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Ferredoxin-dependent glutamate synthase moonlights in plant sulfolipid biosynthesis by forming a complex with SQD1
Archives of Biochemistry and Biophysics 436 (2005) 206–214 www.elsevier.com/locate/yabbi
Ferredoxin-dependent glutamate synthase moonlights in plant sulfolipid biosynthesis by forming a complex with SQD1 Mie Shimojima, Susanne HoVmann-Benning, R. Michael Garavito, Christoph Benning ¤ Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, MI 48824 1319, USA Received 20 January 2005, and in revised form 20 January 2005
Abstract UDP-sulfoquinovose synthase, SQD1, catalyzes the transfer of sulWte to UDP-glucose giving rise to UDP-sulfoquinovose, which is the head group donor for the biosynthesis of the plant sulfolipid sulfoquinovosyldiacylglyerol. The native SQD1 enzyme of spinach exists as a 250 kDa heteroprotein complex with much higher aYnity for the substrate sulWte than the recombinant SQD1 protein itself. The SQD1 protein co-puriWed with nine proteins. Likely binding partners included rubisco activase, HSP70, and ferredoxindependent glutamate synthase (FdGOGAT). While the Wrst two proteins are known to interact with many other proteins, the identiWcation of FdGOGAT was most intriguing because this 160 kDa protein contains an FMN cofactor known to bind sulWte in vitro. Using diVerent constructs expressing recombinant forms of the multidomain protein FdGOGAT, it was demonstrated that the FMN-binding domain of FdGOGAT is essential for speciWc binding of the protein to SQD1. A model suggests that FdGOGAT could channel sulWte to SQD1. 2005 Elsevier Inc. All rights reserved. Keywords: Chloroplast lipid; Glycolipid; Sugar nucleotide; SulWte; Sulfolipid; Sulfoquinovosyldiacylglycerol; Thylakoid lipid; UDP-sulfoquinovose; UDP-glucose
The plant sulfolipid sulfoquinovosyldiacylglycerol is common to most photosynthetic organisms where it is an intricate part of the photosynthetic membrane [1,2]. In plants and bacteria, sulfolipid is essential under phosphate-limiting growth conditions as a substitute for phosphatidylglycerol [3]. For the basic reactions of sulfolipid biosynthesis in plants two proteins are required, SQD1 and SQD2 [4,5]. The SQD1 protein catalyzes the transfer of sulWte to UDP-Glc giving rise to UDP-sulfoquinovose [6], which serves as the sulfoquinovose head group donor. Sulfoquinovose is transferred to diacylglycerol by SQD2, the sulfolipid synthase. SulWte is derived from the sulfate assimilation pathway in plants inside chloroplasts and is generally considered toxic to the cell [7,8]. Its concentration is very low in plant ¤
Corresponding author. Fax: +1 517 353 9334. E-mail address: [email protected] (C. Benning).
0003-9861/$ - see front matter 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.abb.2005.02.005
tissues, i.e., in the picomole per milligram fresh weight range [9]. The turnover number for the transfer of sulWte onto UDP-Glc for the recombinant SQD1 enzyme was found to be very low [6]. Moreover, the crystal structure of the SQD1 dimer of Arabidopsis has been solved and shows a peculiar loop protruding from the otherwise globular monomer, which might facilitate the interaction with other proteins [10]. Overall the protein is similar in structure to other sugar nucleotide modifying enzymes such as UDP-Glc epimerases. It contains a tightly bound NAD+ cofactor which participates in the reaction as hydride acceptor [10,11]. Due to the low turnover number of the recombinant SQD1 protein we hypothesized that the native SQD1 protein exists in vivo as a complex with other proteins to facilitate the transfer of sulWte to the reaction center of SQD1. To test this hypothesis, we partially puriWed the native SQD1 complex from spinach leaves during a previous study [12]. The estimated molecular
M. Shimojima et al. / Archives of Biochemistry and Biophysics 436 (2005) 206–214
mass for the native SQD1 complex was »250 kDa, or »160 kDa in excess of the molecular mass expected for an SQD1 dimer (»90 kDa) and in disagreement with a native tetramer structure. The complex was active and its KM for sulWte was considerably lower than that for the recombinant spinach SQD1 protein [12]. Here, we describe the detailed analysis of the highly puriWed native spinach SQD1 complex, the identiWcation of its components and their interaction in vitro.
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dards (Bio-Rad), and visualized by Coomassie brilliant blue R-250 staining or silver staining (Silver Stain Plus, Bio-Rad). Immuno-blot analysis for SQD1 was done as previously described using a polyclonal antibody raised against Arabidopsis SQD1 [12]. Strep-Tactin alkaline phosphatase (Strep-Tactin AP, IBA, St. Louis, MO, USA) was used to detect recombinant FdGOGAT according to the manufacturer’s instructions. In-gel protein digest and separation of the peptides by reverse-phase HPLC
Materials and methods PuriWcation of the native SQD1 complex from spinach leaves Locally purchased spinach (Spinacia oleracea L.) was used. The extraction and initial puriWcation was performed at 4 °C as previously described [12]. Following ion-exchange chromatography (Macro-Prep HighQ, Bio-Rad, Hercules, CA, USA), the peak fractions were combined, dialyzed against 0.1 M NaCl, 50 mM Tris– HCl, pH 7.5, 10% glycerol, and loaded onto a column (1.2 cm diameter) Wlled with 10 ml of CM AY-Gel Blue Gel (Bio-Rad) equilibrated with 0.1 M NaCl, 50 mM Tris–HCl, pH 7.5, and 10% glycerol. The proteins were eluted by gravity with a linear gradient of 0.15–1.0 M NaCl in the buVer mentioned above. Fractions of 5 ml each were collected. The peak fractions (24 ml total) were concentrated using Centriprep 30 (Millipore, Bedford, MA, USA) to 1.2 ml, which was loaded onto Superdex 200 (FPLC, Amersham Bioscience, Piscataway, NJ, USA). Fractions of 0.3 ml each were collected. Native PAGE analysis All steps were carried out at room temperature. For native PAGE we used Ready Gel Tris–HCl (4–15% linear gradient, Bio-Rad) with a running buVer of 25 mM Tris, pH 8.3, 192 mM glycine. The two peak fractions obtained from CM AY-Gel Blue (7.8 ml) were concentrated with Centriprep 30 and Ultrafree 30 (Millipore) to 120 l and 7.5 l/lane were loaded after mixing with 2£ loading buVer (62.5 mM Tris–HCl, pH 6.8, 40% glycerol, and 0.01% bromophenol blue). Electrophoresis was accomplished at constant 35 V for 17 h. Gel slices containing proteins were macerated with a pestle in a microtube and the proteins were extracted with 20 mM Tris–HCl, pH 8.0, 1% SDS by shaking at 37 °C for 16 h. Proteins were precipitated by adding 4-volumes of acetone and incubation at ¡80 °C for 1 h. SDS–PAGE and Western blot analysis Proteins were separated by SDS–PAGE as described by Laemmli [13], sized using low molecular mass stan-
In-gel digests were performed essentially according to the method of Shevchenko et al. [14]. Tryptic digests were obtained using 13 ng/l trypsin (Promega, Madison, WI, USA) in 100 mM ammonium bicarbonate (pH 8.0) at 37 °C for 16 h. Digestion with endoproteinase Lys-C (Roche, Indianapolis, IN, USA) was performed using 25 ng/l endoproteinase in 25 mM Tris–HCl, pH 8.5, 1 mM EDTA at 37 °C for 16 h. The digested peptides were then separated using reverse-phase HPLC and the N-terminal sequences were analyzed at the Michigan State University Molecular Structure Sequencing and Support Facility. Isolation of a spinach cDNA for FdGOGAT A 32P-labeled 1615-bp fragment (isolated by PCR using the crude cDNA library as template and primers: 5⬘-GCG AAT TCT ATG TTG CGT CTG AGG TG-3⬘; 5⬘-GAT TGG ATC CTC ACC ACC TTC-3⬘) corresponding to the center part of the spinach SoFdGOGAT (GenBank AF061515) was used as a probe to screen a ZAPII spinach leaf cDNA library (Stratagene, La Jolla, CA, USA). Screening was done as previously described [12] but at a hybridization temperature of 65 °C. Two cDNA clones, 6-1 and 1-1 were isolated. Clone 6-1 covers 310 bp of the 5⬘-untranslated region in addition to bases 1–2819 of the published sequence for spinach FdGOGAT (AF061515) [15]. Clone 1-1 covers bp 1733–5000 of the published cDNA sequence. Together, these two clones represent a full-length cDNA with an open reading of 4554 nucleotide frame identical to that published (AF061515). As these two partial clones overlapped, a central BamHI site was used to splice the two sequences in subsequent constructs. Protein expression of SoFdGOGAT fragments in Escherichia coli The four domains of SoFdGOGAT were predicted based on comparison to the amino acid sequence of the cyanobacterial FdGOGAT from Synechocystis sp. PCC6803, for which the crystal structure is known [16]. The three sequences inserted in the expression vector pASK-IBA3 (IBA) were as follows: Full (1–4551 bp,
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AF061515), M2D (1170–3600 bp, AF061515), and L2D (2277–4551 bp, AF061515). In all cases the respective fragments were ampliWed from cDNA clones 6-1 and 1-1, spliced together via a central BamHI site and inserted into the EcoRI and KpnI sites of the vector. Primer pairs and templates were as follows for each of the three constructs as indicated: Full, 6-1 template 5⬘-GCG AAT TCT GTG GTG TTG GAT TTA TTG C-3⬘ and 5⬘GAT TGG ATC CTC ACC ACC TTC-3⬘, 1-1 template 5⬘-GTG AGG ATC CAA TCA GAT GGC-3⬘ and 5⬘GCG GTA CCT GCA GAC TGT AAA CTG GC-3⬘; M2D, 6-1 template 5⬘-GCG AAT TCT ATG TTG CGT CTG AGG TG-3⬘ and 5⬘-GAT TGG ATC CTC ACC ACC TTC-3⬘, 1-1 template 5⬘-GTG AGG ATC CAA TCA GAT GGC-3⬘ and 5⬘-GCG GTA CCA TAG CTT AGA TCA AGA TG-3⬘; LD2, 6-1 template 5⬘-GCG AAT TCA GTG TCT CTA AGA TGG GTG-3⬘ and 5⬘GAT TGG ATC CTC ACC ACC TTC-3⬘, 1-1 template 5⬘-GTG AGG ATC CAA TCA GAT GGC-3⬘ and 5⬘GCG GTA CCT GCA GAC TGT AAA CTG GC-3⬘. The three recombinant proteins were expressed with a Strep-tag (WSHPQFDK) in E. coli XL1-Blue (Stratagene). A 15 ml overnight culture was used to inoculate 500 ml LB with 100 g/ml ampicillin and the culture was incubated at 37 °C under shaking. When the absorption (550 nm) reached 0.5, anhydrotetracycline was added at 0.2 g/ml to induce the protein. The cells were collected after 3 h by centrifugation at 4500g for 10 min. The pellets were stored at ¡80 °C. The cell pellets were homogenized with 2 mg/ml lysozyme (Roche) in buVer A (20 mM potassium phosphate buVer, pH 7.5, 1 mM EDTA, 10 mM -mercaptoethanol, 1 mM glutamine, 1 mM 2-oxoglutarate, and 0.3 M NaCl), incubated on ice for 30 min, and centrifuged at 10,000g for 15 min. The supernatant was Wltered with a 0.45 m Wlter (Millipore). The recombinant protein was puriWed according to the manufacturer’s instructions on 1 ml Strep-Tactin Sepharose per 500 ml culture except that buVer A was used. The proteins were eluted with 2.5 mM desthiobiotin in the buVer A, 1 ml fractions were collected, and were stored at ¡20 °C in 10% glycerol until use. Protein concentrations were assayed throughout using the DC Protein Assay Kit from Bio-Rad.
Results PuriWcation of the SQD1 complex and identiWcation of proteins interacting with SQD1 The SQD1 complex was puriWed from the stroma fraction of spinach chloroplasts using ion-exchange chromatography (High Q), aYnity chromatography (Blue Sepharose), and size exclusion chromatography (Superdex 200) as previously published [12] and described above. The enrichment of the complex was followed throughout by immunodetection of SQD1 on protein blots. SuYcient amount of protein was puriWed for visualization on a silver-stained SDS–PAGE gel (Fig. 1). Any proteins associated with SQD1 should coelute with a similar elution proWle. The SQD1 protein detected by immuno-blotting (Fig. 1A) was predominantly present in fraction 39 of the Superdex 200 column. The corresponding protein band was detected in the silver-stained gel as well (Fig. 1B). Co-eluting with SQD1 in lane 39 (Fig. 1B) were nine other proteins with molecular masses of »160 kDa (a), »97 kDa (b), »75 kDa
Protein–protein interaction assay PuriWed fragments of FdGOGAT were concentrated to eliminate desthiobiotin by centrifugation using Centriprep 30 spin columns (Millipore). SQD1 (54 g in 250 l) and FdGOGAT proteins (37 g in 500 l) were incubated at room temperature for 30 min followed by puriWcation using a 1 ml Strep-Tactin Sepharose column. SQD1 protein was present at approximately Wvefold molar excess. The 300 l of the peak fraction were concentrated by acetone precipitation and subjected to SDS–PAGE and immuno-blot analysis.
Fig. 1. Superdex 200 column elution proWle for the spinach SQD1 complex. (A) Immuno-blot analysis using a polyclonal antibody raised against SQD1 from Arabidopsis. (B) Silver-stained gel after SDS– PAGE. Nine proteins labeled a–i were present in fraction 39. Molecular sizes of markers are indicated to the left of the gel. (C) Enlargement of the regions of the gel in B containing the »160 kDa protein (a) and the SQD1 protein as indicated.
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(c), »70 kDa (d), »60 kDa (e), »55 kDa (f), »46 kDa (g), »43 kDa (h), and »40 kDa, (i). The »160 kDa protein migrated close to a much more prominent protein, but slightly slower and an enlargement of this portion of the gel is shown in Fig. 1C. Of all the proteins present in fraction 39, only this »160 kDa protein showed the same elution proWle as SQD1 consistent with a protein present in the SQD1 complex. Moreover, its estimated molecular mass of »160 kDa added up to »250 kDa, when present in a 1-to-2 ratio with SQD1 (»90 kDa for the dimer) in a protein complex. Alternative puriWcation of the SQD1 complex by native PAGE To Wnd independent corroborating evidence for the »160 kDa protein as a possible component of the SQD1 complex, the eluate containing SQD1 after aYnity chromatography with Blue Sepharose was concentrated and separated by native PAGE (Fig. 2A). Staining of the gel with Coomassie brilliant blue (Fig. 2A, lane I) revealed at least seven proteins or protein complexes as marked. However, immuno-blotting detected the SQD1 protein exclusively in one complex (Fig. 2A). Proteins/complexes 1–7 were excised from the native gel and reanalyzed by denaturing PAGE as shown in Fig. 2B. The only protein other than SQD1 which was exclusively present in gel slice three was again a protein with a molecular mass of »160 kDa. Two other proteins were present in this native gel slice (Fig. 2A, 3), but they were present in other slices of the native gel as well, which would not be expected for speciWc components of the SQD1 complex. The proteins had estimated molecular masses of »75 and »43 kDa, respectively. Because, proteins with comparable masses were also present in the eluate of the Superdex 200 column (Fig. 1B, c and h), we analyzed these further in parallel to the more likely candidate protein with a molecular mass of »160 kDa. IdentiWcation of the »160, »75, and »43 kDa proteins The proteins were digested directly in the gel slices using either trypsin or lysylendopeptidase and the peptides were separated by capillary HPLC and N-ter-
Fig. 2. Analysis of the spinach SQD1 complex by native PAGE. (A) Native PAGE of the partially puriWed spinach SQD1 complex. Lane I, gel stained with Coomassie brilliant blue. Seven prominent proteins/ complexes were present as numbered. Lane II, immuno-blot analysis of the gel in lane I using the polyclonal antiserum against SQD1 from Arabidopsis. (B) Silver-stained gel after SDS–PAGE of proteins/complexes 1–7 isolated from the native gel in (A). Numbers on top of the gel correspond to the numbers assigned to proteins/complexes in (A). Molecular masses of size markers are indicated on the left of the gel.
minally sequenced. The amino acid sequences of the peptides are shown in Table 1. Fortunately, all three protein sequences were known and the proteins were unambiguously identiWed. The »160 kDa protein was spinach ferredoxin-dependent glutamate synthase (Fd-GOGAT). Its sequences and the location of the peptides are shown in Fig. 3. The »75 kDa protein was spinach HSP70, and the »43 kDa protein was spinach rubisco activase. Of
Table 1 N-terminal amino acid sequences of peptides and identiWcation of the proteins Proteins (kDa)
Peptides
Amino acid sequences of N-terminus
Descriptions (Accession No.)
»160
5-1 5-2 9-1 9-2 10
LLENDTILR AQVFKQA(I)DVL RLENFGFIQFRP WRPLTDVVDGYS KQ[V/E][L/V]H[T/M]F[Y/L][P/V]X[L/A]
Ferredoxin-dependent glutamate synthase (Q43155)
»75
8
(K)AVVTVPAYFN
70 kDa heat shock-related protein (Q08080)
»43
7 9
RVPIIVTGNDFS YDIDNMLGDFYI
Rubisco activase (AAD13841)
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Fig. 3. Amino acid sequence for spinach ferredoxin-dependent glutamate synthase. Four domains (1–389, amidotransferase domain; 390–758, central domain; 759–1200, FMN-binding domain; 1201–1517, C-terminal domain) as underlined with alternating solid and broken lines were predicted from the alignment of spinach FdGOGAT with the ortholog from Synechocystis sp. PCC6803, for which the crystal structure is known. The Wve peptide sequences derived by peptide sequencing from the »160 kDa protein are indicated by boxes.
the three, HSP70 and rubisco activase are known to readily interact with other proteins and could have been co-puriWed with the SQD1 complex for this reason. As the »160 kDa protein was the prime candidate based on the analysis described above, we focused on FdGOGAT as complex partner of SQD1. Furthermore, this particular glutamate synthase isoform is present in the stroma of chloroplasts [17], and is therefore resident in the same cellular compartment as SQD1 [4,12]. Equally relevant is the fact that FdGOGAT has a Xavin mononucleotide cofactor (FMN)1 in its active site, which has been shown to react with sulWte [18,19]. Being able to bind sulWte, FdGOGAT in a complex with the sulWte-utilizing enzyme SQD1 seemed reasonable, and led to the hypothesis (see below) that FdGOGAT assists SQD1 in vivo by providing sulWte to SQD1. Expression of recombinant spinach FdGOGAT fragments in E. coli To probe the interaction between FdGOGAT and SQD1 and to reconstitute the complex in vitro, we used His-tagged SoSQD1 recombinant protein [12] and produced the Strep-tagged full-length protein and frag1
Abbreviation used: FMN, Xavin mononucleotide.
ments of spinach FdGOGAT in E. coli. The crystal structure of Fd-GOGAT from Synechocystis sp. PCC6803 is known [16] and this protein consists of four distinct globular domains, the amidotransferase domain, the central domain, the FMN-binding domain, and the C-terminal domain. Given that the two FdGOGAT orthologous amino acid sequences from spinach and the cyanobacterium share 58% identical or 72% similar residues, we assumed that their structures must be similar as well and deduced the diVerent domains for spinach FdGOGAT as indicated in Fig. 3. Based on the cyanobacterial domain structure and taking into account that sulWte binds to the FMN cofactor in the FMN-binding domain presumably making it the most critical domain to interact with SQD1, we constructed three diVerent plasmids expressing the full-length (Full), the FMNbinding and central domains (M2D), and the FMNbinding and C-terminal domains (L2D) as shown in Fig. 4A. The recombinant proteins were tagged at the C-terminus with a Strep-tag permitting the puriWcation on Strep-Tactin Sepharose columns, which bind the Strep-tag. Extracts from the three respective E. coli strains, which were partially puriWed on the Strep-Tactin Sepharose columns are shown in Fig. 4B. The Coomassie brilliant blue-staining and visualization using Strep-Tactin AP conjugate revealed in each case proteins of the
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Fig. 4. Recombinant protein fragments of spinach FdGOGAT. (A) Schematic view of plasmid constructs delineating the diVerent FdGOGAT domains and the Strep-tag. Full, contains all of the four domains (1–1517 aa), M2D the middle 2 domains (390–1200 aa), and L2D the last 2 domains (759–1517 aa). (B) SDS–PAGE of Strep-Tactin Sepharose puriWed proteins. Lanes designated 1, Coomassie brilliant blue stained SDS–PAGE gel. Lanes designated 2, protein blot of the same gel incubated with Strep-Tactin alkaline phosphatase conjugate to detect proteins with Strep-tag. Proteins of the expected molecular masses »160, »89, and »83 kDa, respectively, are indicated.
correct size. In addition, truncated products were present, which were particularly abundant in the extract from the E. coli strain harboring the full-length construct (Fig. 4B). Interaction of recombinant FdGOGAT and SQD1 To demonstrate the interaction of FdGOGAT and SQD1 in vitro, FdGOGAT fragments and SQD1 were partially puriWed by aYnity chromatography based on their respective tags and were mixed in the test tube. Reisolation of the FdGOGAT fragments on Strep-Tactin Sepharose columns (pull-down assay) should, therefore, lead to the co-puriWcation of SQD1, if SQD1 and FdGOGAT form a complex in vitro. Proteins eluting from the Strep-Tactin Sepharose columns were separated by SDS–PAGE and analyzed for FdGOGAT fragments on protein blots using the Strep-Tactin AP conjugate or for SQD1 using a polyclonal SQD1-speciWc antibody. For control purposes the two respective proteins were individually analyzed prior to re-isolation on the Strep-Tactin Sepharose columns. As shown in Fig. 5A, the SQD1 protein by itself does not bind to the Strep-Tactin Sepharose column. When the interaction of the M2D fragment (the central and the FMN-binding domains) of FdGOGAT and SQD1 was tested (Fig. 5B) it was striking that the presence of SQD1 prevented the re-isolation of the FdGOGAT fragment (Fig. 5B, left panel third lane). In addition, SQD1 was not recovered from the mixture following re-isolation of the complex. As will be discussed further below, the direct contact between
Fig. 5. Reconstitution of diVerent spinach FdGOGAT/SQD1 complexes in vitro. (A) Lack of binding of SQD1 to Strep-Tactin Sepharose in the absence of FdGOGAT Strep-tagged proteins. (B–D) Interaction of SQD1 with M2D, Full, and L2D, respectively. Anti SQD1, immunoblot using the polyclonal antiserum against SQD1 from Arabidopsis; Strep-Tactin AP, protein blot incubated with Strep-Tactin alkaline phosphatase conjugate to detect the FdGOGAT fragments with Streptag; control, individual FdGOGAT or SQD1 proteins as indicated by +/¡; pull-down, mixtures of SQD1 and FdGOGAT fragments or FdGOGAT fragments alone as indicated by +/¡ following re-puriWcation on Strep-Tactin Sepharose columns. Expected diagnostic protein bands are indicated to the right of each gel. For additional details and rationale refer to the text.
SQD1 and the FMN-binding domain of FdGOGAT prevents the Strep-tag from interacting with the column in this particular construct. Therefore, this result indi-
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rectly conWrmed the interaction between SQD1 and FdGOGAT. Using full-length FDGOGAT (Fig. 5C) SQD1 was detected following the re-isolation of the complex. This result was consistent with a direct interaction between FdGOGAT and SQD1. The L2D fragment containing the FMN-binding and C-terminal domains was bound to the Strep-Tactin Sepharose column irrespective of the presence of SQD1 (unlike M2D), and SQD1 did not co-purify with this fragment of FdGOGAT (Fig. 5D). Taken together, the results of the pull-down assay presented in Fig. 5 suggest that SQD1 forms a complex with FdGOGAT in vitro and speciWcally binds to the FMN-binding and central domains of FdGOGAT.
Discussion The native SQD1 protein from spinach was previously puriWed as a »250 kDa complex and showed altered substrate binding for sulWte compared to the recombinant SQD1 dimer of »90 kDa [12]. In essence the Km for sulWte decreased from 20 to less than 5 M and the sulWte concentration versus velocity Michaelis– Menten plot changed from hyperbolic for the recombinant SQD1 dimer to sigmoidal for the native complex [12]. In general, this eVect has been attributed to cooperativity among the individual subunits of a given enzyme complex. Because of these earlier observations, we expected to Wnd a protein or proteins in association with SQD1, which could aVect the sulWte availability for the SQD1 reaction center. Initially, it was surprising that ferredoxin-dependent glutamate synthase (FdGOGAT) emerged as the protein partner in the native SQD1 complex. However, the evidence was striking: (1) During the
isolation of the native SQD1 complex, of the diVerent proteins present in SQD1-containing fractions only a single protein of »160 kDa molecular mass equivalent to the molecular mass of FdGOGAT closely co-puriWed with the SQD1 protein using two independent procedures (Figs. 1 and 2). (2) Multiple peptide sequences derived from the »160 kDa protein were identical to the corresponding sequence published for spinach FdGOGAT (Table 1 and Fig. 3), a »160 kDa protein. (3) Two dimers of SQD1 and a monomer of FdGOGAT give rise to a complex of »250 kDa, the same molecular mass as determined for the native SQD1 complex [12]. (4) The two proteins FdGOGAT and SQD1 are present in the same subcellular compartment, the chloroplast stroma [4,12,17]. (5) Direct interaction between SQD1 and FdGOGAT was demonstrated (Fig. 5). (6) A plausible role for FdGOGAT in a complex can be derived from the fact that the FMN cofactor of FdGOGAT reversibly binds sulWte [18,19]. While this property of some Xavoproteins has been primarily used in vitro to probe the molecular environment of the Xavin cofactor, the discovery of FdGOGAT in the native SQD1 complex hints at a possible role for sulWte binding by the FMN cofactor of FdGOGAT in vivo. Based on the available data, it is proposed that the FMN-binding domain of FdGOGAT mediates the delivery of sulWte to the reaction center of SQD1. For this process to occur, one would expect that SQD1 is bound close to the FMN-binding domain of FdGOGAT in a way that exchange of sulWte between the two proteins is facilitated. This hypothesis was testable, because the crystal structures for SQD1 from Arabidopsis [10] and for FdGOGAT from Synechococcus sp. PCC6803 [16] were available. The two proteins were suYciently similar to the respective spinach orthologs that a meaningful
Fig. 6. Model of the FdGOGAT/SQD1 complex. The model depicts the published crystal structures for FdGOGAT from Synechococcus sp. PCC6803 (PDB 1OFE) and Arabidopsis SQD1 (PDB 1QRR). The green and blue domains are the FMN-domain and central domain, respectively. The glutamate amidotransferase domain is shown in magenta and the C-terminal domain in orange. The arrow indicates the position where the FMN-domain was truncated in the M2D construct and the Strep-tag was attached. SQD1 domains are light grey in color with UDP-Glc shown in cyan and NAD+ in red. (A) Left side view, (B) front view, (C) right side view.
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FdGOGAT/SQD1 complex could be modeled as shown in Fig. 6. The SQD1 dimer structure has two overlapping, protruding loops, which neatly Wt into a groove on top of the central domain such that two globular SQD1 monomers are positioned adjacent to the FMN-binding and central domains of FdGOGAT, respectively. This arrangement is experimentally supported by the in vitro binding between the recombinant SQD1 protein and the three diVerent fragments of FdGOGAT (Fig. 5), which showed that the FMN-binding and central domains of FdGOGAT are critical for the SQD1 binding. The equivalent position of the Strep-tag in the M2D fragment is indicated in Fig. 6 (arrow). Incubation of the M2D fragment with SQD1 prevented M2D binding to the Strep-Tactin Sepharose column (Fig. 5B). Based on the model (Fig. 6) this observation is readily understandable, because the Strep-tag would be close to the binding surface between SQD1 and the FMN-binding domain of FdGOGAT preventing it from interacting with the Strep-Tactin Sepharose column. Finally the model also shows that SQD1 is oriented in such a way that its predicted substrate channel (Wlled with water molecules in the structure) is pointed at the FMN cofactor in the FMN-binding domain of FdGOGAT. Thus, the hypothesis that FdGOGAT is involved in providing sulWte to the SQD1 reaction center is supported by the model and the available data. The amount of SQD1 protein in chloroplasts was estimated to be less than 1% (7 g/mg chlorophyll) [12], while FdGOGAT represents up to 1% of the total leaf protein [20,21]. Given the excess of FdGOGAT, one would expect that most of the SQD1 protein is bound to FdGOGAT. Indeed, throughout the puriWcation of the native SQD1 protein complex using diVerent chromatographic techniques, we never observed diVerent forms of SQD1 than the »250 kDa complex. These data and observations suggest that SQD1 exists primarily bound to FdGOGAT in vivo.
Conclusions The analysis of the »250 kDa native SQD1 complex from spinach led to the identiWcation of FdGOGAT as the second component. This large multidomain protein is normally thought of as a key enzyme of nitrogen metabolism [17] or photorespiration [22] in plants. Therefore, it was initially surprising to Wnd FdGOGAT moonlighting in sulfolipid biosynthesis as auxiliary protein for SQD1. However, in view of the reversible binding of sulWte by the FMN cofactor of FdGOGAT, the biochemical properties of the native SQD1 complex and the structural model of the FdGOGAT/SQD1 interaction such a moonlighting function for FdGOGAT makes sense. Finding proteins with known biochemical functions participating in unexpected reactions or pro-
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cesses is increasingly being recognized as a widespread phenomenon in diVerent model organisms [23,24]. The native FdGOGAT/SQD1 complex adds just another example. Plants coordinate their sulfate and nitrate assimilation [25,26]. As SQD1 and FdGOGAT are involved in S- and N-metabolism, respectively, one might wonder whether their interaction also provides opportunities for the coordinated regulation between these two essential processes. This possibility remains to be explored.
Acknowledgments We thank Joseph Leykam and Stacy Trzos (Michigan State University Molecular Structure Sequencing and Support Facility) for protein sequencing and Todd Lydic for technical support. Financial support was provided in parts by a grant from the US National Science Foundation (MCB-0109912).
References [1] C. Benning, Annu. Rev. Plant Physiol. Plant Mol. Biol. 49 (1998) 53–75. [2] C. Benning, in: P.A. Siegenthaler, N. Murata (Eds.), Lipids in Photosynthesis: Structure Function and Genetics, Kluwer Academic Publishers, Boston, 1998, pp. 83–101. [3] M. Frentzen, Curr. Opin. Plant Biol. 7 (2004) 270–276. [4] B. Essigmann, S. Güler, R.A. Narang, D. Linke, C. Benning, Proc. Natl. Acad. Sci. USA 95 (1998) 1950–1955. [5] B. Yu, C. Xu, C. Benning, Proc. Natl. Acad. Sci. USA 99 (2002) 5732–5737. [6] S. Sanda, T. Leustek, M. Theisen, M. Garavito, C. Benning, J. Biol. Chem. 276 (2001) 3941–3946. [7] R. Hell, Planta 202 (1997) 138–148. [8] T. Leustek, K. Saito, Plant Physiol. 120 (1999) 637–644. [9] G. Tsakraklides, M. Martin, R. Chalam, M.C. Tarczynski, A. Schmidt, T. Leustek, Plant J. 32 (2002) 879–889. [10] A.M. Mulichak, M.J. Theisen, B. Essigmann, C. Benning, R.M. Garavito, Proc. Natl. Acad. Sci. USA 96 (1999) 13097–13102. [11] B. Essigmann, B.M. Hespenheide, L.A. Kuhn, C. Benning, Arch. Biochem. Biophys. 369 (1999) 30–41. [12] M. Shimojima, C. Benning, Arch. Biochem. Biophys. 413 (2003) 123–130. [13] U.K. Laemmli, Nature 227 (1970) 680–685. [14] A. Shevchenko, M. Wilm, O. Vorm, M. Mann, Anal. Chem. 68 (1996) 850–858. [15] H.B. Dincturk, D.B. KnaV, Mol. Biol. Rep. 27 (2000) 141–148. [16] R.H. van den Heuvel, D. Ferrari, R.T. Bossi, S. Ravasio, B. Curti, M.A. Vanoni, F.J. Florencio, A. Mattevi, J. Biol. Chem. 277 (2002) 24579–24583. [17] A. Weber, U.I. Flugge, J. Exp. Bot. 53 (2002) 865–874. [18] V. Massey, F. Muller, R. Feldberg, M. Schuman, P.A. Sullivan, L.G. Howell, S.G. Mayhew, R.G. Matthews, G.P. Foust, J. Biol. Chem. 244 (1969) 3999–4006. [19] S. Ravasio, L. Dossena, E. Martin-Figueroa, F.J. Florencio, A. Mattevi, P. Morandi, B. Curti, M.A. Vanoni, Biochemistry 41 (2002) 8120–8133. [20] K.A. Loulakakis, N.I. Primikirios, M.A. Nikolantonakis, K.A. Roubelakis-Angelakis, Planta 215 (2002) 630–638.
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[21] P. Pajuelo, E. Pajuelo, B.G. Forde, A.J. Marquez, Planta 203 (1997) 517–525. [22] C.R. Somerville, W.L. Ogren, Nature 286 (1980) 257–259. [23] S.D. Copley, Curr. Opin. Chem. Biol. 7 (2003) 265–272. [24] C.J. JeVery, Trends Genet. 19 (2003) 415–417.
[25] S. Kopriva, M. Suter, P. von Ballmoos, H. Hesse, U. Krahenbuhl, H. Rennenberg, C. Brunold, Plant Physiol. 130 (2002) 1406– 1413. [26] A. Koprivova, M. Suter, R.O. den Camp, C. Brunold, S. Kopriva, Plant Physiol. 122 (2000) 737–746.
Ferredoxin-dependent glutamate synthase moonlights in plant sulfolipid biosynthesis by forming a complex with SQD1 Mie Shimojima, Susanne HoVmann-Benning, R. Michael Garavito, Christoph Benning ¤ Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, MI 48824 1319, USA Received 20 January 2005, and in revised form 20 January 2005
Abstract UDP-sulfoquinovose synthase, SQD1, catalyzes the transfer of sulWte to UDP-glucose giving rise to UDP-sulfoquinovose, which is the head group donor for the biosynthesis of the plant sulfolipid sulfoquinovosyldiacylglyerol. The native SQD1 enzyme of spinach exists as a 250 kDa heteroprotein complex with much higher aYnity for the substrate sulWte than the recombinant SQD1 protein itself. The SQD1 protein co-puriWed with nine proteins. Likely binding partners included rubisco activase, HSP70, and ferredoxindependent glutamate synthase (FdGOGAT). While the Wrst two proteins are known to interact with many other proteins, the identiWcation of FdGOGAT was most intriguing because this 160 kDa protein contains an FMN cofactor known to bind sulWte in vitro. Using diVerent constructs expressing recombinant forms of the multidomain protein FdGOGAT, it was demonstrated that the FMN-binding domain of FdGOGAT is essential for speciWc binding of the protein to SQD1. A model suggests that FdGOGAT could channel sulWte to SQD1. 2005 Elsevier Inc. All rights reserved. Keywords: Chloroplast lipid; Glycolipid; Sugar nucleotide; SulWte; Sulfolipid; Sulfoquinovosyldiacylglycerol; Thylakoid lipid; UDP-sulfoquinovose; UDP-glucose
The plant sulfolipid sulfoquinovosyldiacylglycerol is common to most photosynthetic organisms where it is an intricate part of the photosynthetic membrane [1,2]. In plants and bacteria, sulfolipid is essential under phosphate-limiting growth conditions as a substitute for phosphatidylglycerol [3]. For the basic reactions of sulfolipid biosynthesis in plants two proteins are required, SQD1 and SQD2 [4,5]. The SQD1 protein catalyzes the transfer of sulWte to UDP-Glc giving rise to UDP-sulfoquinovose [6], which serves as the sulfoquinovose head group donor. Sulfoquinovose is transferred to diacylglycerol by SQD2, the sulfolipid synthase. SulWte is derived from the sulfate assimilation pathway in plants inside chloroplasts and is generally considered toxic to the cell [7,8]. Its concentration is very low in plant ¤
Corresponding author. Fax: +1 517 353 9334. E-mail address: [email protected] (C. Benning).
0003-9861/$ - see front matter 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.abb.2005.02.005
tissues, i.e., in the picomole per milligram fresh weight range [9]. The turnover number for the transfer of sulWte onto UDP-Glc for the recombinant SQD1 enzyme was found to be very low [6]. Moreover, the crystal structure of the SQD1 dimer of Arabidopsis has been solved and shows a peculiar loop protruding from the otherwise globular monomer, which might facilitate the interaction with other proteins [10]. Overall the protein is similar in structure to other sugar nucleotide modifying enzymes such as UDP-Glc epimerases. It contains a tightly bound NAD+ cofactor which participates in the reaction as hydride acceptor [10,11]. Due to the low turnover number of the recombinant SQD1 protein we hypothesized that the native SQD1 protein exists in vivo as a complex with other proteins to facilitate the transfer of sulWte to the reaction center of SQD1. To test this hypothesis, we partially puriWed the native SQD1 complex from spinach leaves during a previous study [12]. The estimated molecular
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mass for the native SQD1 complex was »250 kDa, or »160 kDa in excess of the molecular mass expected for an SQD1 dimer (»90 kDa) and in disagreement with a native tetramer structure. The complex was active and its KM for sulWte was considerably lower than that for the recombinant spinach SQD1 protein [12]. Here, we describe the detailed analysis of the highly puriWed native spinach SQD1 complex, the identiWcation of its components and their interaction in vitro.
207
dards (Bio-Rad), and visualized by Coomassie brilliant blue R-250 staining or silver staining (Silver Stain Plus, Bio-Rad). Immuno-blot analysis for SQD1 was done as previously described using a polyclonal antibody raised against Arabidopsis SQD1 [12]. Strep-Tactin alkaline phosphatase (Strep-Tactin AP, IBA, St. Louis, MO, USA) was used to detect recombinant FdGOGAT according to the manufacturer’s instructions. In-gel protein digest and separation of the peptides by reverse-phase HPLC
Materials and methods PuriWcation of the native SQD1 complex from spinach leaves Locally purchased spinach (Spinacia oleracea L.) was used. The extraction and initial puriWcation was performed at 4 °C as previously described [12]. Following ion-exchange chromatography (Macro-Prep HighQ, Bio-Rad, Hercules, CA, USA), the peak fractions were combined, dialyzed against 0.1 M NaCl, 50 mM Tris– HCl, pH 7.5, 10% glycerol, and loaded onto a column (1.2 cm diameter) Wlled with 10 ml of CM AY-Gel Blue Gel (Bio-Rad) equilibrated with 0.1 M NaCl, 50 mM Tris–HCl, pH 7.5, and 10% glycerol. The proteins were eluted by gravity with a linear gradient of 0.15–1.0 M NaCl in the buVer mentioned above. Fractions of 5 ml each were collected. The peak fractions (24 ml total) were concentrated using Centriprep 30 (Millipore, Bedford, MA, USA) to 1.2 ml, which was loaded onto Superdex 200 (FPLC, Amersham Bioscience, Piscataway, NJ, USA). Fractions of 0.3 ml each were collected. Native PAGE analysis All steps were carried out at room temperature. For native PAGE we used Ready Gel Tris–HCl (4–15% linear gradient, Bio-Rad) with a running buVer of 25 mM Tris, pH 8.3, 192 mM glycine. The two peak fractions obtained from CM AY-Gel Blue (7.8 ml) were concentrated with Centriprep 30 and Ultrafree 30 (Millipore) to 120 l and 7.5 l/lane were loaded after mixing with 2£ loading buVer (62.5 mM Tris–HCl, pH 6.8, 40% glycerol, and 0.01% bromophenol blue). Electrophoresis was accomplished at constant 35 V for 17 h. Gel slices containing proteins were macerated with a pestle in a microtube and the proteins were extracted with 20 mM Tris–HCl, pH 8.0, 1% SDS by shaking at 37 °C for 16 h. Proteins were precipitated by adding 4-volumes of acetone and incubation at ¡80 °C for 1 h. SDS–PAGE and Western blot analysis Proteins were separated by SDS–PAGE as described by Laemmli [13], sized using low molecular mass stan-
In-gel digests were performed essentially according to the method of Shevchenko et al. [14]. Tryptic digests were obtained using 13 ng/l trypsin (Promega, Madison, WI, USA) in 100 mM ammonium bicarbonate (pH 8.0) at 37 °C for 16 h. Digestion with endoproteinase Lys-C (Roche, Indianapolis, IN, USA) was performed using 25 ng/l endoproteinase in 25 mM Tris–HCl, pH 8.5, 1 mM EDTA at 37 °C for 16 h. The digested peptides were then separated using reverse-phase HPLC and the N-terminal sequences were analyzed at the Michigan State University Molecular Structure Sequencing and Support Facility. Isolation of a spinach cDNA for FdGOGAT A 32P-labeled 1615-bp fragment (isolated by PCR using the crude cDNA library as template and primers: 5⬘-GCG AAT TCT ATG TTG CGT CTG AGG TG-3⬘; 5⬘-GAT TGG ATC CTC ACC ACC TTC-3⬘) corresponding to the center part of the spinach SoFdGOGAT (GenBank AF061515) was used as a probe to screen a ZAPII spinach leaf cDNA library (Stratagene, La Jolla, CA, USA). Screening was done as previously described [12] but at a hybridization temperature of 65 °C. Two cDNA clones, 6-1 and 1-1 were isolated. Clone 6-1 covers 310 bp of the 5⬘-untranslated region in addition to bases 1–2819 of the published sequence for spinach FdGOGAT (AF061515) [15]. Clone 1-1 covers bp 1733–5000 of the published cDNA sequence. Together, these two clones represent a full-length cDNA with an open reading of 4554 nucleotide frame identical to that published (AF061515). As these two partial clones overlapped, a central BamHI site was used to splice the two sequences in subsequent constructs. Protein expression of SoFdGOGAT fragments in Escherichia coli The four domains of SoFdGOGAT were predicted based on comparison to the amino acid sequence of the cyanobacterial FdGOGAT from Synechocystis sp. PCC6803, for which the crystal structure is known [16]. The three sequences inserted in the expression vector pASK-IBA3 (IBA) were as follows: Full (1–4551 bp,
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AF061515), M2D (1170–3600 bp, AF061515), and L2D (2277–4551 bp, AF061515). In all cases the respective fragments were ampliWed from cDNA clones 6-1 and 1-1, spliced together via a central BamHI site and inserted into the EcoRI and KpnI sites of the vector. Primer pairs and templates were as follows for each of the three constructs as indicated: Full, 6-1 template 5⬘-GCG AAT TCT GTG GTG TTG GAT TTA TTG C-3⬘ and 5⬘GAT TGG ATC CTC ACC ACC TTC-3⬘, 1-1 template 5⬘-GTG AGG ATC CAA TCA GAT GGC-3⬘ and 5⬘GCG GTA CCT GCA GAC TGT AAA CTG GC-3⬘; M2D, 6-1 template 5⬘-GCG AAT TCT ATG TTG CGT CTG AGG TG-3⬘ and 5⬘-GAT TGG ATC CTC ACC ACC TTC-3⬘, 1-1 template 5⬘-GTG AGG ATC CAA TCA GAT GGC-3⬘ and 5⬘-GCG GTA CCA TAG CTT AGA TCA AGA TG-3⬘; LD2, 6-1 template 5⬘-GCG AAT TCA GTG TCT CTA AGA TGG GTG-3⬘ and 5⬘GAT TGG ATC CTC ACC ACC TTC-3⬘, 1-1 template 5⬘-GTG AGG ATC CAA TCA GAT GGC-3⬘ and 5⬘GCG GTA CCT GCA GAC TGT AAA CTG GC-3⬘. The three recombinant proteins were expressed with a Strep-tag (WSHPQFDK) in E. coli XL1-Blue (Stratagene). A 15 ml overnight culture was used to inoculate 500 ml LB with 100 g/ml ampicillin and the culture was incubated at 37 °C under shaking. When the absorption (550 nm) reached 0.5, anhydrotetracycline was added at 0.2 g/ml to induce the protein. The cells were collected after 3 h by centrifugation at 4500g for 10 min. The pellets were stored at ¡80 °C. The cell pellets were homogenized with 2 mg/ml lysozyme (Roche) in buVer A (20 mM potassium phosphate buVer, pH 7.5, 1 mM EDTA, 10 mM -mercaptoethanol, 1 mM glutamine, 1 mM 2-oxoglutarate, and 0.3 M NaCl), incubated on ice for 30 min, and centrifuged at 10,000g for 15 min. The supernatant was Wltered with a 0.45 m Wlter (Millipore). The recombinant protein was puriWed according to the manufacturer’s instructions on 1 ml Strep-Tactin Sepharose per 500 ml culture except that buVer A was used. The proteins were eluted with 2.5 mM desthiobiotin in the buVer A, 1 ml fractions were collected, and were stored at ¡20 °C in 10% glycerol until use. Protein concentrations were assayed throughout using the DC Protein Assay Kit from Bio-Rad.
Results PuriWcation of the SQD1 complex and identiWcation of proteins interacting with SQD1 The SQD1 complex was puriWed from the stroma fraction of spinach chloroplasts using ion-exchange chromatography (High Q), aYnity chromatography (Blue Sepharose), and size exclusion chromatography (Superdex 200) as previously published [12] and described above. The enrichment of the complex was followed throughout by immunodetection of SQD1 on protein blots. SuYcient amount of protein was puriWed for visualization on a silver-stained SDS–PAGE gel (Fig. 1). Any proteins associated with SQD1 should coelute with a similar elution proWle. The SQD1 protein detected by immuno-blotting (Fig. 1A) was predominantly present in fraction 39 of the Superdex 200 column. The corresponding protein band was detected in the silver-stained gel as well (Fig. 1B). Co-eluting with SQD1 in lane 39 (Fig. 1B) were nine other proteins with molecular masses of »160 kDa (a), »97 kDa (b), »75 kDa
Protein–protein interaction assay PuriWed fragments of FdGOGAT were concentrated to eliminate desthiobiotin by centrifugation using Centriprep 30 spin columns (Millipore). SQD1 (54 g in 250 l) and FdGOGAT proteins (37 g in 500 l) were incubated at room temperature for 30 min followed by puriWcation using a 1 ml Strep-Tactin Sepharose column. SQD1 protein was present at approximately Wvefold molar excess. The 300 l of the peak fraction were concentrated by acetone precipitation and subjected to SDS–PAGE and immuno-blot analysis.
Fig. 1. Superdex 200 column elution proWle for the spinach SQD1 complex. (A) Immuno-blot analysis using a polyclonal antibody raised against SQD1 from Arabidopsis. (B) Silver-stained gel after SDS– PAGE. Nine proteins labeled a–i were present in fraction 39. Molecular sizes of markers are indicated to the left of the gel. (C) Enlargement of the regions of the gel in B containing the »160 kDa protein (a) and the SQD1 protein as indicated.
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(c), »70 kDa (d), »60 kDa (e), »55 kDa (f), »46 kDa (g), »43 kDa (h), and »40 kDa, (i). The »160 kDa protein migrated close to a much more prominent protein, but slightly slower and an enlargement of this portion of the gel is shown in Fig. 1C. Of all the proteins present in fraction 39, only this »160 kDa protein showed the same elution proWle as SQD1 consistent with a protein present in the SQD1 complex. Moreover, its estimated molecular mass of »160 kDa added up to »250 kDa, when present in a 1-to-2 ratio with SQD1 (»90 kDa for the dimer) in a protein complex. Alternative puriWcation of the SQD1 complex by native PAGE To Wnd independent corroborating evidence for the »160 kDa protein as a possible component of the SQD1 complex, the eluate containing SQD1 after aYnity chromatography with Blue Sepharose was concentrated and separated by native PAGE (Fig. 2A). Staining of the gel with Coomassie brilliant blue (Fig. 2A, lane I) revealed at least seven proteins or protein complexes as marked. However, immuno-blotting detected the SQD1 protein exclusively in one complex (Fig. 2A). Proteins/complexes 1–7 were excised from the native gel and reanalyzed by denaturing PAGE as shown in Fig. 2B. The only protein other than SQD1 which was exclusively present in gel slice three was again a protein with a molecular mass of »160 kDa. Two other proteins were present in this native gel slice (Fig. 2A, 3), but they were present in other slices of the native gel as well, which would not be expected for speciWc components of the SQD1 complex. The proteins had estimated molecular masses of »75 and »43 kDa, respectively. Because, proteins with comparable masses were also present in the eluate of the Superdex 200 column (Fig. 1B, c and h), we analyzed these further in parallel to the more likely candidate protein with a molecular mass of »160 kDa. IdentiWcation of the »160, »75, and »43 kDa proteins The proteins were digested directly in the gel slices using either trypsin or lysylendopeptidase and the peptides were separated by capillary HPLC and N-ter-
Fig. 2. Analysis of the spinach SQD1 complex by native PAGE. (A) Native PAGE of the partially puriWed spinach SQD1 complex. Lane I, gel stained with Coomassie brilliant blue. Seven prominent proteins/ complexes were present as numbered. Lane II, immuno-blot analysis of the gel in lane I using the polyclonal antiserum against SQD1 from Arabidopsis. (B) Silver-stained gel after SDS–PAGE of proteins/complexes 1–7 isolated from the native gel in (A). Numbers on top of the gel correspond to the numbers assigned to proteins/complexes in (A). Molecular masses of size markers are indicated on the left of the gel.
minally sequenced. The amino acid sequences of the peptides are shown in Table 1. Fortunately, all three protein sequences were known and the proteins were unambiguously identiWed. The »160 kDa protein was spinach ferredoxin-dependent glutamate synthase (Fd-GOGAT). Its sequences and the location of the peptides are shown in Fig. 3. The »75 kDa protein was spinach HSP70, and the »43 kDa protein was spinach rubisco activase. Of
Table 1 N-terminal amino acid sequences of peptides and identiWcation of the proteins Proteins (kDa)
Peptides
Amino acid sequences of N-terminus
Descriptions (Accession No.)
»160
5-1 5-2 9-1 9-2 10
LLENDTILR AQVFKQA(I)DVL RLENFGFIQFRP WRPLTDVVDGYS KQ[V/E][L/V]H[T/M]F[Y/L][P/V]X[L/A]
Ferredoxin-dependent glutamate synthase (Q43155)
»75
8
(K)AVVTVPAYFN
70 kDa heat shock-related protein (Q08080)
»43
7 9
RVPIIVTGNDFS YDIDNMLGDFYI
Rubisco activase (AAD13841)
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Fig. 3. Amino acid sequence for spinach ferredoxin-dependent glutamate synthase. Four domains (1–389, amidotransferase domain; 390–758, central domain; 759–1200, FMN-binding domain; 1201–1517, C-terminal domain) as underlined with alternating solid and broken lines were predicted from the alignment of spinach FdGOGAT with the ortholog from Synechocystis sp. PCC6803, for which the crystal structure is known. The Wve peptide sequences derived by peptide sequencing from the »160 kDa protein are indicated by boxes.
the three, HSP70 and rubisco activase are known to readily interact with other proteins and could have been co-puriWed with the SQD1 complex for this reason. As the »160 kDa protein was the prime candidate based on the analysis described above, we focused on FdGOGAT as complex partner of SQD1. Furthermore, this particular glutamate synthase isoform is present in the stroma of chloroplasts [17], and is therefore resident in the same cellular compartment as SQD1 [4,12]. Equally relevant is the fact that FdGOGAT has a Xavin mononucleotide cofactor (FMN)1 in its active site, which has been shown to react with sulWte [18,19]. Being able to bind sulWte, FdGOGAT in a complex with the sulWte-utilizing enzyme SQD1 seemed reasonable, and led to the hypothesis (see below) that FdGOGAT assists SQD1 in vivo by providing sulWte to SQD1. Expression of recombinant spinach FdGOGAT fragments in E. coli To probe the interaction between FdGOGAT and SQD1 and to reconstitute the complex in vitro, we used His-tagged SoSQD1 recombinant protein [12] and produced the Strep-tagged full-length protein and frag1
Abbreviation used: FMN, Xavin mononucleotide.
ments of spinach FdGOGAT in E. coli. The crystal structure of Fd-GOGAT from Synechocystis sp. PCC6803 is known [16] and this protein consists of four distinct globular domains, the amidotransferase domain, the central domain, the FMN-binding domain, and the C-terminal domain. Given that the two FdGOGAT orthologous amino acid sequences from spinach and the cyanobacterium share 58% identical or 72% similar residues, we assumed that their structures must be similar as well and deduced the diVerent domains for spinach FdGOGAT as indicated in Fig. 3. Based on the cyanobacterial domain structure and taking into account that sulWte binds to the FMN cofactor in the FMN-binding domain presumably making it the most critical domain to interact with SQD1, we constructed three diVerent plasmids expressing the full-length (Full), the FMNbinding and central domains (M2D), and the FMNbinding and C-terminal domains (L2D) as shown in Fig. 4A. The recombinant proteins were tagged at the C-terminus with a Strep-tag permitting the puriWcation on Strep-Tactin Sepharose columns, which bind the Strep-tag. Extracts from the three respective E. coli strains, which were partially puriWed on the Strep-Tactin Sepharose columns are shown in Fig. 4B. The Coomassie brilliant blue-staining and visualization using Strep-Tactin AP conjugate revealed in each case proteins of the
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Fig. 4. Recombinant protein fragments of spinach FdGOGAT. (A) Schematic view of plasmid constructs delineating the diVerent FdGOGAT domains and the Strep-tag. Full, contains all of the four domains (1–1517 aa), M2D the middle 2 domains (390–1200 aa), and L2D the last 2 domains (759–1517 aa). (B) SDS–PAGE of Strep-Tactin Sepharose puriWed proteins. Lanes designated 1, Coomassie brilliant blue stained SDS–PAGE gel. Lanes designated 2, protein blot of the same gel incubated with Strep-Tactin alkaline phosphatase conjugate to detect proteins with Strep-tag. Proteins of the expected molecular masses »160, »89, and »83 kDa, respectively, are indicated.
correct size. In addition, truncated products were present, which were particularly abundant in the extract from the E. coli strain harboring the full-length construct (Fig. 4B). Interaction of recombinant FdGOGAT and SQD1 To demonstrate the interaction of FdGOGAT and SQD1 in vitro, FdGOGAT fragments and SQD1 were partially puriWed by aYnity chromatography based on their respective tags and were mixed in the test tube. Reisolation of the FdGOGAT fragments on Strep-Tactin Sepharose columns (pull-down assay) should, therefore, lead to the co-puriWcation of SQD1, if SQD1 and FdGOGAT form a complex in vitro. Proteins eluting from the Strep-Tactin Sepharose columns were separated by SDS–PAGE and analyzed for FdGOGAT fragments on protein blots using the Strep-Tactin AP conjugate or for SQD1 using a polyclonal SQD1-speciWc antibody. For control purposes the two respective proteins were individually analyzed prior to re-isolation on the Strep-Tactin Sepharose columns. As shown in Fig. 5A, the SQD1 protein by itself does not bind to the Strep-Tactin Sepharose column. When the interaction of the M2D fragment (the central and the FMN-binding domains) of FdGOGAT and SQD1 was tested (Fig. 5B) it was striking that the presence of SQD1 prevented the re-isolation of the FdGOGAT fragment (Fig. 5B, left panel third lane). In addition, SQD1 was not recovered from the mixture following re-isolation of the complex. As will be discussed further below, the direct contact between
Fig. 5. Reconstitution of diVerent spinach FdGOGAT/SQD1 complexes in vitro. (A) Lack of binding of SQD1 to Strep-Tactin Sepharose in the absence of FdGOGAT Strep-tagged proteins. (B–D) Interaction of SQD1 with M2D, Full, and L2D, respectively. Anti SQD1, immunoblot using the polyclonal antiserum against SQD1 from Arabidopsis; Strep-Tactin AP, protein blot incubated with Strep-Tactin alkaline phosphatase conjugate to detect the FdGOGAT fragments with Streptag; control, individual FdGOGAT or SQD1 proteins as indicated by +/¡; pull-down, mixtures of SQD1 and FdGOGAT fragments or FdGOGAT fragments alone as indicated by +/¡ following re-puriWcation on Strep-Tactin Sepharose columns. Expected diagnostic protein bands are indicated to the right of each gel. For additional details and rationale refer to the text.
SQD1 and the FMN-binding domain of FdGOGAT prevents the Strep-tag from interacting with the column in this particular construct. Therefore, this result indi-
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rectly conWrmed the interaction between SQD1 and FdGOGAT. Using full-length FDGOGAT (Fig. 5C) SQD1 was detected following the re-isolation of the complex. This result was consistent with a direct interaction between FdGOGAT and SQD1. The L2D fragment containing the FMN-binding and C-terminal domains was bound to the Strep-Tactin Sepharose column irrespective of the presence of SQD1 (unlike M2D), and SQD1 did not co-purify with this fragment of FdGOGAT (Fig. 5D). Taken together, the results of the pull-down assay presented in Fig. 5 suggest that SQD1 forms a complex with FdGOGAT in vitro and speciWcally binds to the FMN-binding and central domains of FdGOGAT.
Discussion The native SQD1 protein from spinach was previously puriWed as a »250 kDa complex and showed altered substrate binding for sulWte compared to the recombinant SQD1 dimer of »90 kDa [12]. In essence the Km for sulWte decreased from 20 to less than 5 M and the sulWte concentration versus velocity Michaelis– Menten plot changed from hyperbolic for the recombinant SQD1 dimer to sigmoidal for the native complex [12]. In general, this eVect has been attributed to cooperativity among the individual subunits of a given enzyme complex. Because of these earlier observations, we expected to Wnd a protein or proteins in association with SQD1, which could aVect the sulWte availability for the SQD1 reaction center. Initially, it was surprising that ferredoxin-dependent glutamate synthase (FdGOGAT) emerged as the protein partner in the native SQD1 complex. However, the evidence was striking: (1) During the
isolation of the native SQD1 complex, of the diVerent proteins present in SQD1-containing fractions only a single protein of »160 kDa molecular mass equivalent to the molecular mass of FdGOGAT closely co-puriWed with the SQD1 protein using two independent procedures (Figs. 1 and 2). (2) Multiple peptide sequences derived from the »160 kDa protein were identical to the corresponding sequence published for spinach FdGOGAT (Table 1 and Fig. 3), a »160 kDa protein. (3) Two dimers of SQD1 and a monomer of FdGOGAT give rise to a complex of »250 kDa, the same molecular mass as determined for the native SQD1 complex [12]. (4) The two proteins FdGOGAT and SQD1 are present in the same subcellular compartment, the chloroplast stroma [4,12,17]. (5) Direct interaction between SQD1 and FdGOGAT was demonstrated (Fig. 5). (6) A plausible role for FdGOGAT in a complex can be derived from the fact that the FMN cofactor of FdGOGAT reversibly binds sulWte [18,19]. While this property of some Xavoproteins has been primarily used in vitro to probe the molecular environment of the Xavin cofactor, the discovery of FdGOGAT in the native SQD1 complex hints at a possible role for sulWte binding by the FMN cofactor of FdGOGAT in vivo. Based on the available data, it is proposed that the FMN-binding domain of FdGOGAT mediates the delivery of sulWte to the reaction center of SQD1. For this process to occur, one would expect that SQD1 is bound close to the FMN-binding domain of FdGOGAT in a way that exchange of sulWte between the two proteins is facilitated. This hypothesis was testable, because the crystal structures for SQD1 from Arabidopsis [10] and for FdGOGAT from Synechococcus sp. PCC6803 [16] were available. The two proteins were suYciently similar to the respective spinach orthologs that a meaningful
Fig. 6. Model of the FdGOGAT/SQD1 complex. The model depicts the published crystal structures for FdGOGAT from Synechococcus sp. PCC6803 (PDB 1OFE) and Arabidopsis SQD1 (PDB 1QRR). The green and blue domains are the FMN-domain and central domain, respectively. The glutamate amidotransferase domain is shown in magenta and the C-terminal domain in orange. The arrow indicates the position where the FMN-domain was truncated in the M2D construct and the Strep-tag was attached. SQD1 domains are light grey in color with UDP-Glc shown in cyan and NAD+ in red. (A) Left side view, (B) front view, (C) right side view.
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FdGOGAT/SQD1 complex could be modeled as shown in Fig. 6. The SQD1 dimer structure has two overlapping, protruding loops, which neatly Wt into a groove on top of the central domain such that two globular SQD1 monomers are positioned adjacent to the FMN-binding and central domains of FdGOGAT, respectively. This arrangement is experimentally supported by the in vitro binding between the recombinant SQD1 protein and the three diVerent fragments of FdGOGAT (Fig. 5), which showed that the FMN-binding and central domains of FdGOGAT are critical for the SQD1 binding. The equivalent position of the Strep-tag in the M2D fragment is indicated in Fig. 6 (arrow). Incubation of the M2D fragment with SQD1 prevented M2D binding to the Strep-Tactin Sepharose column (Fig. 5B). Based on the model (Fig. 6) this observation is readily understandable, because the Strep-tag would be close to the binding surface between SQD1 and the FMN-binding domain of FdGOGAT preventing it from interacting with the Strep-Tactin Sepharose column. Finally the model also shows that SQD1 is oriented in such a way that its predicted substrate channel (Wlled with water molecules in the structure) is pointed at the FMN cofactor in the FMN-binding domain of FdGOGAT. Thus, the hypothesis that FdGOGAT is involved in providing sulWte to the SQD1 reaction center is supported by the model and the available data. The amount of SQD1 protein in chloroplasts was estimated to be less than 1% (7 g/mg chlorophyll) [12], while FdGOGAT represents up to 1% of the total leaf protein [20,21]. Given the excess of FdGOGAT, one would expect that most of the SQD1 protein is bound to FdGOGAT. Indeed, throughout the puriWcation of the native SQD1 protein complex using diVerent chromatographic techniques, we never observed diVerent forms of SQD1 than the »250 kDa complex. These data and observations suggest that SQD1 exists primarily bound to FdGOGAT in vivo.
Conclusions The analysis of the »250 kDa native SQD1 complex from spinach led to the identiWcation of FdGOGAT as the second component. This large multidomain protein is normally thought of as a key enzyme of nitrogen metabolism [17] or photorespiration [22] in plants. Therefore, it was initially surprising to Wnd FdGOGAT moonlighting in sulfolipid biosynthesis as auxiliary protein for SQD1. However, in view of the reversible binding of sulWte by the FMN cofactor of FdGOGAT, the biochemical properties of the native SQD1 complex and the structural model of the FdGOGAT/SQD1 interaction such a moonlighting function for FdGOGAT makes sense. Finding proteins with known biochemical functions participating in unexpected reactions or pro-
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cesses is increasingly being recognized as a widespread phenomenon in diVerent model organisms [23,24]. The native FdGOGAT/SQD1 complex adds just another example. Plants coordinate their sulfate and nitrate assimilation [25,26]. As SQD1 and FdGOGAT are involved in S- and N-metabolism, respectively, one might wonder whether their interaction also provides opportunities for the coordinated regulation between these two essential processes. This possibility remains to be explored.
Acknowledgments We thank Joseph Leykam and Stacy Trzos (Michigan State University Molecular Structure Sequencing and Support Facility) for protein sequencing and Todd Lydic for technical support. Financial support was provided in parts by a grant from the US National Science Foundation (MCB-0109912).
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