a plasma-membrane-bound nitrate reductase involved in nitrate uptake of Chlorella sorokiniana R. Tischner*, M.R. Ward**, and R.C. Huffaker *** Pflanzenphysiologisches Institut, Universit/it G6ttingen, Untere Karspiile 2, D-3400 G6ttingen, Federal Republic of Germany

Abstract. Anti-nitrate-reductase (NR) immunoglobulin-G (IgG) fragments inhibited nitrate uptake into ChIorella cells but had no affect on nitrite uptake. Intact anti-NR serum and preimmune IgG fragments had no affect on nitrate uptake. Membrane-associated NR was detected in plasma-membrane (PM) fractions isolated by aqueous twophase partitioning. The PM-associated NR was not removed by sonicating PM vesicles in 500 mM NaC1 and i mM ethylenediaminetetraacetic acid and represented up to 0.8% of the total Chlorella NR activity. The PM NR was solubilized by Triton X-100 and inactivated by ChIorella NR antiserum. Plasma-membrane NR was present in ammoniumgrown Chlorella cells that completely lacked soluble NR activity. The subunit sizes of the PM and soluble NRs were 60 and 95 kDa, respectively, as determined by sodium-dodecyl-sulfate electrophoresis and western blotting. Key words: Chlorella - Nitrate reductase (plasma membrane bound) - Nitrate uptake - Plasma membrane

Introduction Nitrate reductase (NR) and NO 3- transport have several characteristics in common. Induction of each requires NO~ (Jackson et al. 1973; Oaks and * To whom correspondence should be addressed Department of Genetics, Harvard Medical School and Deartment of Molecular Biology, Massachusetts General Hospital, Boston, MA 02114, USA *** P r e s e n t address: Plant Growth Laboratory, University of California, Davis, CA 95616, USA ** P r e s e n t address:

E D T A = ethylenediaminetetraacetic acid; F A D = flavine-adenine dinucleotide; IgG = immunoglobulin G; N R = nitrate reductase; PM = plasma membrane; TX-100 Triton X-100 Abbreviations:

Hirel 1985) and is inhibited by inhibitors of RNA and protein synthesis (Jackson et al. 1973; Klobus et al. 1988). Derepression of the NO~--uptake system in NH2-grown Chlorella cells in N-free medium requires protein synthesis (Knobloch 1987). Although the kinetics of NO 3 transport have been extensively characterized (Tischner and Lerenzen 1979; Ullrich 1983; Deane-Drummond 1984; Goyal and Huffaker 1986) an NOr-transport protein has not been identified from plant roots. The similarities of induction between NR and NO;- transport caused Butz and Jackson (1977) to propose that a membrane-associated NR could also function as a transporter for NO~-. Their proposal conflicted with the generally accepted assumption that NR is a soluble cytosolic enzyme in eukaryotic organisms (Oaks and Hirel 1985; Oelmuller et al, 1988). Several reports have suggested that a portion of the total NR was associated with the membranes of various organelles, microbodies (Lips and Avissar 1972), chloroplasts (Kamachi et al. 1987) and the pyrenoid of green algae (Lopez-Ruiz et al. 1985). Evidence for a plasma-membrane (PM)-bound NR in the diatom Thalassio sira was reported by Jones and Morel (1988). Recently, Ward et al. (1988a, b) reported finding an NR associated with a PM fraction isolated from barley and corn roots. In addition, Ward et al. (1988 b) found that fragments prepared from anti-soluble NR inhibited NO~ transport by barley roots. The purpose of this research was to determine if there is some unity to the above findings by comparing a more divergent species with those already investigated. We show that Chlorella cells also contain an NR associated with a PM fraction and fragments prepared from anti-soluble-NR immunoglobulin G specifically inhibit NO3 transport by Chtorella cells.

20

Material and methods Plant material. Chlorella sorokin&na (strain 211-8k of the algae collection of the Pflanzenphysiologisches Institut, University of G6ttingen, F R G ) was used. Algae were cultivated and synchronized as described by Tischner (1976). Antiserum preparation. Antiserum to Chlorella N R was prepared as described by Tischner (1984). Immunoglobulin G (IgG) was purified from the serum by protein A-Sepharose chromatography and hydrolyzed with papain (Lifter and Choi 1978). The cleaved IgG fragments were separated from papain by gel filtration on a Sephadex G-150 column, concentrated and used in the uptake experiments. Nitrate uptake. Cells were pretreated with intact anti-NR serum, preimmune IgG fragments or anti-NR IgG fragments (protein (IgG)/chlorophyll ratio =0.8) in darkness at 39 ~ C for 3 min prior to nitrate-uptake measurements. Nitrate uptake was determined as described by Tischner and Lorenzen (1979). Cells treated with anti-NR IgG fragments were washed three times with phosphate buffer and a post-wash uptake rate was also determined. Plasma-membrane isolation. Chlorella ceils were harvested and extracted as described by Tischner (1976). The extraction buffer (buffer 1) was 50 m M 2-amino-2-(hydroxymethyl)-l,3-propanediol-2(N-morpholino) ethanesulfonic acid (Tris-Mes; pH 7.8), 250 mM sorbitol, 23 m M ethylenediaminetetraacetic acid (EDTA), 1 gM sodium molybdate, 5 gM flavine-adenine dinucleotide (FAD), 1 mM phenylmethylsulfonyl fluoride (PMSF), 1 gM leupeptin, 2 m M dithiothreitol (DTT), 0.1% polyvinylpolypyrrolidone and 0.1% bovine serum albumin (BSA). The homogenate was centrifuged at 10000.g for 10 min. The supernatant was centrifuged at 100000.g to obtain a microsome and a soluble fraction. The microsome fraction was resuspended in 1 mM TrisMes (buffer 2) containing 250 mM sorbitol, 1 gM sodium molybdate, 5 gM F A D and partitioned on an aqueous polymer two-phase system as described by Larsson (1985) and Hodges and Mills (1986). The phase system contained 6.5% Dextran T 500, 6.5% polyethylene glycol, 0.33 M sucrose and 4 mM KC1. The contents of the tube were mixed by inversion 40-50 times, and centrifuged in a swinging-bucket rotor at 1200.g for 5 min. The upper phase was collected and partitioned on a fresh lower phase. This was repeated twice to remove all of the chlorophyll-containing material from the upper phase. The first lower phase and the final upper phase (U3) were collected, diluted with buffer 2 lacking sorbitol and centrifuged at 100000-g for 1 h. The pellets were resuspended and again centrifuged at 100000.g for 1 h. The final pellets were suspended in buffer 2 and used for enzyme analysis. Salt wash. The U3 pellet was resuspended in buffer 2 and brought to a final concentration of 500 mM NaC1 and 1 m M E D T A in 0.5 ml and placed on ice. After 20 min, the fractions were centrifuged at 100000-g for 1 h. Nitrate-reductase activities were determined in the resuspended pellet and soluble fractions.

R. Tischner et al, : Plasma-membrane-bound nitrate reductase described by Tischner (1976) except that 0.1% TX-100 (Triton/ protein ratio = 10) was included in the assay mix for the membrane fractions.

Electrophoresbs and blotting. Polyacrylamide gel electrophoresis in the presence of 10% sodium dodecyl sulfate (SDS) was performed according to Laemmli (1970). Electrophoretic transfer to nitrocellulose was performed as described by Clausen (1974). Peptide molecular weights were estimated with biotinylated molecular-weight markers (Biorad Laboratories, Richmond, Cal,, USA). After electrophoretic transfer, the nitrocellulose sheets were air-dried and then incubated with blocking solution consisting of 2 0 m M Tris-HC1 (pH 7.5), 500 mM NaCI and 1% BSA (buffer 3) for at least 1 h and then incubated in the same solution lacking BSA (buffer 4) containing anti-NR serum (1:1000) for 2 h. The blot was then washed two times for 30 rain in buffer 3 and once in buffer 4 containing 0.05% (v/v) Tween 20, polyoxyethylene sorbitan monolaurate. After rinsing in buffer 3, the blot was incubated with horseradish-peroxidase (HRP)-conjugated, goat anti-rabbit IgG, and HRP-avidin-conjugate, diluted 1 : 1000 in buffer 3 for 1 h. Anti-NR cross-reactive bands and molecular-weight markers were detected by development of the blot with 30 mg of 4-chloro-l-naphthol (in 10 ml of methanol) and 40 gl of 30% H202 in 100 m[ of buffer 3. Autoclaving the blots to destroy endogenous peroxidase activity did not affect the results (Rohringer and Holden 1985).

Results

Immunoglobulin-G fragments purified from Chlorella N R antiserum inhibited nitrate uptake by 95% but had no affect on nitrite uptake by Chlorella cells (Table 1). The inhibition of nitrate uptake by IgG fragments was only partially reversible; uptake recovered to about 50% of the control after two rinses in fresh uptake solution. Intact anti-NR serum and preimmune-serum IgG fragments did not affect nitrate uptake. Since the plasma membrane (PM) is the primary barrier of ions (and IgG fragments) to the cell cytosol, the inhibition of nitrate uptake by anti-NR IgG fragments indicated that NR or an antigenically related protein involved in nitrate transport

Table 1. Effect of anti-NR IgG fragments on nitrate and nitrite

uptake into intact Chlorella cells ( + SD) Treatment

Uptake rate (nmol. (rag chlorophyll)- t. min 1) Nitrate

Nitrite

64.3_+0.5 63.1 _+0.7 64.0 • 0.7 2.6+_0.4 29.3 _+0.3

47.0_+0.3 47.2 _+0.1 46.8 _+0.4 47.1 +_0,5 46.7 _+0.4

Inactivation of PM NR. The U3 pellet was solubilized with Triton X-100 (TX-100; octylphenoxypolyethoxyethanol) in buffer 2, incubated in the presence of anti-NR serum or preimmune serum for 2 h, after which N R activity was determined.

None Preimmune fragments Intact anti-NR serum Anti-NR IgG fragments Anti-NR IgG fragments wash"

Enzyme analysis. Marker enzymes were determined as described by Quail (1979). Nitrate-reductase activity was determined as

~ Cells were resuspended in fresh phosphate buffer twice after treatment with anti-NR IgG fragments

21

R. Tischner et al. : Plasma-membrane-bound nitrate reductase Table 2. Marker-enzyme activities in ChIorella cell fractions Marker enzyme a

Crude extract b

Inosinediphosphatase (Golgi) NADH-cytochrome-c-reductase (endoplasmic reticulum) Cytochrome-c-oxidase (mitochondria) Phosphoenol pyruvate carboxylase (cytoplasm) NADPH-glyceraldehyde-3-phosphate dehydrogenase (stroma) NO~- sensitive ATPase (tonoplast) VO] sensitive ATPase (plasmalemma)

0.059 1.05 0.1 0.8 60.8 0.2 0.3

Microsomal pellet

U3 Fraction

0.003 0.2 0.02 0.01 0.8 0.15 0.75

n.d. r 0.03 n.d. n.do n.d. n.d. 2.3

All activities are given in gmol substrate. (mg protein)- 1. min- 1 b 10000.g supernatant Not detected a

Table 3. Distribution of NR activity in Chlorella celt fractions Fraction

Vol Protein NR NR-total (ml) (mg.ml 1) specific activity b activity"

10000.g supernatant 20 100000.g supernatant 36 100000. g microsomal pellet 6 Lower phase d 8 Upper phase d 3

5.4 2.7 1.2 0.5 0.8

16.2 13.8 2.0 r 0.048 ~ 1.8 ~

1749 1341 14.4 c 0.192 c 4.32 ~

" gmol NOz. (mg protein)- 1. h 1 b p,mol NO2.ml -* c Activity determined after extraction with TX-100 a Phase partitioning carried out three times (Ua fraction)

was present in the P M . To d e t e r m i n e if N R was p r e s e n t in the P M a highly purified P M f r a c t i o n was isolated b y a q u e o u s t w o - p h a s e p a r t i t i o n i n g ( L a r s s o n 1985; Table 2). A threefold e n r i c h m e n t o f the P M - A T P a s e activity was detected in the U3 f r a c t i o n o v e r the m i c r o s o m e fraction. T h e P M A T P activity was increased f o u r to eightfold b y T X - 1 0 0 ( d a t a n o t shown) indicating t h a t the vesicles were sealed a n d right-side o u t ( R o b i n s o n et al. 1988). E v a l u a t i o n o f m a r k e r s for the G o l g i a p p a r a tus, e n d o p l a s m i c reticulum, m i t o c h o n d r i a , cytoplasm, chloroplast (stroma) and tonoplast demonstrated t h a t each o f these activities was significantly r e d u c e d in the U3 f r a c t i o n c o m p a r e d to the mic r o s o m e fraction. T h e m a r k e r - e n z y m e d a t a indicate t h a t the U3 f r a c t i o n consisted essentially o f PM. T h e distribution o f N R activity in Chlorella cell fractions is s h o w n in Table 3. M o s t o f the N R activity was soluble as is well established; however, a p p r o x . 0.8% o f the total N R activity (based u p o n the 1 0 0 0 0 - g s u p e r n a t a n t ) was detected in the m e m b r a n e fraction. This N R activity was n o t r e m o v e d

Table 4. Characterization of plasma-membrane-bound nitrate reductase Treatment PM fraction (U3) +0.5 M NaC1, 1 mM EDTA, ultrasonification pellet" supernatant" PM fraction (U3) + TX- 100 b before centrifugation + TX-100 u after centrifugation pellet supernatant PM fraction (U3) + TX-] 00 + preimmune serum + anti-NR serum

gmol NOy(mg protein) 1.h- 1

0.68 0.03 0.73 0.07 0.70 0.80 0.03

Activity after centrifugation at 105 .g and extraction with TX100 b TX-100/protein =0.85

f r o m the m e m b r a n e by several washes with low c o n c e n t r a t i o n s (1.0 m M ) o f buffer. To d e t e r m i n e the extent o f the association o f N R a n d the P M , P M vesicles were treated with 0.5 M NaC1, / m M E D T A , sonicated a n d then centrifuged (Table 4). T h e salt-chelate-sonication t r e a t m e n t did n o t r e m o v e N R f r o m the P M fraction, however, t r e a t m e n t with 0.1% T X - 1 0 0 (Trit o n / p r o t e i n r a t i o = 0 . 8 5 ) solubilized N R f r o m the P M (Table 4). T h e P M - a s s o c i a t e d N R was inactiv a t e d b y N R a n t i s e r u m but was n o t affected by p r e i m m u n e s e r u m (Table 4). P l a s m a - m e m b r a n e N R activity was reduced only 5 0 % in a m m o n i u m g r o w n Chlorella cells (Table 5). In contrast, soluble N R activity was n o t detected. To d e t e r m i n e the subunit structure o f the P M N R , P M a n d soluble fractions were s e p a r a t e d by s o d i u m dodecyl s u l f a t e - p o l y a c r y l a m i d e gel electro-

22

R. Tischner et al. : Plasma-membrane-bound nitrate reductase

Table 5. Nitrate-reductase activity in ammonium- and nitrategrown Chlorella cells

Plasmalemma-bound N R " Soluble N R (10 s g supernatant)

NH,~-grown cells

NO~-grown cells

0.40 n.d. b

0.75 14.8

" Activity determined after extraction with TX-100 as gmol N O ~ - ( m g protein) - 1 - h 1 u Not detected

Fig. 1. Western blot of Chlorella soluble and P M fractions. Soluble (aliquot from 100000-g centrifugation) and P M fractions (U3) were isolated, separated by SDS-PAGE, transferred to nitrocellulose and probed with N R antiserum as described in Material and methods. Lane 1 : P M N R ; lane 2: soluble N R

phoresis (SDS-PAGE) then electrophoretically transferred to nitrocellulose. The blot was probed with antiserum prepared to soluble Chlorella NR. A single 60-kDa subunit was detected in the PM fraction, whereas 95- and 60-kDa bands were detected in the soluble fraction (Fig. 1). No peptides of molecular weight greater than 60 kDa were detected in the PM fraction.

Discussion

Plasma-membrane-bound NR. Highly enriched PM fractions devoid of cytoplasmic contamination (Table 2) isolated from Chlorella microsome preparations contained 0.8% of the total N R activity (Table 3). It was particularly noteworthy that the PM fraction was devoid of cytoplasmic contamination, since N R is generally considered to be localized in the cytosol. Sonicating PM fractions with 500 m M NaC1 and 1 m M E D T A (Table 4) did not remove N R activity, indicating that N R was tightly associated with the PM. Plasma-membrane N R was solubilized by TX-100 (Triton/protein ratio = 0.85) similar to corn and barley root PM N R (Ward et al. 1988a, b). Characteristics of P M and soluble NRs. Both soluble N R and PM N R were inhibited by antibody raised against the soluble form, indicating that the two NRs are antigenically related. Although much is known about the soluble N R in Chlorella, little is known about the PM form. The soluble form is a complex protein containing F A D (Howard and Solomonson 1981, 1982; Solomonson and Barber 1986), cytochrome b557, and molybdenum (Solomonson et al. 1975). Soluble N R consists of four identical 95-kDa subunits (Howard and Solomonson 1981, 1982). The PM N R has a subunit size of 60 kDa (Fig. 1). Both 95- and 60-kDa bands were detected in western blots of the soluble fraction with the Chlorella N R antibody: Sotomonson and Barber (1986) have reported that the 60-kDa band in the soluble fraction is a breakdown product of the soluble N R that lacks the F A D portion of the protein moiety. The 60-kDa band may also represent PM N R that copurified with the soluble fraction. It is unlikely that the 60-kDa PM N R band is a breakdown product of the soluble form since as yet no evidence has been found for highermolecular-weight peptides in the PM fractions. The PM fractions were also devoid of cytoplasmic contamination (Table 2). Regulation of P M NR. The PM N R appears to be under a regulatory control that is different from that of the soluble form. Plasma-membrane N R is detected in Chlorella cells grown in the presence of ammonium whereas the soluble form is completely repressed (Table 5). Similarly, the diaphorase activity of putative PM N R in the diatom Thalassio sira was not repressed by ammonium in contrast to the soluble form (Jones and Morel 1988). Funkhouser and Ramadoss (1980) reported that ammonium-grown Chlorella cells contain proteins

R. Tischner et al.: Plasma-membrane-bound nitrate reductase that cross-react with a n t i - N R . Jones and M o r e l (1988) p r o p o s e d that these cross-reactive proteins m a y be constitutively synthesized N R subunits. Perhaps the a n t i - N R cross-reactive material detected in a m m o n i u m - g r o w n Chlorella cells was f r o m the P M N R .

Relationship of P M N R to nitrate uptake. A n t i - N R I g G fragments inhibited nitrate u p t a k e but h a d no affect on nitrite u p t a k e by Chlorella cells (Table 1). I n t a c t a n t i - N R molecules did n o t affect nitrate u p t a k e p r e s u m a b l y because they are m u c h larger (150 k D a ) t h a n the cleaved fragments (50 k D a ) a n d c o u l d n o t m o v e as easily t h r o u g h the ChlorelIa cell wall to bind to n i t r a t e - t r a n s p o r t sites (Jeanjean et al. 1984).

The inactivation of P M N R and the inhibition of nitrate uptake by anti-NR present the possibility that nitrate transport and the PM-associated N R may be related. Butz a n d J a c k s o n (1977) p r o p o s e d that N R m a y be a p a r t o f an e n z y m e complex that b o t h t r a n s p o r t s a n d reduces nitrate. Alternatively, nitrate t r a n s p o r t and nitrate reduction m a y be carried o u t by separate but antigenically related systems in the P M o f Chlorella cells. O t h e r possible functions have been p r o p o s e d for N A D H - o x i d i z i n g activities b o u n d to the m e m brane ( P a n t o j a a n d Willmer 1988; M o r r 6 e t a l . 1986) such as N R . These activities m a y f u n c t i o n to acidify the c y t o p l a s m ( M o r t 6 et al. 1986) or as an alternative p r o t o n p u m p to supply energy for active nutrient t r a n s p o r t (Crane et al. 1985). It has also been p r o p o s e d that t r a n s m e m b r a n e redox enzymes such as N R m a y transfer electrons to Fe 3 +, m a k i n g it available to plants (Campbell and Redinb a u g h 1984; Castignetti a n d Smarelli 1984). We thank Mrs. C. Rabe for her skillful technical assistance.

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R. Tischner et al. : Plasma-membrane-bound nitrate reductase Tischner, R., Lorenzen, H. (1979) Nitrate uptake and nitrate reduction in synchronous Chlorella. Planta 146, 287-292 Tischner, R. (1984) A comparison of the high active and low active form of nitrate reductase in synchronous Chlorella sorokiniana. Planta 160, I 5 Ullrich, W.R. (1983) Uptake and reduction of nitrate: Algae and fungi. In: Encyclopedia of plant physiology, N.S. vol. 15 A: Inorganic plant nutrition, pp. 376 397, L/iuchli, A., Bieleski, R.L., eds. Springer, Berlin Heidelberg New York London Paris Tokyo Ward, M.R., Grimes, H.D., Huffaker, R.C. (1989a) Latent nitrate reductase activity is associated with the plasma membrane of corn roots. Planta (in press) Ward, M.R., Tischner, R., Huffaker, R.C. (1989b) Inhibition of nitrate transport by anti-nitrate reductase IgG fragments and the identification of plasma membrane associated nitrate reductase in roots of barley seedlings. Plant Physiol. (in press)

Received 30 July; accepted 6 December 1988