The ferredoxin-thioredoxin system of a green alga,Chlamydomonas reinhardtii

Planta (1990)180:341-351 P l a n t a

�9 Springer-Verlag 1990

The ferredoxin-thioredoxin system of a green alga, Chlam ydomonas reinhardtii

Identification and characterization of thioredoxins and ferredoxin-thioredoxin reductase components

Heather C. Huppe 1 , , Fr6d6ric de Lamotte-Gu6ry 2, Jean-Pierre Jacquot 2, and Bob B. Buchanan 1 **

i Division of Molecular Plant Biology, Hilgard Hall, University of California, Berkeley, CA 94720, USA 2 Laboratoire de Physiologie V6g6tale Mol6culaire, B~itiment 430, Universitb de Paris Sud, F-91405 Orsay C6dex, France

Abstract. The components of the ferredoxin-thioredoxin (FT) system of Chlamydomonas reinhardtii have been purified and characterized. The system resembled that of higher plants in consisting of a ferredoxin-thioredoxin reductase (FTR) and two types of thioredoxin, a single f and two rn species, ml and m2. The Chlamydomonas m and f thioredoxins were antigenically similar to their higher-plant counterparts, but not to one another. The m thioredoxins were recognized by antibodies to both higher-plant m and bacterial thioredoxins, whereas the thioredoxin f was not. Chlamydomonas thioredoxin f reacted, although weakly, with the antibody to spinach thioredoxin f The algal thioredoxin f differed from thioredoxins studied previously in behaving as a basic protein on ion-exchange columns. Purification revealed that the algal thioredoxins had molecular masses (Mrs) typical of thioredoxins from other sources, ml and m2 being 10700 a n d f l 1 500. Chlamydomonas FTR had two dissimilar subunits, a feature common to all FTRs studied thus far. One, the 13-kDa ("similar") subunit, resembled its counterpart from other sources in both size and antigenicity. The other, 10-kDa ("variable") subunit was not recognized by antibodies to any FTR tested. When combined with spinach, (Spinacia oleracea L.) thylakoid membranes, the components of the FT system functioned in the light activation of the standard target enzymes from chloroplasts, corn (Zea mays L.) NADP-malate dehydrogenase (EC 1.1.1.82) and spinach fructose 1,6-bisphosphatase (EC 3.1.3.11) as well as the

*Present address: Laboratoire de Physiologie Vbg6tale Mol6culaire, Bfitiment 430, Universit6 de Paris Sud, F-91405 Orsay C+dex, France ** To whom correspondence should be addressed

Abbreviations: DEAE = diethylaminoethyl; ELISA = enzyme- linked immunosorption assay; FBPase=fructose 1,6-bisphosphatase; Fd = ferredoxin; FPLC = fast protein liquid chromatography; FTR=ferredoxin-thioredoxin reductase; FT system=ferredoxin- thioredoxin system; kDa = kilodaltons; M~ = relative molecular mass; NADP-MDH=NADP-malate dehydrogenase; SDS- PAGE = sodium dodecyl sulfate-polyacrylamide gel electrophoresis

chloroplast-type fructose 1,6-bisphosphatase from Chla- mydomonas. Activity was greatest if ferredoxin and other components of the FT system were from Chlamydomon- as. The capacity of the Chlamydomonas FT system to activate autologous FBPase indicates that light regulates the photosynthetic carbon metabolism of green algae as in other oxygenic photosynthetic organisms.

Key words: Chlamydomonas- Chlorophyta - Ferredox- in-thioredoxin reductase - Light and thioredoxin - Thioredoxin

Introduction

Light, absorbed by chlorophyll, functions in the post- translational regulation of enzymes associated with the photosynthetic conversion of carbon dioxide to carbohy- drates (see Buchanan 1980; Cs6ke and Buchanan 1986). One means by which light acts in this capacity is through the ferredoxin-thioredoxin (FT) system - which converts light to a sulfhydryl signal (reduced thioredoxin) by thylakoid membranes, ferredoxin (Fd), and the novel enzyme, ferredoxin-thioredoxin reductase (FTR) (Droux et al. 1987a-c). Reduced thioredoxin, in turn, reduces a disulfide site on each target enzyme, thereby converting the enzyme from inactive to active form, in the case of biosynthetic enzymes (Buchanan 1980; Jacquot 1984; Cs~ke and Buchanan 1989; Scheibe 1987; Ford et al. 1987; Holmgren 1985; Crawford et al. 1989 and references therein), and to an inactive form in the case of degradatory enzyme (glucose-6-phosphate dehydrogenase, 3.1.1.1.49) (Anderson and Avron 1976; Cossar et al. 1984). By this differential effect of light, starch synthesis and degradation can take place in a single com- partment (the chloroplast or the prokaryotic cell) with a minimum of futile cycling (for reviews, see Bassham 1979; Buchanan 1980).

Chloroplasts have two types of Fd-linked thioredoxins, m and f, designated according to their respective

342 H.C. Huppe et al. : The ferredoxin-thioredoxin system of Chlamydomonas

selective regulation of the target enzymes, NADP-malate dehydrogenase (NADP-MDH; EC 1.1.1.82) and fructose 1,6-bisphosphatase (FBPase; EC 3.1.3.11) (Jacquot et al. 1978; Wolosiuk et al. 1979; Crawford et al. 1986). The amino-acid sequences of the regulatory sites have recently been identified for several thioredoxin-linked enzymes: NADP-MDH (Decottignies etal. 1988); FBPase (Marcus et al. 1988; Raines et al. 1988); phosphoribulokinase (EC 2.7.1.19; Porter et al. 1988); and CF1-ATPase (EC 3.6.1.34; Miki et al. 1988). At present there is no correlation between the structure of the target site and its ability to interact preferentially with a partic- ular thioredoxin.

As a group, the m thioredoxins, with a relative molecular mass (Mr) of approx. 12 kilodaltons (kDa), are highly conserved among different photosynthetic organisms, resembling bacterial thioredoxins in their amino- acid sequence, activity and antigenic properties (Johnson etal. 1984; Crawford etal. 1986; Maeda et al. 1986; Kamo et al. 1989). Though less studied, thioredoxin f appears to represent a more heterogeneous group with reported Mrs ranging from 10.5 kDa (spinach: Crawford et al. 1986; Kamo et al. 1989) to 28 kDa (Scenedesmus obliquus: Langlotz et al. 1986a). Another large protein showing thioredoxin f activity (26 kDa) has been isolated from the cyanobacterium, Anabaena sp. 7119 (Whittaker and Gleason 1984). The presence of the thioredoxin active site (Cys-Gly-Pro-Cys) has been con- firmed for only one member of this group, the thioredoxinf f rom spinach (Tsugita et al. 1983; Kamo et al. 1989). Aside from the active site, however, spinach thioredoxin f shows little homology to either the m or bacterial-type thioredoxins (Kamo et al. 1989).

The eukaryotic algae represent a major group that has been relatively neglected with respect to studies of thioredoxins and their role in enzyme regulation. Certain features have been described in earlier work with other members of this group, the green alga Scenedesmus obliquus (Langlotz et al. 1986 a, b) and Chlorella pyrenoidosa (Tsang 1981), but it is not known whether the complete FT system is present or whether the properties of the thioredoxins described apply to other algae. Thus, it is not known how thioredoxin, recently shown to be essen- tial for the activity of a chloroplast-type FBPase from the green alga Chlamydomonas reinhardtii (Huppe and Buchanan 1989) is reduced. To gain information on these points, we have examined Chlamydomonas reinhardtii for the components in question and found evidence for the presence of a complete FT system. Certain properties of the system are described below.

Materials and methods

Plant material

Chlamydomonas reinhardtii (Levine) cc-124 mt-, subsequently called Chlamydomonas, was obtained from Dr. Elizabeth Harris, Duke University, Durham, N.C., USA and grown in 20-1 carboys as described by Huppe and Buchanan (1989). Ceils were harvested by continuous centrifugation and stored at - 8 0 ~ C.

Reagents, assay proteins and buffers

Reagents used in electrophoresis, Western blot, and the enzyme- linked immunosorption assay (ELISA) were purchased from Bio- Rad Laboratories (Richmond, Cal., USA). Diethylaminoethyl (DEAE) cellulose from Whatman (Clifton, N.J., USA), and all other column materials from Pharmacia LKB Biotechnology (Piscataway, N.J., USA). Spinach (Spinacia oleracea L.) FBPase was prepared according to the method of Nishizawa et al. (1982) and NADP-MDH according to Jacquot et al. (1981). Spinach-chloroplast thioredoxinsfand m were purified as previously (Crawford et al. 1986). The purified components of the spinach FT system were a gift from Dr. M. Droux, antibodies to spinach, corn (Zea mays L.) and Escherichia coli proteins were gifts from N. Crawford, and spinach-leaf thioredoxin h (Florencio et al. 1988) was provided by B. Yee (all of this laboratory). Dr. D. Le Master (Yale University New Haven, Conn., USA) provided the thioredoxin from E. coli. Other reagents and supplies were purchased from commercial sources and were of the highest quality available.

Buffers used were: A=30 mM 2-amino-2-(hydroxymethyl)-l,3-propanediol (Tris)- HC1 (pH 7.9), 3 mM MgCI2 ; B = 30 mM Tris-HCl (pH 8.3), 3 mM MgClz ; C = 50 mM sodium acetate (pH 5.5), 3 mM MgClz ; D = 50 mM sodium acetate (pH 4.8), 3 mM MgC12 ; E=50 mM 2(N-morpholino)ethanesulfonic acid (Mes) (pH 6.0), 3 mM MgC12 ; F = 50 mM Tris-HCl, pH 7.9

Initial purification procedures

Unless otherwise noted, previously described procedures were used for the preparation of buffers and for the dialysis, ultrafiltration and fast liquid protein chromatography (FPLC) (see Crawford et al. 1986). Centrifugations were at 15000.g for the indicated time.

I Initial separation of FT-system proteins

(1) Extraction and ammonium-sulfate fractionation. Approximately 200 g of frozen cells were thawed overnight and suspended in 600 ml of 30 mM Tris-HC1 containing 3 mM MgC12, pH 7.9 (Buffer A) and broken using a Biospec Products (Bartlesville, Okla., USA) Bead Beater (Huppe and Buchanan 1989). The sus- pension was filtered through Miracloth (Calbiochem, San Diego, Cal., USA) to remove beads and then centrifuged (20 min). The clarified solution was fractionated by precipitation with solid ammonium sulfate. The 45-85% precipitate, containing the thioredoxins, Fd and FTR, was resuspended and dialyzed against Buffer A.

(2) Sephadex G-IO0 filtration. The dialyzed sample was clarified by centrifugation (10 min), concentrated to 70 ml by ultrafiltration and applied to a Sephadex G-100 column (5 cm diameter, 90 cm long) which was developed with Buffer A supplemented with 200 mM NaC1. Thioredoxin f and m activities eluted together with Fd. These fractions were combined and dialyzed against Buffer A. As interfering activities prevented assaying FTR at this step, fractions containing proteins of the approximate M, of FTR (20- 50 kDa) were saved for further purification (see below).

(3) Diethylaminoethyl-cellulose chromatography (pH 7.9). The dialyzed fraction containing the thioredoxins and Fd was applied to a DEAE-cellulose column (2.5 cm diameter, 15 cm long) which had been equilibrated with Buffer A, and developed with a linear gradient of 0-500 mM NaC1 in Buffer A. Three thioredoxin activities were separated and recovered as follows: a single thioredoxin

H.C. Huppe et al. : The ferredoxin-thioredoxin system of Chlamydomonas 343

f(recovered in the pass-through) and two m thioredoxins (ml eluting in the pass-through and m2 at approx. 100 mM NaCI). Frac- tions containing each thioredoxin were combined and further purified separately. The Fd, which was separated from the other components on the DEAE column, was purified as described below (Step V. 1).

I I P u r i f i c a t i o n o f t h i o r e d o x i n s m l

Thioredoxin m! was purified as given below from the DE-52 pass- through fraction described above (Step 1.3).

(1) Diethylaminoethyl-cellulose chromatography (pH8.3). The non-adherent thioredoxin m (ml) was dialyzed against Buffer B (the same solution as Buffer A except the pH was 8.3). At this higher pH the protein adhered to DEAE, eluting at approx. 50 mM NaCI in Buffer B.

(2) Heat. The active fractions from the above column were combined, heated with frequent mixing to 70 ~ C, and then immediately transferred to an ice-water bath. The cooled sample was centrifuged (15 min) to remove denatured protein.

(3) S-Sepharose fast-flow chromatography (pH 5.5). The thioredoxin ml sample was dialyzed against 50 mM sodium acetate containing 3 mM MgC12 (pH 5.5) (Buffer C) and applied to an S- Sepharose Fast-Flow column (1.5 cm diameter, 10 cm long). The column was eluted with a 0-300 mM NaC1 gradient in Buffer C. The activity eluted early in the gradient as a broad peak.

(4) S-Sepharose fast-flow chromatography (pH 4.8). The active fractions from above were combined and dialyzed against Buffer D (the same solution as Buffer C except the pH was 4.8). The sample was applied to the above column equilibrated with Buffer D and eluted with a 0-300 mM NaC1 gradient in Buffer D. The thioredoxin m! activity was recovered as a single tight peak from this column.

(5) Fast protein liquid Mono Q chromatography (pH 8.3). Final purification of thioredoxin ml was accomplished by FPLC using a Mono Q column equilibrated with Buffer B and a flow rate of 1 ml/min. After loading and washing with three volumes of buffer, the column was eluted with an NaC1 concentration gradient (0-300 mM NaCI, increasing 10 mM/ml in Buffer B). This was done at room temperature and the samples were collected in tubes in an ice-water bath.

I I I P u r i f i c a t i o n o f t h i o r e d o x i n m 2

The thioredoxin retained on DE-52 (m2) was purified from the fraction eluting at 100 mM NaC1 described above in Step 1.3.

(1) Sephadex G-75 filtration. The adherent thioredoxin m (m2) was concentrated by ultrafiltration to 6 ml and applied to a Sepha- dex G-75 column (2.5 cm diameter, 90 em long) which was equilibrated and developed with Buffer A containing 200 mM NaC1. Active fractions were pooled and dialyzed against Buffer D.

(2) S-Sepharose fast-flow chromatography (pH 4.8). After centrifugation (10 min) to remove the precipitated material, the dialyzed sample was applied to an S-Sepharose column (1.5 em diameter, 10 cm long), equilibrated with Buffer D and eluted with a linear gradient from 0-300 mM NaC1 in Buffer D. Active fractions were pooled and dialyzed against Buffer A.

(3) Q-Sepharose fast-flow chromatography (pH 7.9). The dialyzed fraction from Step 2 was applied to a Q-Sepharose column (1.5 em diameter, 5 cm long), equilibrated with Buffer A, and eluted with

a 0-300 mM gradient of NaC1 in Buffer A. The thioredoxin eluted as a single peak with a small shoulder. Active fractions were combined and dialyzed against Buffer B.

(4) Fast protein liquid Mono Q chromatography (pH 8.3). The dialyzed sample from above was applied to a Mono Q column equilibrated with Buffer B. The rate and gradient were as described above (II.5). This column allowed a more complete separation of thioredoxin m2 from a minor contaminant still present after Step 111.3.

IV P u r i f i c a t i o n o f t h i o r e d o x i n f

Thioredoxinfwas purified from the DE-52 pass-through described in Step L3 above.

(1) S-Sepharose fast-flow chromatography (pH 6.0). The DEAE- cellulose column fractions containing thioredoxin f activity were pooled and dialyzed against 50 mM 2(N-morpholino)ethanesulfonic acid (Mes) (pH 6.0) containing 3 mM MgC12 (Buffer E). This sample was applied to an S-Sepharose column (1.5 cm diameter, 15 cm long), which had been equilibrated with Buffer E, and was eluted with a 0-500 mM NaC1 gradient in Buffer E. Thioredoxin fe lu ted at approx. 150 mM NaC1.

(2) Heat. The active fractions from the previous column were pooled, concentrated by ultrafiltration, heated, and clarified as described for thioredoxin m! (Step 11.2).

(3) S-Sepharose fast-flow chromatography (pH 5.5). The heat- treated sample was dialyzed against Buffer C and applied to an S-Sepharose column (1.5 cm diameter, 5 cm long) equilibrated with the same buffer. The active fractions from this column were pooled. At this stage, the sample still contained an orange pigment and required further fractionation. Based on its absorption spectrum, the pigment in question was a c-type cytochrome (ct peak at 550 nm).

(4) Phenyl-Sepharose chromatograph),. The sample was divided and one half was diluted with an equal volume of Buffer A which was 60% saturated with ammonium sulfate (the diluted sample was 30% saturated). This portion was applied to a phenyl-Sephar- ose column (1 cm diameter, 7 cm long) previously equilibrated with Buffer A that was 30% saturated with ammonium sulfate. The column was washed and then eluted with a gradient of decreasing ammonium-sulfate saturation (30% to 0%). The cytochrome pigment passed through this column and the thioredoxin was retained. Because ammonium sulfate interferes with thioredoxin-linked activation of FBPase, the entire gradient was pooled, concentrated by ultrafiltration and dialyzed against Buffer A before measurable thioredoxin f activity was found. This step was repeated with the second half of the sample, and the activities from both columns were combined and dialyzed against Buffer C.

(5) S-Sepharose fast-flow chromatography (p H 5.5 ). The dialyzed thioredoxinfsample was applied to an S-Sepharose column (1 cm diameter, 7 cm long) which was equilibrated and eluted with Buffer C. Thioredoxin f was eluted on this column as a single protein peak. These fractions were pooled, dialyzed against Buffer A, and stored at - 2 0 ~ C.

V P u r i f i c a t i o n o f F T R

This enzyme was purified from the Sephadex G-100 fractions described in Step L2 above.

(1) Diethylaminoethyl-cellulose chromatography (pH 7.9). Frac- tions from the Sephadex G-100 column corresponding to an Mr


of 30-50 kDa, were combined and dialyzed against 50 mM Tris- HCI, pH 7.9 (Buffer F). The sample was applied to a DEAE-cellulose column (2.5 cm diameter, 15 cm long) equilibrated with Buffer F and eluted with a gradient of 0-300 mM NaC1 in this buffer. The colorimetric orthophosphate (Pi) assay for FTR, in which FBPase serves as the target enzyme, revealed a small shoulder of specific activity trailing a large non-specific phosphatase peak. NADPH-oxidase activity prevented confirmation of the presence of FTR with the NADP-MDH assay at this stage. Fractions showing FTR activity in the FBPase assay were combined, concentrated by ultrafiltration and dialyzed against Buffer C.

(2) Ferredoxin-Sepharose 4B affinity and Mono Q chromatography. The dialyzed FTR sample was divided into three aliquots for application to a Chlamydomonas Fd-Sepharose 4B affinity column (0.5 cm diameter, 5 cm long) (see below). Following application of each part, the column was washed with five volumes of Buffer C and then eluted with a 0-200 mM NaC1 gradient in Buffer C to remove a single protein peak containing both FTR and ferredoxin-NADP reductase activities. Ferredoxin-thioredoxin reductase eluted at approx. 200 mM NaC1, free of interfering oxidase and phosphatase activities. The non-adherent fractions from all three profiles were combined and then reapplied to the column which was developed as before. The fractions showing FTR activity were combined, concentrated by ultrafiltration, and dialyzed against Buffer A. At this stage, FTR showed insignificant background activity and appeared to be about 50% pure upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Purifica- tion to homogeneity was accomplished for part of the sample using the FPLC Mono Q step (II.5) as described for thioredoxin ml above except that Buffer F was used. As more than half of the activity was lost at this step, most assays were carried out with the 50%-pure sample obtained from the ferredoxin affinity column.

VI Other Chlamydomonas proteins

Chlamydomonas Fd fractions from the DEAE-cellulose column (Step L3) were further purified according to Schmitter et al. (1988). The final sample showed a single protein band in SDS-PAGE. Chlamydomonas FBPase was partially purified according to Huppe and Buchanan (1989). The enzyme fraction contained insignificant background activities and, thus, was sufficiently pure for our studies.

Preparation of Chlamydomonas Fd affinity column

A Chlamydomonas Fd affinity column was prepared as outlined in the Pharmacia Fine Chemicals manual (1979) using approx. 45 mg of purified Fd and 5 ml of swollen CNBr-activated Sephar- o s e .

Results

Identification of thioredoxins. When cell-free extracts from Chlamydomonas were chromatographed on DEAE cellulose at pH 7.9, we observed two thioredoxin m peaks, m! and m2, and one thioredoxin f peak (Fig. 1). Thioredoxin ml and thioredoxin f did not adhere to the column at pH 7.9, thus differing from a variety of plant thioredoxins previously studied (Yee et al. 1981; Hutcheson and Buchanan 1983; Crawford et al. 1986). When the pH of the column was raised to 8.3, thioredoxin ml adhered but thioredoxinfdid not. The thioredoxin fwas retained on cation-exchange columns up to pH 7.5.

Chlamydomonas thioredoxins showed specificities similar to those of their higher-plant counterparts (Table 1). That is, neither of the m algal thioredoxins sufficient to activate corn NADP-MDH activated spinach FBPase. Likewise, the thioredoxinfspecifically activated the FBPase. Thus, despite the unusual behavior of Chla-

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Fig. 1. Separation of different thioredoxins from Chlamydomonas reinhardtii on DEAE cellulose (pH 7.9). Thioredoxin m andfactivi- ties reflect the increase in the activity of corn NADP-MDH and spinach FBPase, respectively, upon incubation with 100 lal of the designated fraction

Analytical procedures

Protein estimations, SDS-PAGE and Western blotting were as described by Huppe and Buchanan (1989) unless indicated otherwise. Enzyme-linked immunosorption assays (ELISA) were as described in Crawford et al. (1986).

Assays

Previously described procedures were used for the assay of thioredoxins m andf (Crawford et al. 1986) and for FTR using FBPase or NADP-MDH as target enzymes (Droux et al. 1987b). Unless stated otherwise, Fd and FTR present in the assays were from the same source.

Table 1. Specificity of Chlamydomonas thioredoxins for target enzymes

Thioredoxins a NADP-MDH FBPase (Ixmol NADPH (gmol Pi �9 min 1.m1-1) "min-l-m1-1)

Chlamydomonas ml 1.41 0.01 Chlamydomonas m2 2.32 0.01 Chlamydomonas f 0.08 2.94

" Each thioredoxin was assayed following separation on DEAE cellulose and further fractionation on Sephadex G-100. The amounts of thioredoxin used were in the range in which activity was proportional to thioredoxin added


mydomonas ml a n d f o n ion-exchange columns, the algal thioredoxins showed classical specificity for target enzymes - a specificity that was retained throughout their purification.

Purification of the thioredoxins. As shown in Tables 2 and 3, approx. 45% of the thioredoxin m activity (i.e. ml) adhered to DEAE cellulose. The ratio of pass- through (m2) to retention (ml) thioredoxin varied among different preparations, but a physiological basis for this variation was not evident. Typical of most thioredoxins, both thioredoxin m! and m2 were stable to heat. A heat step was used in the ml preparation, but was not necessary for the purification of m2. Both m-

type thioredoxins were also stable to acidic pH at the initial stage of purification. In later steps of purification, however, the two proteins showed differing stabilities to acidic pH. Thioredoxin ml fractions showed a de- crease in specific activity when adjusted to pH 4.8 prior to chromatography on S-Sepharose (Table 2), whereas the specific activity of thioredoxin m2 was markedly en- hanced by this treatment (Table 3). Final purification of both thioredoxin ml and m2 was achieved using FPLC Mono Q.

Like its counterpart from higher plants (Wolosiuk et al. 1979; Hutcheson and Buchanan 1983; Crawford et al. 1986), Chlamydomonas thioredoxin f required MgC12 for stabilization. Even with M g C I E , the sample

Table 2. Purification of Chlamydomonas thioredoxin ml

Treatment Total protein Total activity Specific acitivity Recovery Purification (mg) (U) a (U/mg) a (%) (fold)

Ammonium-sulfate purification 2 772 G-100 gel filtration 763 DEAE-cellulose (pH 7.9) ion exchange 51.3 Q-Sepharose (pH 8.3) ion exchange 26.5 70 ~ C heat treatment 21.9 S-Sepharose (pH = 5.5) ion exchange 6.7 S-Sepharose (pH 4.8) ion exchange 1.5 FPLC mono Q (pH 8.5) ion exchange 0.39

33.6 0.01 - 1 88.6 0.20 100 16 40.1 0.78 45 b 65 26.6 0.97 17 81 26.2 1.19 17 99 21 3.13 14 260

3.5 2.30 2.3 191 1.7 4.36 1.1 363

" U = Ixmol NADPH oxidized' min- 1 b Thioredoxin m activity was separated into m! and m2 at this step

Table 3. Purification of Chlamydomonas thioredoxin m2

Treatment Total protein Total activity Specific activity Recovery Purification (mg) (U) a (U/mg) a (%) (fold)

Ammonium-sulfate purification 2 772 G-100 gel filtration 763 DEAE-cellulose (pH 7.9) ion exchange 345 G-75 gel filtration 66.1 S-Sepharose (pH 4.8) ion exchange 7.8 Q-Sepharose (pH 7.9) ion exchange 4.0 FPLC mono Q (pH 8.3) ion exchange 0.15

33.6 0.01 - 1 88.6 0.2 100 9.6 53.0 0.15 60 b 12.5 47.0 0.71 53 59.3 34.0 4.38 39 365 15.3 3.83 7.3 318 3.5 23.1 4.0 1920

a U = ~tmol NADPH oxidized- min- 1 b Thioredoxin m activity was separated into m! and m2 at this step

Table 4. Purification of Chlamydomonas thioredoxin f

Treatment Total protein Total activity Specific activity Recovery Purification (mg) (U)" (U/mg) a (%) (fold)

Ammonium-sulfate purification 2 772 G-100 gel filtration 763 DE-52 ion exchange 89.6 S-Sepharose (pH = 6.0) ion exchange 46.4 70 ~ C heat treatment 31.9 S-Sepharose (pH = 5.5) ion exchange 21.1 Phenyl-Sepharose 2.7 S-Sepharose (pH = 6.0) ion exchange 0.63

4095 1.5 1 7739 10.1 100 6.7 5 562 62.1 72 41.4 3010 64.9 39 43.3 3 097 97.1 40 64.7 1429 67.7 18.5 45

257 95.3 3.3 63.5 477 757 6.2 505

a U = ~tmol FBP hydrolyzed.min


lost activity during purification (Table 4) especially at the phenyl-Sepharose step that removed a persistent c- type cytochrome contaminant of similar size. Once purified, the Chlamydomonas th ioredoxinfsamples lost sub- stantial activity when stored at - 2 0 ~ (50% loss in several weeks). Similar to its spinach counterpart (Pla and Lopez-Gorge 1981 ; Buc et al. 1984; Crawford et al. 1986), purified Chlamydomonas thioredoxin f formed a complex with spinach FBPase attached to an affinity column, eluting between 0 and 50 mM NaC1 (data not shown).

Estimation of molecular mass o f f and m thioredoxins. The behavior of purified Chlamydomonas m andf th io re - doxins on Sephadex G-100 was similar to that of the spinach counterparts, indicative of a molecular mass of approx. 12 kDa (data not shown). Because an atypically large (28 kDa) thioredoxin has been reported for Scene- desmus (Langlotz et al. 1986a), we consistently assayed for t h io redox in fand m activities throughout the molecular-sieving columns. However, no large thioredoxins were detected in Chlamydomonas preparations. The Chlamydomonas ml , m2 andf th ioredoxins showed similar Mrs on SDS-polyacrylamide gels (approx. 11 kDa) (Fig. 2). A gel system designed to be more accurate in the low-molecular-weight region (Hashimoto et al. 1983) showed that ml and m2 ran at approx. 10700 whereas thioredoxin f ran slightly larger, approx. 11 500 (data not shown).

Immunological properties of Chlamydomonas thioredoxins. We tested the immunological similarity of the algal thioredoxins to each other and to other thioredoxins by Western-blot analysis. The antibodies to corn thioredoxin m recognized a single protein band in both mixed and partially purified samples of Chlamydomonas m l and m2 thioredoxins, indicating that both proteins are antigenically related to their higher-plant counterparts (Fig. 3). Since a single band was recognized both before and after separation on DEAE cellulose, these results

Chlamy Fig. 3. Demonstration of antigenic similarity among thioredoxins by Western-blot analysis. Approximately 1 gg each of purified E. coil (Ec) thioredoxin, spinach thioredoxin rn (Sm) and spinach thioredoxinf(SJ) was used. The m-type thioredoxins from Chlamy- domonas (Chlamy) and spinach and the thioredoxin from E. coli were probed with antiserum to corn thioredoxin m. Chlamydomon- as thioredoxin m was identified in a crude extract (Crude) and thioredoxins ml and m2 were identified after separation on DEAE cellulose. The amounts of Chlamydomonas thioredoxin m used were: crude, 100 Ixg partially purified thioredoxin ml and m2 fractions, activity equivalent to 1 Ixg of spinach thioredoxin m. For Chlamydomonas thioredoxinf, a highly purified preparation showing five times the activity of 1 gg spinach protein was used. The antisera to corn thioredoxin m and spinach f were diluted 1 : 2500 and 1 : 1500, respectively

also confirm that thioredoxin ml and m2 have the same molecular mass. Antibodies to spinach th ioredoxinfrec- ognized highly purified Chlamydomonas thioredoxin f ; the reaction, however, was weak (Fig. 3).

Fig. 2. Photograph of SDS-PAGE separation demonstrating the size similarity among thioredoxins ml, m2 and f from Chlamydomonas, thioredoxins f, h and m from spinach, and E. coli thioredoxin. The lanes contained approx. 3 gg of each protein


The antigenic similarity of the different thioredoxins was determined by ELISA. Figure 4 shows that thioredoxins m/ and m2 reacted to the same extent with the antibodies to spinach thioredoxin m and E. eoli thioredoxin. The strongest reaction was with the antibody to the spinach m. The antibody to E. coli thioredoxin

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/ n L ~ - - A ~ ., Chlamydomonas f o

10 100 1000

Thioredoxin (ng) Fig. 4. Immunological cross reactions of Chlamydomonasf, m! and m2, spinach m and E. coli thioredoxins with antisera to spinach m and E. coli thioredoxins. Proteins tested were homogeneous. Both antisera were diluted 1:2000

f

= 0 . ~ .

1.0

reacted more with algal ml and m2 than with the spinach m.

When tested with an antibody to spinach thioredoxin f, the thioredoxin f from Chlamydomonas reacted, but as expected from prior experience with Western blots (see above), the reaction was weak (Fig. 5). That the reaction was specific was indicated by the finding that Chlamydomonas thioredoxin f did not react with antibodies to either spinach thioredoxin m or to E. coli thioredoxin (Fig. 4). Studies o f f thioredoxins from several higher-plant sources also showed little reaction with antibodies to m-type thioredoxins, while they showed the expected reaction with the antibody against spinach thioredoxinf(Hutcheson and Buchanan 1983; Crawford et al. 1986).

Purification of FTR from Chlamydomonas. As noted above, it was not possible to detect FTR in crude Chla- mydomonas preparations because of interfering oxidase and phosphatase activities. Therefore, during the initial purification on Sephadex G-100 and DEAE cellulose, fractions were combined based on the size of native FTR from other sources (approx. 30000 kDa) (Droux et al. 1987b). Sephadex G-100 fractions of the Chlamydomon- as preparation corresponding to an Mr between 20 and 50 kDa were combined and applied to DEAE cellulose - a step that partially separated specific (light-activated) FBPase activity from non-specific phosphatase.

As FTR forms a stable complex with Fd, an Fd affinity column has been used as a key step in purification of FTR from several sources (Hutcheson and Buchanan 1983; Droux et al. 1987b). Early results indicated that Chlamydomonas FTR adheres poorly to spinach Fd columns, thus prompting use ofa Chlamydomonas Fd affinity column. Chlamydomonas FTR adhered to this column and eluted at approx. 200 mM NaC1, free of interfering activities. Because of activity loss at the final purification step (Mono Q), the fraction purified through the preceding Fd affinity column was generally used for activity assays.

0,8

.7. ~ 0.6 ~ Spinach f

< 0.4

0.2 ~ ~.AChlamydomonasf

0 / . - A. �9 ~. ~ Chlamydomonas ml & m2 i f , . ~ ~ i ~ ~ - - i �9

0 10 100 1000

Thioredoxin (ng)

Fig. 5. Immunological cross reactions of spinach f and Chlamydo- monas f, ml, and m2 thioredoxins with antiserum to spinach f thioredoxin. Proteins tested were homogeneous and antiserum was diluted 1 : 1000

General properties of the Chlamydomonas FTR. As with the enzyme from other sources (Droux et al. 1987b), Chlamydomonas FTR was yellow-brown and showed a visible absorption peak at 410 nm and a protein peak at 278 nm (ratio of A41o :Azvs =0.42) (Fig. 6). Chlamy- domonas FTR consisted of two dissimilar subunits which showed Mrs of 13 kDa and 10 kDa upon SDS-PAGE (Fig. 7). All FTRs studied thus far also have a 13-kDa "similar" subunit and a second "variable" subunit which differs in size with each species (Droux et al. 1987 b). The 13-kDa similar subunit contains a catalytically active thiol group (Droux et al. 1987a, c), but the function of the variable subunit is not known.

In previous studies only the 13-kDa subunits were found to show immunological cross reactivity with the FTRs from corn, spinach and Nostoc (Droux et al. 1987 b). The antibody to corn FTR, which had previously been found to react well with the similar subunits


t- O r~ 3 ~D

0.50 I I I

278nm

0"I:)5 ~ 4 ~ O n m

0.00 I I I 250 345 430 515 600

Wovelength,nm

Fig. 6. Absorption spectrum of Chlamydomonas FTR. The spectrum was measured in 30 mM Tris-HC1 buffer, pH 7.9

Fig. 8. Western-blot analysis of FTR from Nostoc (N) (5 p.g), Chla- mydomonas (C) (15 ~tg) and spinach (S) (5 Ixg) using antisera to corn FTR (diluted 1 : 1000) and spinach FTR (diluted 1 : 1 500)

Table 5. Activation of target enzymes of components of the ferredoxin-thioredoxin system purified from Chlamydomonas

Corn Spinach Chlamydomonas NADPH-MDH" FBPase b FBPase b

Light Complete 4.1 703 823 -ferredoxin 0.3 473 335 -thioredoxin 0.2 182 275 -FTR 0.3 158 267 -enzyme 0.3 95 98

Dark Complete 0.2 73 183

a tzmol NADPH oxidized- rain- 1. (mg Chl)- 1 b lamol FBP hydrolyzed.min- 1. (mg Chl)-

Fig. 7. Comparison of FTR from Chlamydomonas (C), Nostoe (N), and spinach (S) on SDS-PAGE. Lanes contained 3-5 ~tg of each protein. Molecular masses of proteins standards (Stds) are indicated on the left

f rom both spinach and Nostoc and to recognize both subunits of the corn enzyme (Droux etal . 1987b), reacted with the 13-kDa subunit in Chlamydomonas (Fig. 8). An ant ibody made to spinach F T R enriched in the 13-kDa subunit gave a similar response (Fig. 8). Antibody to Nostoc F T R reacted very weakly with the Chlamydomonas 13-kDa subunit (data not shown). None of the antibodies recognized the smaller (variable) subunit of Chlamydomonas.

Light-activation studies. Once interfering phosphatase and oxidase activities were removed, Chlarnydomonas F T R effectively activated corn N A D P - M D H and spin-

ach FBPase in the presence of photoreduced Fd and thioredoxins rn and f , respectively (Table 5). The chloroplast-type FBPase f rom Chlamydomonas was also acti- viated by this system and required the same components as the spinach enzyme. The data demonstrate that Chla- mydomonas has a complete FT system that functions in the activation of chloroplast FBPase, a key regulatory enzyme of photosynthetic carbon assimilation. The green algae, like higher plants, can thus use the FT system to link light to the regulation of photosynthetic carbon flow.

Specificity of the Fd-FTR interaction. As mentioned above, Chlamydomonas F T R adhered better to a Chla- mydomonas than to a spinach Fd affinity column. To determine whether this behavior was a reflection of differential Fd affinity, light-activation assays were carried out using varying amounts of each of the two Fds. The max imum activity (apparent Vmax) attained did not differ significantly between the two Fds; however, to reach


Table 6. Ferredoxin specificity of Chlamydomonas FTR a

Ferredoxin NADP-MDH activity

Apparent Vmax (ktmo1 NADPH ox" min- x. (mg Chl) - 1)

Apparent Km (nmol Fd)

Chlamydomonas 3.0 0.21 Spinach 2.6 3.33

a FTR was assayed by its ability to reduce thioredoxin m that is monitored by the NADP-MDH assay

half-maximal activity required at least 10-fold more spinach than Chlamydomonas Fd - i.e. the apparent Km for spinach Fd was at least 10 times that of Chlamydo- monas Fd (Table 6). These data imply that there is a structural difference between the two Fds which affects their interaction with Chlamydomonas FTR.

Discussion

We have purified and characterized components of the FT system of a green alga, Chlamydomonas reinhardtii, and found them to resemble those of higher plants and cyanobacteria. The algal FT system consisted of an FTR and two types of thioredoxins, m and f, in addition to Fd. Together, these components functioned in the light activation of both of the standard target enzymes from chloroplasts of higher plants, corn NADP-MDH and spinach FBPase, and, importantly, the chloroplast-type FBPase from Chlamydomonas. The capacity of the Chla- mydomonas FT system to activate an autologous target enzyme supports the idea that in green algae, as in other oxygenic photosynthetic organisms, light regulates photosynthetic carbon assimilation by way of the FT system.

Three thioredoxins were identified, a single f and two m species, m! and m2. The behavior of the algal thioredoxins f and ml on ion-exchange columns indicates that these proteins are more positively charged than thioredoxins studied previously. On the basis of both Western-blot and ELISA analyses, the Chlamydo- monas thioredoxins m and f were antigenically related to their higher-plant counterparts. As is the case with higher-plant thioredoxins, the f and m thioredoxins of Chlamydomonas were antigenically unrelated. The relatively weak reaction of Chlamydomonas thioredoxin f with the antibody to its spinach counterpart indicates that the f-type thioredoxins are a more heterogeneous group than the m-type proteins. Further characterization o f f thioredoxins will be of interest especially since they appear to have a novel evolutionary history (Buchanan et al. 1989). In addition, it becomes timely to document the intracellular location of the thioredoxins identified in this study.

The algal thioredoxins had Mrs similar to those of thioredoxins from other sources; ml and m2 were the same size, 10.7 kDa, whereas thioredoxin f w a s slightly

larger, 11.5 kDa. Although typical of thioredoxins, the Mr of the Chlamydomonas thioredoxinfdiffers consider- ably from that found for the 28- and 26-kDa proteins showing thioredoxin f activity isolated, respectively, from Scenedesmus (Langlotz et al. 1986a) and Anabaena (Whittaker and Gleason 1984). Further investigation of the Scenedesmus and Chlamydomonasfthioredoxins will be valuable in determining the structural similarities between these two proteins which, despite a great difference in apparent size, are both able to activate chloroplast FBPase. Such a comparison may also yield information relevant to the mechanism of ribonucleotide re- duction in green algae - a reaction previously concluded to be linked to thioredoxin (Wagner and Follmann 1977).

The FTR from Chlamydomonas resembled the enzyme from other sources in having two dissimilar subunits, one 13 kDa and the other 10 kDa. The 13-kDa subunit resembled the corresponding (similar) subunit of spinach, corn and Nostoc FTR both in size and antigenicity. The 10-kDa variable subunit resembled the corresponding subunit from cyanobacteria in size, but was not recognized by antibodies to this or the other FTRs tested.

Chlamydomonas FTR formed an electrostatic complex with Fd bound to Sepharose affinity columns, ad- hering better to Chlamydomonas Fd than to spinach Fd. This difference in binding was also reflected in the 10- fold greater affinity for the autologous Fd when Chlamy- domonas FTR functioned in the light activation of NADP-MDH. These data imply that there is a structural difference between the two Fds which affects their interaction with Chlamydomonas FTR. At present we are unable to identify the pertinent domain on the basis of primary-structure analysis.

The observed difference in affinity of Chlamydomon- as FTR for the two Fds was unexpected for two reasons. First, it was reported earlier that the FTRs from both Nostoc and spinach showed similar activity with the op- posing Fds of both sources (Yee et al. 1981). The current study demonstrates that the amount of Fd used in those studies (7 nM) was probably above that required for maximum activity. Second, spinach and Chlamydomonas Fds showed behavior in NADP + photoreduction by spinach ferredoxin-NADP reductase opposite to that seen here - i.e. a similar apparent Km but a different V . . . . with the algal Fd showing lower maximal activity (Schmitter et al. 1988). This contrasting behavior is ex- plained by differences in the nature of the Fd-FTR and Fd-ferredoxin-NADP reductase complexes deduced from chemically modified Fd (Hirasawa et al. 1988).

In conclusion, the FT system in the green alga, Chla- mydomonas reinhardtii resembles its counterpart in higher plants. The components show differences which may prove useful in understanding the structural and functional relationships of the proteins as well as their interaction with each other. The availability of mutants of Chlamydomonas make it a good organism to investigate further how the FT system functions in the metabolic adaptation of plants to changes in their environment.


This work was supported in part by a grant from the National Aeronautics and Space Administration. We would like to thank Don Carlson and Jacqueline Girard for their assistance with cell cultures.

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Received 24 July; accepted 9 October 1989

The ferredoxin-thioredoxin system of a green alga,Chlamydomonas reinhardtii

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