Expression and Assembly of Spectrally Active Recombinant Holophytochrome (Plant Photoreceptor/Phytochrome Biosynthesis/Yeast/Eschenchia Coli) JILL A

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Proc. Natl. Acad. Sci. USA Vol. 88, pp. 10387-10391, December 1991 Biochemistry Expression and assembly of spectrally active recombinant holophytochrome (plant photoreceptor/phytochrome biosynthesis/yeast/Eschenchia coli) JILL A. WAHLEITHNER, LIMING LI, AND J. CLARK LAGARIAS Department of Biochemistry and Biophysics, University of California, Davis, CA 95616 Communicated by Winslow R. Briggs, August 9, 1991

ABSTRACT To develop an in vitro phytochrome assembly translation fails to yield sufficient material for structural system, we have expressed an oat phytochrome cDNA in both and/or spectrophotometric analyses (4). Here we show that the yeast Saccharomyces cerevisiae and the bacterium Esche- the recombinant apophytochromes produced in both yeast richia col. Analysis of soluble protein extracts showed that the and a bacterium are soluble and full-length and can assemble recombinant apophytochromes were full-length and capable of with bilins to produce spectrally active holophytochromes. covalently attaching the phytochrome chromophore analogue phycocyanobilin. Difference spectra indicated that in vitro- MATERIALS AND METHODS assembled holophytochrome species were photoreversible; however, maxima and minima difference absorption values Plasmids. Standard protocols utilizing Escherichia coli were blue-shifted relative to those of the native photoreceptor. DH5a were used for all clone constructions (7). An E. coli Extracts containing the recombinant apophytochromes were expression plasmid containing the oat apophytochrome also incubated with phytochromobilin, the natural chro- phyA3 coding region (8) was constructed as follows. Plasmid mophore synthesized from biliverdin by cucumber etioplast pPC3 (4), which contains the full-length oat phyA3 cDNA, preparations. In these experiments, the difference spectrum was linearized with Pvu I. Plasmid pAQE58 (9), which obtained was identical to that of native oat holophytochrome. contains the genes for the a and 83 subunits of phycocyanin These results suggest that the recombinant apophytochromes from Synechococcus sp. PCC 7002, was linearized with adopt a structure similar to that of the apoprotein biosynthe- BstEII. An equal molar ratio mixture of the two linearized sized in vivo. ELISAs were used to quantitate phytochrome plasmids was then treated with mung bean nuclease to expression levels in both yeast and E. coil extracts. These remove single-stranded overhangs, digested with Kpn I, measurements show that 62-75% of the phytochrome apopro- ligated, and transformed into E. coli. From the resulting tein in the soluble protein extract was competent to assemble transformants, clone pAQ3'PC-18 was selected. This clone with bilins to form spectrally active holophytochrome. contained a 3.2-kilobase Kpn I-Pvu I fragment of pPC3, comprising the 3' end of the phyA3 cDNA, which was The effect of light on many growth and developmental inserted into the Kpn I-BstEII sites of pAQE58. In this processes of plants is mediated by the photoreceptor phy- construction, phyA3 sequences were inserted within the tochrome (1). The functionally active photoreceptor mole- phycocyanin operon replacing most of the a and ( subunit cule is comprised of a large apoprotein of =1100 amino acids genes. To construct a full-length phyA3 cDNA clone, the to which the linear tetrapyrrole (bilin) chromophore phy- plasmid pAQ3'PC-18 was digested with the enzymes Sma I tochromobilin (P46B) is covalently bound (2). Synthesis ofthe and Nsi I. Plasmid pPC3 was also digested with Xba I and the holoprotein, therefore, involves the convergence of two overhangs were removed with mung bean nuclease and then biosynthetic pathways-one for the apoprotein and the other digested with Nsi I. A 7.8-kilobase-pair Sma I-Nsi I fragment for the chromophore. Holophytochrome can be assembled in from pAQ3'PC-18 and a 2492-base-pair Xba I-Nsi I fragment vitro by the incubation of plant-derived apophytochrome from pPC3 containing the 5' end of the phytochrome gene preparations with purified bilins (3). In addition, an in vitro were gel purified and ligated together. After transformation, transcription and translation system has been used to show the plasmid pAQPC-5 (L27-5), which contained the complete that holophytochrome assembly is autocatalytic, requiring phytochrome coding region, was isolated. For the final only apophytochrome and a free bilin (4). cloning step, a 5.0-kilobase HindIII-EcoRI fragment derived In vitro synthesis of phytochrome is a useful tool for the from plasmid pAQPC-5, which contained the entire phy- structural and functional analysis of this important photo- tochrome coding region, flanking cyanobacterial phycobili- receptor. For this reason, the objective of this study was to protein promoter, and transcription termination signals, was produce apophytochrome using recombinant expression in cloned into the HindIII-EcoRI sites of pGEM-4. The result- yeast and bacteria. Both of the presently available experi- ing oat phytochrome construct, pGphyA3 (Fig. 1A), should mental systems for apophytochrome production have their express apophytochrome as a full-length polypeptide, not a limitations. Plant-derived apophytochrome preparations con- fusion protein, since a stop codon lies between the phyco- tain significant levels of spectrally active holophytochrome cyanin and phytochrome translation initiation sites. A control due to incomplete inhibition of chromophore synthesis by plasmid, pGphy-10, was also constructed. This construct tetrapyrrole synthesis inhibitors (3, 5). Apophytochrome lacks a 900-base-pair HindIII-BamHI fragment that contains heterogeneity also arises from the expression of multiple the promoter sequences of the phycobiliprotein operon. phytochrome genes (6) and from posttranslational and arti- A yeast expression plasmid containing the oat phyA3 factual posthomogenization modifications of the protein. sequences was prepared by insertion of the phytochrome Apophytochrome produced by in vitro transcription and coding region from plasmid pAQPC-5 (described above) into the yeast-E. coli shuttle vector pMAC105, a derivative of the vector pAC1 (10). The plasmid pMAC105 The publication costs of this article were defrayed in part by page charge yeast expression payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. Abbreviations: PCB, phycocyanobilin; P4B, phytochromobilin.

10387 Downloaded by guest on September 27, 2021 10388 Biochemistry: Wahleithner et al. Proc. Natl. Acad. Sci. USA 88 (1991) A Hindldl aXn AIz-l with phytochrome sense and antisense orientations were named pMphyA3 and pMphy-11, respectively. The structure /~~~~ ~~~~~Kpril!i < < of plasmid pMphyA3 is shown in Fig. 1B. Apophytochrome Expression and Extraction from E. coli Cells. Single colonies containing either the sense (pGphyA3) or the control (pGphy-10) plasmids were inoculated into 5 ml of 2YT medium [Bacto tryptone (16 g/liter)/Bacto yeast extract (10 g/liter)/NaCl (5 g/liter)] containing ampicillin (100 ,ug/ml) and grown for 5 hr at 37TC. The cultures were pGphyA3 then added to 750 ml of 2YT medium plus ampicillin (100 7750 bp > ,ug/ml) and shaken vigorously at room temperature until the OD6w was 1.0. These cultures were chilled to 40C and centrifuged for 5 min at 2000 x g at 40C. The cell pellet was washed with 300 ml of 20 mM Tris'HCl (pH 8.0) containing 20 mM NaCl and 1 mM EDTA. Washed cell pellets were frozen in liquid nitrogen and stored at -80°C until needed. All subsequent steps of the protein isolation were performed at l0-arll~l+'-~~~X~DC"A~ ~~/ ~ C i/I-/ 4°C or on ice. Frozen cells from a single 750-ml culture were thawed by resuspension in 1:3 (wt/vol) homogenization buffer [50 mM Tris HCl, pH 7.2/0.1 M NaCl/1% dimethyl sulfoxide/1 mM EDTA/1 mM EGTA/2 mM phenylmethylsulfonyl fluoride/1 mM benzamidine/leupeptin (1.5 ,ug/ ml)/10 mM dithiothreitol] and immediately lysed by two passages through a French pressure cell at 10,000 psi (1 psi = 6.9 kPa) using a cell with a 3/8-inch diameter (1 inch = 2.54 cm). The lysate was cleared by centrifugation for 15 min at 22,000 x g and 0.23 g of (NH4)2SO4 was added per ml of the resulting supernatant to precipitate the apophytochrome. After incubation for 10 min, the precipitate was collected by centrifugation for 30 min at 22,000 x g. The resulting pellet was dissolved in 0.25 ml ofTEGE [25 mM Tris-HCl, pH 7.2/2 mM EDTA/25% (vol/vol) ethylene glycol/2 mM phenylmethylsulfonyl fluoride/1 mM dithiothreitol] per g (fresh cell weight). This protein solution was cleared by ultracentrifu- ---TNCA gation for 30 min at 200,000 x g and directly used for BallI holophytochrome assembly experiments. Apophytochrome Expression and Extraction from Yeast Cells. Sense (pMphyA3) and antisense (pMphy-11) plasmids were transformed into lithium acetate-treated Saccharomy- ces cerevisiae 29A (MATa leu2-3 leu2-112 his3-AJ adel-101 trpl-289) using published protocols (11). Single colonies were inoculated into 3 ml of liquid medium containing 0.67% yeast nitrogen base without amino acids, 2% (wt/vol) galactose, FIG. 1. E. coli (A) and yeast (B) expression plasmids containing and the full-length oat phytochrome cDNA sequences. For both plas- 0.02% amino acid "drop out" mixture (minus leucine) mids, phytochrome sequences are indicated with open boxes. Pro- and grown for 2-3 days at 30°C. Two of these cultures were moter initiation sites and direction of transcription are indicated with then combined, transferred to 1 liter of the same medium in an arrow. ATG translation start and TGA stop codons for phy- a 2-liter Fernbach flask, and shaken vigorously at 30°C until tochrome are indicated. The shaded boxes on the E. coli plasmid the cells reached an OD580 of 0.5-1.5. This typically took 2-3 pGphyA3 (A) represent sequences originating from the cyanobacte- days. The cells were harvested by centrifugation for 5 min at rium Synechococcus sp. 7002(9). Sequences on the yeast expression 500 x g. All subsequent steps of the protein isolation were plasmid pMphyA3 (B) that are derived from the yeast enoI structural performed at 4°C or on ice. Cell pellets were resuspended in gene including the "TATA box" and transcription initiation and ice-cold distilled water, recentrifuged, and then termination sites are labeled with shaded boxes. The solid black box resuspended on this plasmid denotes the yeast galactose-inducible upstream in 1.25 ml of homogenization buffer per g (fresh cell weight). activating sequence (GAL UAS). Selectible marker genes, origins of The cell suspension was transferred into chilled 2-ml poly- plasmid replication, and selected restriction sites are also indicated ethylene vials (Biospec Products, catalogue no. 1083, Bar- on both plasmids. tlesville, OK) containing 0.5 vol of acid-washed 0.5-mm glass beads. Cell lysates were prepared using five 1-min pulses was chosen to place phytochrome expression under regula- with a Mini-bead beater (Biospec Products) with cooling on tory control of the yeast enoI promoter and a galactose- ice between each pulse. The crude homogenate was cleared inducible upstream activator sequence (GAL UAS). The by ultracentrifugation for 1 hr at 100,000 x g and 0.23 g of unique HindIII site in pMAC105 was first converted to a (NH4)2SO4 was added per ml of the resulting supernatant. BamHI site by insertion of a 12-base-pair BamHI linker and After a 15-min incubation, the precipitate was collected by the resulting plasmid was named pMAC105(H -* B). A centrifugation for 30 min at 16,000 x g. The resulting pellet gel-purified 4.0-kilobase-pairBamHI fragment containing the was resuspended in 1.0-1.5 ml of TEGE per g (fresh cell phytochrome sequences was then excised from plasmid weight). The apophytochrome preparation was either di- pAQPC-5, gel-purified, and ligated to BamHI-linearized rectly used for holophytochrome assembly experiments or pMAC1O5(H -- B). Clones containing both the sense and frozen in liquid nitrogen and stored at -80°C. antisense orientations of apophytochrome sequences were Holophytochrome Assembly. The following procedures identified by restriction enzyme analysis and both plasmids were performed under a green safelight (12). Apophy- were subsequently isolated. The yeast expression vectors tochrome preparations from E. coli and yeast were divided Downloaded by guest on September 27, 2021 Biochemistry: Wahleithner et al. Proc. Natl. Acad. Sci. USA 88 (1991) 10389 into three 200-,ul samples. One sample [termed + phycocy- room temperature. Color development utilized a freshly anobilin (+PCB)] was diluted with 400 /l of TEGE or prepared solution (100 /l per well) of 0.01% 3,3',5,5'- assembly buffer (20 mM Tes/10 mM Hepes-NaOH, pH tetramethylbenzidine (added as a 1% stock solution in di- 7.7/500 mM sorbitol/1 mM phenylmethylsulfonyl fluoride/ methyl sulfoxide) and 0.3% H202 in 0.1 M NaOAc adjusted 0.5 mM dithiothreitol/2 ,uM leupeptin) to which PCB was to pH 6.0 with citric acid. After a 20-min incubation, the added from a 1.0 mM stock solution in dimethyl sulfoxide to reaction was stopped by the addition of 25 /l of 0.2 M H2SO4 give a final concentration of 4 ,uM PCB. A second sample per well. Absorbance at 450 nm was read using a Molecular (+P4B) was diluted with 400 Al of a P4)B-containing mixture Devices kinetic microtiter plate reader. Unless otherwise in assembly buffer. The P4B solution was obtained after a indicated, measurements were performed in triplicate and 30-min incubation of cucumber etioplast preparations with each microtiter plate contained a standard dilution series of biliverdin and a NADPH-regenerating system (13). The third oat phytochrome of known concentration. sample (-PCB control) was diluted with 400 /l of TEGE or Protein Assays. Protein assays were performed using the assembly buffer only. All three samples were then incubated BCA method (Pierce) with bovine serum albumin as the at 280C for 30 min. After incubation, the suspensions were protein standard (22). cooled to 40C and clarified by ultracentrifugation for 15 min at 200,000 x g prior to holophytochrome photoassay. SDS/PAGE, Zinc-Blot, and Immunoblot Analyses. SDS/ RESULTS AND DISCUSSION PAGE analyses were performed using 1- or 1.5-mm-thick Recombinant Apophytochrome Expression. Expression of minigels according to Laemmli (14). After electrophoresis, objective of these proteins were transferred to poly(vinylidene difluoride) mem- full-length apophytochrome was the first branes (Immobilon P, Millipore) for 1 hr at 100 V. After studies. After constructing the expression plasmids pG- soaking the membranes in 100 ml of 1 M Zn(OAc)2 for 5-30 phyA3 and pMphyA3 for E. coli and yeast, respectively (see min, transblotted biliproteins were visualized with a Foto- Fig. 1), we examined protein extracts from transformed cell dyne UV transilluminator (model 3-3000) and photographed cultures for the presence of phytochrome polypeptides using with Technical Pan film (Kodak type 4415) using a Schott two immunoassays, Western blot and quantitative ELISA. RG-630 red cutoff filter and a 2-min exposure (15, 16). The Western blot analyses were performed on whole-cell SDS same membranes were immunostained as specified by Bir- extracts and on crude and (NH4)2SO4-fractionated soluble kett et al. (17) using the following sequence of antibodies: 10 protein extracts. Fig. 2 A and B shows immunoblots of antibody, affinity-purified oat phytochrome polyclonal rabbit (NH4)2SO4-fractionated soluble protein extracts from yeast antibody (52 ng/ml) (18); bridging antibody, goat anti-rabbit and E. coli cultures, respectively, that contain phyA3 sense IgG fraction (Boehringer Mannheim, catalogue no. 605-200, plasmids (lane 6) or control plasmids (lane 8). These blots 1:7000 dilution); 20 antibody, swine anti-goat IgG alkaline- demonstrate that both recombinant systems produce apo- phosphatase conjugate (Boehringer Mannheim, catalogue phytochromes with electrophoretic mobility similar to puri- no. 605-280, 1:7000 dilution). Blots were typically developed fied oat phytochrome (compare lanes 5 and 6). Similar results for 3-10 min. After immunostaining, blots were also stained were obtained using whole-cell SDS extracts and crude with Coomassie blue according to instructions of the poly- soluble protein extracts from both organisms (data not (vinylidene difluoride) membrane manufacturer. shown). These results indicate that recombinant apophy- Holophytochrome Photoassay. Holophytochrome concen- tochromes produced in both yeast and E. coli systems are trations were estimated using an absorbance difference assay predominantly full length. with a HP8450A UV-visible spectrophotometer (19). For PCB experiments, a 636-nm interference filter was substi- Zinc Blot Immunoblot Coomassie Blue A 1 2 3 5 6 7 8 9 10 11 12 tuted for the 660-nm filter because of the blue-shifted differ- 180 ence maxima of the PCB-apophytochrome adduct (3). The 116 - 84 concentration estimate for the PCB-apophytochrome adduct 58 was made with the assumption that the molar absorption 48 coefficients and photoequilibrium values were identical with 36 those ofnative oat phytochrome except for the position ofthe absorption maxima. 56 7 8 9 ~~~~~~10 1 1 12 180 Quantitative Immunoassays. Phytochrome concentrations 116 were estimated using a double-antibody sandwich ELISA 84 protocol (20) with the following modifications. A 96-well --58 flat-bottom microtiter plate (Coming no. 25805-96) was 48 coated with 50 jml of affinity-purified oat phytochrome polyclonal rabbit antibody (5 ,g/ml) (18) in 50 mM Na2CO3 (pH 9.6) for 2 hr at 4°C. Wells were then blocked with 200 ,l of FIG. 2. Zinc-blot and immunostaining analyses of phytochrome albumin in phosphate-buffered from recombinant strains of yeast (A) and E. coli (B). Soluble protein 2% (wt/vol) bovine serum extracts were prepared from yeast and E. coli cultures containing saline (PBS = 20 mM potassium phosphate, pH 7.4/150 mM sense (pMphyA3 and pGphyA3) and control (pMphy-11 and pGphy- NaCl) overnight at 4°C. Phytochrome-containing samples 10) plasmids, respectively, and fractionated with (NH4)2S04. Two and all subsequent antibody incubation media were diluted samples from each sense plasmid extract were removed and incu- with 1% bovine serum albumin in PBS/Tween (PBS/0.05% bated with PCB or buffer only and analyzed by SDS/PAGE, zinc- Tween 20). Samples of 50 ,l per well were- used for these blot, and immunostaining protocols. For the control plasmid exper- incubations. Between each step, the wells were washed for iments, only the PCB incubation was performed. Zinc-blot, immu- three 10-min periods with PBS/Tween. Three dilutions of nostaining, and Coomassie blue-staining analyses for yeast (A) and each sample were prepared, transferred to the appropriate E. coli (B) samples are as indicated. Lanes: 1, 5, and 9, purified oat and for 2 hr at 4°C. Immu- holophytochrome; 2, 6, and 10, -PCB, sense construct; 3, 7, and 11, antibody-coated well, incubated +PCB, sense construct; 4, 8, and 12, +PCB, control construct. nodevelopment involved incubation with Oat-25 or Pea-25 (1 Lanes 1 contain 98 ng (A) or 25 ng (B) of purified oat phytochrome. ,g/ml), two phytochrome-directed mouse monoclonal anti- For yeast lysates 70 ,ug of protein or for E. coli lysates 87 ,ug of bodies (21), followed by incubation with a 1:7000 dilution of protein was added per lane. Molecular mass markers with sizes in horseradish peroxidase-conjugated goat anti-mouse IgG kDa are indicated on the right. The arrow indicates the position ofthe (Boehringer Mannheim, catalogue no. 605-250) for 1 hr at 124-kDa phytochrome polypeptide. Downloaded by guest on September 27, 2021 10390 Biochemistry: Wahleithner et al. Proc. Natl. Acad. Sci. USA 88 (1991) Results from the quantitative immunoassay analysis, sum- tochrome polypeptide is not found in the control extracts marized in Table 1, reveal that a significant amount of (Fig. 2, lanes 8). In addition, the lack ofother zinc-dependent immunochemically cross-reactive apophytochrome is pro- fluorescent bands in the control samples indicates that PCB duced in both cell systems. Control experiments were per- attachment is specific for the recombinant apophytochrome. formed to ascertain that the antigenicity of the recombinant Indeed, the- Coomassie blue-stained blots reveal that both apophytochromes was similar to that of phytochrome puri- extracts contain many other proteins that do not covalently fied from etiolated oat seedlings, and that yeast and E. coli bind to this bilin (Fig. 2, lanes 10-12). proteins did not interfere with immunoquantitation (data not To determine whether the interactions between both re- shown). Under the respective experimental conditions used combinant apophytochromes and bilins yield photoreversible for cell culture, the level of apophytochrome expression in holoproteins, the +PCB and -PCB samples described above yeast cells is -3-fold greater than that found in E. coli cells were spectrophotometrically assayed for holophytochrome. on a per g fresh weight basis (Table 1). Immunochemical The resulting difference spectra, shown in Fig. 3, indicate estimates of apophytochrome recoveries in soluble protein that the addition of PCB to the soluble-protein extracts from extracts after (NH4)2SO4 fractionation were 28% for E. coli both the recombinant yeast and E. coli cell lines leads to the and 33% for yeast. In both systems, these results were production of photoreversible holophytochrome adducts. obtained under conditions of constitutive expression. In the For both systems, the maximum and minimum absorbance vector pGphyA3, apophytochrome transcription is under the regions of the difference spectra, 650-652 nm and 716-720 control ofthe cyanobacterial phycocyanin promoter that also nm, respectively, are blue-shifted from those of the native functions in E. coli (23). Growth of the yeast cultures in galactose leads to constitutive expression of apophytochrome in cells containing pMphyA3. Although the yeast 0.008 expression vector pMphyA3 contains regulatory sequences for induction of apophytochrome expression, such growth conditions did not lead to enhanced yields or improved recoveries of apophytochrome in the soluble protein fraction 0.004 (data not shown). Holophytochrome Assembly. To determine if recombinant apophytochromes could covalently attach bilins, zinc-blot 0.000 analyses were performed. In these analyses, polypeptides with covalently bound bilins can be visualized on blots in the presence of Zn2+ ions by UV transillumination (15, 16). -0.004 These experiments were performed with the phytochrome CD chromophore analogue PCB, whose assembly with apophy- a) tochrome in vitro has been demonstrated (3, 4). Fig. 2 shows 1.4 that a single orange-fluorescing band at 124 kDa appears in -0.008 protein extracts prepared from both yeast and E. coli cultures -.4 that are expressing phyA3 sequences only when PCB was a) added [compare lanes 2 with lanes 3 for yeast (Fig. 2A) and 0) E. coli (Fig. 2B) samples]. For both yeast and E. coli samples, 0.002 the molecular mass ofthe zinc-dependent fluorescent band is 0 identical to that of phytochrome isolated from oats (Fig. 2, ,0 lanes 1). This fluorescent band was not detected in the soluble protein fraction obtained from either yeast or E. coli cultures 0.001 that were transformed with control plasmids and subsequently incubated with PCB (Fig. 2, lanes 4). These results demonstrate that both yeast and E. coli apophytochrome 0.000 covalently attach to the bilin pigment PCB under our experimental conditions. After zinc-blot analysis, immunostaining of the -same membrane was performed. As was described above, these analyses confirm that apophytochrome ex- -0.001 tracted from both yeast and E. coli cells is predominantly full length (Fig. 2, lanes 6 and 7) and that the 124-kDa phy- -0.002 Table 1. Recombinant apophytochrome and holophytochrome yields Yield, ;Lg/g (fresh weight) 500 600 700 800 Source Yeast E. coli wavelength (nm) Apophytochrome FIG. 3. Holophytochrome photoassay of in vitro-assembled ho- Whole cells 28.54 ± 4.83 9.51 ± 0.% lophytochromes. Apophytochrome-containing protein extracts from Soluble extract 11.01 ± 0.93 7.74 ± 0.67 yeast (A) and E. coli (B) cells containing sense plasmids pMphyA3 (NH4)2SO4 fraction 9.50 ± 0.75 2.73 ± 0.19 and pGphyA3, respectively, were prepared and fractionated with Holophytochrome (NH4)2SO4. Spectrophotometric difference assays of samples incu- Spectral estimate 7.1 ± 0.3 1.62 ± 0.35 bated with PCB (solid line), P#B (dashed lines), and buffer only % ligatable 74.7 ± 7.3 62.3 ± 3.51 (dotted line) are indicated. Difference maxima and minima (in nm) were taken from mathematically smoothed plots. Data points for the Phytochrome recoveries were determined by ELISA. Each value four absorbance values from 652 to 658 nm were omitted due to a represents an average of three experiments with at least three light-scattering artifact arising from masking of these diodes in the replicas of each data point. Holophytochrome recoveries were HP8450A spectrophotometer. The absorbance values at these wave- determined by spectrophotometric assay ofthe +PCB samples. Each lengths represent interpolated values from the measured values at value represents an average of three experiments. 650 and 660 nm. Downloaded by guest on September 27, 2021 Biochemistry: Wahleithner et al. Proc. Natl. Acad. Sci. USA 88 (1991) 10391 chromoprotein, which lie at 668 and 730 nm (19). These using well-established recombinant DNA methodologies. In blue-shifted spectra are similar to that of the PCB- vitro mutagenesis experiments will be especially useful to apophytochrome adduct obtained from tetrapyrrole-deficient elucidate the structural basis for bilin-apophytochrome in- oat seedlings (3, 18). The control spectra, shown in Fig. 3, teraction and to address the structural requirements for the also demonstrate that the apophytochrome present in both molecule's photoactivity. This approach, in conjunction with yeast and E. coli extracts is not photochromic in the, absence in vitro or in vivo assays for recombinant holophytochrome of added bilin. Based on immunochemical estimates of the function, will also enable experimental -dissection of the amount of apophytochrome present in these extracts and the molecular basis of phytochrome action. assumptions that the molar absorption coefficients and quan- tum yield of the PCB adduct are similar to those of native oat We thank Dr. M. A. Innis (Cetus, Emeryville, CA) for the gift of phytochrome, an estimate of percent bilin-apophytochrome the pMAC105 plasmid, Dr. M. J. Holland (University of California, ligation yield was obtained. Table 1 shows that the majority Davis, CA) for S. cerevisiae 29A, Drs. M.-M. Cordonnier and L. H. Pratt (University of Georgia, Athens) for monoclonal antibodies ofthe apoprotein in the final soluble-protein extract, 62-75%, Pea-25 and Oat-25, Dr. A. N. Glazer for PCB, and Dr. Matthew is competent to form photoreversible holophytochrome for Terry for providing P4B samples. This work was funded by U.S. both yeast and E. coli expression systems. It is significant to Department of Agriculture Grant GAM89-001162. note that the E. coli holophytochrome assembly experiments described above were performed using apophytochrome 1. Furuya, M., ed. (1987) Phytochrome and Photoregulation in preparations obtained from cells grown at temperatures be- Plants (Academic, Tokyo). low 30'C. Although apophytochrome is expressed from pG- 2. Vierstra, R. D. & Quail, P. H. (1986) in Photomorphogenesis in phyA3 in E. coli cultures grown at 37TC, this apophy- Plants, eds. Kendrick, R. E. & Kronenberg, G. H. M. (Nij- tochrome failed to assemble with PCB to form a photoactive hoff, Dordrecht, The Netherlands), pp. 35-60. holoprotein (data not shown). In this regard, improved yields 3. Elich, T. D. & Lagarias, J. C. (1989) J. Biol. Chem. 264, 12902-12908. of functional proteins that are expressed in E. coli grown at 4. Lagarias, J. C. & Lagarias, D. M. (1989) Proc. Natl. Acad. Sci. lower temperatures have been well documented (24). USA 86, 5778-5780.- The observed blue-shifted difference spectra ofthe recom- 5. Elich, T. D. & Lagarias, J. C. (1987) Plant Physiol. 84, 304- binant apophytochrome-PCB adducts (described above) 310. could arise from the attachment of a nonnatural bilin chro- 6. Tomizawa, K.-I., Nagatani, A. & Furuya, M. (1990) Photo- mophore or from structural alterations of the apoprotein. To chem. Photobiol. 52, 265-275. distinguish between these two possibilities, we performed 7. Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989) Molecular similar assembly experiments with both recombinant apo- Cloning:A Laboratory Manual (Cold Spring Harbor Lab., Cold phytochromes using the natural phytochrome chromophore Spring Harbor, NY), 2nd Ed. 8. Hershey, H. P., Barker, R. F., Idler, K. B., Murray, M. G. & P4B. For these experiments, P4B was obtained by the Quail, P. H. (1987) Gene 61, 339-348. enzymatic conversion of the -bilin precursor biliverdin in 9. de Lorimier, R., Wang, Y.-J. & Yeh, M.-L. (1988) in Light isolated plastids (13). Difference spectra produced by the Energy Transduction in Photosynthesis: Higher Plant and incubation of recombinant yeast and E. coli apophytochrome Bacterial Models, eds. Stevens, S. E., Jr., & Bryant, D. A. preparations with P4B are shown in Fig. 3. In both cases, the (Am. Soc. Plant Physiol., Rockville, MD), pp. 332-336. difference maxima at 666-668 nm and difference minima at 10. Innis, M. A., Holland, M. J., McCabe, P. C., Cole, G. E., 730 nm are nearly indistinguishable from those of native oat Wittman, V. P., Tal, R., Watt, K. W. K., Gelfand, D. H., phytochrome preparations (19). In addition, the AAm/AAm Holland, J. P. & Meade, J. H. (1985) Science 228, 21-26. ratios of 1.15 (yeast) and 1.04 (E. coli) are also similar with 11. Rose, M. D., Winston, F. & Hieter, P. (1989) Laboratory Course Manual for Methods in Yeast Genetics (Cold Spring the native photoreceptor from oats. Based on these results, Harbor Lab., Cold Spring Harbor, NY). we conclude that both recombinant apophytochromes adopt 12. Litts, J. C., Kelly, J. M. & Lagarias, J. C. (1983) J. Biol. Chem. protein structures that are similar to the natural phytochrome 258, 11025-11031. polypeptide synthesized in planta. These observations reaf- 13. Terry, M. J. & Lagarias, J. C. (1991) J. Biol. Chem., in press. firm the conclusions of our earlier in vitro transcription- 14. Laemmli, U. K. (1970) Nature (London) 227, 680-685. translation studies (4) that plant-specific posttranslational 15. Berkelman, T. R. & Lagarias, J. C. (1986) Anal. Biochem. 156, modifications are not required for proper folding of the 194-201. apophytochrome polypeptide, for subsequent bilin attach- 16. Jones, A. M. & Quail, P. H. (1989) Planta 178, 147-156. ment, or for photoreversibility of the newly assembled ho- 17. Birkett, C. R., Foster, K. E., Johnson, L. & Gull, K. (1985) FEBS Lett. 187, 211-218. lophytochrome. 18. Elich, T. D., McDonagh, A. F., Palma, L. A. & Lagarias, J. C. Concluding Remarks. The development ofE. coliand yeast (1989) J. Biol. Chem. 264, 183-189. experimental systems to express and assemble photoactive 19. Lagarias, J. C., Kelly, J. M., Cyr, K. L. & Smith, W. O., Jr. holophytochrome represents a significant advance in the (1987) Photochem. Photobiol. 46, 5-13. study of this important plant photoreceptor. Recently, the 20. Thomas, B., Crook, N. E. & Penn, S. E. (1984) Physiol. Plant. expression of high levels of pea apophytochrome in E. coli 60, 409-415. was reported; however, this protein was both truncated and 21. Cordonnier, M.-M. (1989) Photochem. Photobiol. 49, 821-831. insoluble (25). The ability of this recombinant apophy- 22. Smith, P. K., Krohn, R. I., Hermanson, G. T., Mallia, A. K., tochrome to assemble with bilins was not described. We Gartner, F. H., Provenzano, M. D., Fujimoto, E. K., Goeke, N. M., Oson, B. J. & Klenk, D. C. (1985) Anal. Biochem. 150, believe that the lower constitutive level expression of our 76-85. constructions contributed to the ability to produce soluble 23. Bryant, D. A., Dubbs, J. M., Field, P. I., Porter, R. D. & de ligation-competent apophytochromes. Lorimier, R. (1985) FEMS Microbiol. Lett. 29, 343-349. In conjunction with the holophytochrome assembly sys- 24. Schein, C. H. (1989) BiolTechnology 7, 1141-1147. tems described here, site-specific alterations can now be 25. Tomizawa, K., Ito, N., Komeda, Y., Uyeda, T. Q. P., Takio, readily introduced into the apophytochrome polypeptide K. & Furuya, M. (1991) Plant Cell Physiol. 32, 95-102. Downloaded by guest on September 27, 2021