Proc. Natl. Acad. Sci. USA Vol. 91, pp. 12535-12539, December 1994 Plant Biology Phytochrome assembly in living cells of the yeast Saccharomyces cerevisiae (plant photoreceptor/in vivo reconstitution/linear tetrapyrrole) LIMING LI AND J. CLARK LAGARIAS Section of Molecular and Cellular Biology, University of California, Davis, CA 95616 Communicated by Eric E. Conn, September 6, 1994 (receivedfor review July 25, 1994)

ABSTRACT The biological activity of the plant photore- (10-12). In vitro holophytochrome assembly has been par- ceptor phytochrome requires the specific association ofa linear ticularly useful for biochemical characterization ofthe struc- tetrapyrrole prosthetic group with a large apoprotein. As an tural features of both bilin chromophore and apophy- initial step to develop an in vivo assay system for structure- tochrome, which are necessary for pigment attachment and function analysis of the phytochrome photoreceptor, we un- photoreversibility (10, 13). dertook experiments to reconstitute holophytochrome in the While the expression ofrecombinant apophytochrome and yeast Saccharomyces cerevisiae. Here we show that yeast cells the in vitro assembly of holophytochrome is an invaluable expressing recombinant oat apophytochrome A can take up research tool, the ability to reconstitute functionally active exogenous linear tetrapyrroles, and, in a time-dependent man- recombinant phytochrome in living cells will facilitate appli- ner, these pigments combine with the apoprotein to form cation of genetic methodologies to the analysis of photore- photoactive holophytochrome in situ. Cell viability measure- ceptor structure and function. In this regard, the yeast ments indicate that holophytochrome assembly occurs in living Saccharomyces cerevisiae has proven particularly useful for cells. Unlike phytochrome A in higher plant tissue, which is genetic dissection of the structure and function of the mam- rapidly degraded upon photoactivation, the reconstituted pho- malian steroid hormone receptor family (14-19) as well as the toreceptor appears to be light stable in yeast. Reconstitution of mammalian heterotrimeric G-protein coupled P2-adrenergic photoactive phytochrome in yeast cells should enable us to receptor (20). Like phytochrome, neither of these mamma- exploit the power of yeast genetics for structure-function lian hormone receptor families occurs naturally in yeast, and dissection of this important plant photoreceptor. their agonists are not known to regulate endogenous pro- cesses in this organism. The present studies were undertaken as the first step to develop an in vivo assay for phytochrome Sensing and adapting to the light environment are essential in yeast. Our studies show that yeast cultures expressing for optimal plant growth and development. Plants therefore apophytochrome can assimilate exogenous PFB and phyco- possess a number of photoreceptors that enable the percep- cyanobilin (PCB), a chemically-related analog from algae, tion ofthe direction, intensity, and/or spectral quality oflight and incorporate either pigment into a photoactive holophy- (1). These include photoreceptors for UV-B, UV-A/blue, tochrome in living cells. The in vivo spectrophotometric and red/far-red regions of the light spectrum. The phy- properties of these holophytochromes as well as their stabil- tochrome photoreceptor mediates numerous photomorpho- ity in yeast cells are also examined. These studies indicate genetic responses to red and far-red light (2). Through its that genetic approaches to the analysis of phytochrome ability to reversibly photointerconvert between red- and structure and function in yeast are now practicable. far-red-light-absorbing forms, Pr and Pfr, phytochrome influ- ences nearly every stage of plant development, from germi- nation to floral induction and ultimately to senescence. To MATERIALS AND METHODS function as a receptor of visible light, the phytochrome Apophytochrome Expression in Yeast. The oat phyto- molecule contains a linear tetrapyrrole pigment that is cova- chrome A3 expression plasmid, pMphyA3, which contains a lently bound to a large polypeptide via a thioether linkage (3). leu2 selection marker, was expressed in the yeast S. cerevi- Light absorption by the phytochrome chromophore effects a siae strain 29A (MATa leu2-3 leu2-112 his3-1 adel-101 trpl- protein conformational change that initiates a signal- 289) as described previously with the following modifications transduction pathway(s) in the plant, the details of which are (11). Several individual colonies of yeast cells containing poorly understood at present. pMphyA3 were inoculated into 12 ml of synthetic raffinose Considerable progress has been made in recent years on (SR) medium supplemented with adenine, histidine, and the biosynthesis of both apophytochrome and phytochromo- tryptophan (each at 40 mg/liter) and grown overnight at 30°C bilin (POB), the linear tetrapyrrole precursor of the phy- with shaking at 300 rpm. The composition of SR is the same tochrome chromophore (reviewed in ref. 4). The observation as synthetic dextrose (SD) medium except that glucose is that apophytochrome could be isolated from chromophore- replaced with raffinose (21). SR and SD both represent deficient plants indicates that the synthesis of both compo- selective media for pMphyA3 since leucine is omitted. The nents is not coupled in vivo (5-7). Subsequent in vitro studies 12-ml overnight cultures were used to inoculate 1 liter of SR have revealed that the assembly of photoactive holophy- medium in a 2-liter Fembach flask. This culture was incu- tochrome occurs spontaneously when POB and apophy- bated at 30°C for 10-12 h with shaking at 300 rpm. When the tochrome are coincubated (8, 9). This feature ofphytochrome OD580 of the culture reached 0.3-0.6, 1% (wt/vol) galactose biosynthesis has permitted many investigators to reconstitute was added to induce apophytochrome expression. After a holophytochromes in vitro using recombinant apophy- 24-h induction period, cells were utilized for in vivo holo- tochrome and linear tetrapyrrole chromophore precursors phytochrome assembly (see the next section) or for apophy-

The publication costs ofthis article were defrayed in part by page charge Abbreviations: PCB, phycocyanobilin; P4'B, phytochromobilin; Pr, payment. This article must therefore be hereby marked "advertisement" red-light-absorbing form ofphytochrome; Pfr, far-red-light-absorbing in accordance with 18 U.S.C. §1734 solely to indicate this fact. form of phytochrome; DMSO, dimethyl sulfoxide. 12535 Downloaded by guest on September 30, 2021 12536 Plant Biology: Li and Lagarias Proc. Natl. Acad Sci. USA 91 (1994) tochrome extraction according to the protocol ofWahleithner containing DMSO-treated control cells (i.e., apophy- et al. (11). tochrome control) was irradiated with continuous red light. In Vivo Holophytochrome Assembly. Yeast cultures grown The far-red light source used for actinic irradiation consisted and induced as described above were collected by centrifu- of two Sylvania F48T12/660 nm/VHO fluorescent tubes gation for 5 min at 3000 rpm in an SS34 rotor. The resulting filtered with 0.125-inch-thick far-red FRS700 Plexiglas cell pellet was resuspended in fresh SR medium at a ratio of (Rohm & Haas dye no. 58015), whereas the red light source 0.5 g offresh weight cells per ml. PCB or P(¶B [as 1 mM stock consisted of six Sylvania (Electric Products, Fall River, MA) solutions in dimethyl sulfoxide (DMSO)] was then added to F20T12 cool white fluorescent tubes, which were filtered the cell suspension under a green safelight to give the desired through 0.125-inch-thick red Plexiglas (Rohm & Haas dye no. final pigment concentration (see Results and Discussion). 2423). For these experiments, flasks were placed 6 inches For in vivo spectrophotometric analysis and in vivo phy- (15.2 cm) from the light source on a rotary shaker and tochrome stability studies, cell incubation mixtures were incubated at room temperature with a shaking speed of 200 gently shaken for 5 h under a green safelight at room rpm. After various incubation periods, 3-ml aliquots were temperature or at 30°C. For the time course experiments of removed from each culture for total protein isolation, and in vivo phytochrome assembly, a 280-,ul aliquot of each cell 10-ml aliquots were removed for preparation of (NH4)2SO4- incubation mixture was removed at various times and trans- fractionated protein extracts as described by Wahleithner et ferred into a 50-ml polypropylene Falcon tube containing 30 al. (11). ml ofice-cold wash buffer (50 mM NaCl/150 mM NaCl/1 mM Whole-Cell Protein Isolation from Yeast Cells. Yeast sus- EDTA/1 mM EGTA/1% DMSO). The resulting cell suspen- pension cultures (3 ml) were transferred into a 13 x 100 mm sion was vortexed, and cells were collected by centrifugation glass test tube and centrifuged for 5 min at 1000 x g. After at 3000 rpm for 5 min. After four such washes to remove free removing the supernatant, 100 ,ul of 2x SDS sample buffer pigment, whole-cell SDS protein extracts and (NH4)2SO4- [125 mM Tris-HCl, pH 6.8/5.6% SDS/15% glycerol/5% fractionated protein extracts were prepared as described (11). 2-mercaptoethanol] and an equal volume of 0.5-mm acid- The amount of holophytochrome that had assembled during washed glass beads were added to the cell pellet. After the the in vivo incubation period was determined using both addition of 1 ,l4 of 200 mM phenylmethylsulfonyl fluoride in spectrophotometric and zinc blot assays (see the following ethanol, the cell mixture was vortexed three times for 20 sec sections). each. Between each 20-sec homogenization pulse, test tubes Spectrophotometric Phytochrome Assays. In vivo spectro- were kept on ice for at least 20 sec. After the final 20-sec photometric assay of holophytochrome in yeast cells was vortexing step, an additional 100-,l aliquot of2x SDS sample performed at 4°C using an Aviv/Cary 14DS UV/visible buffer was added to the mixture. After a brief mixing, spectrophotometer equipped with a light-scattering sample homogenates were centrifuged for 2 min at 1000 x g, and the compartment. Saturating red and far-red light irradiations (15 supernatants were removed and stored at -20°C prior to min for each) of stirred cell suspensions in 5-ml cuvettes were SDS/PAGE analysis. provided by a custom-made actinic light source [250-W, SDS/PAGE, Zinc Blot, Immunoblot, and Coomassie Blue 24-V, Osram (Berlin) Xenophot HLX lamp] and red inter- Analyses. Yeast protein extracts were analyzed by SDS/ ference filters with a 10-nm band pass (650 nm for PCB- PAGE using 10% polyacrylamide minigels according to apophytochrome adduct and 660 nm for P4B-apophy- Laemmli (22). After electrophoresis, proteins were electro- tochrome adduct; Ditric Optics, Hudson, MA) or a far-red phoretically transferred to poly(vinylidene difluoride) mem- plastic filter (FRS 720 Plexiglas, Rohm & Haas dye no. 58015, branes (Immobilon P; Millipore) for 1 h at 100 V. After 0.125 inch thick), respectively. The following parameters transblotting, the same membrane was used for zinc blot, were employed for in vivo spectra: 2-nm wavelength incre- immunoblot, and Coomassie blue staining analyses as de- ments, 10-nm band width, and 4.0-sec dwell time per reading. scribed (11). Holophytochrome concentrations in soluble protein extracts were estimated with a HP8450A UV/visible spectrophotom- eter using the absorbance difference assay described (13). RESULTS AND DISCUSSION Stability of Recombinant Holophytochrome in Yeast. These Photoreversible Holophytochrome Adduct Assembly in experiments were performed with S. cerevisiae strains 29A Yeast. It is well established that yeast cells deficient in heme and Y14 (MATa adel his3 leu2-3 leu 2-112 trp 1-la), which biosynthesis can grow normally in medium supplemented had been transformed with the plasmid pMphyA3 (11). A with heme (23, 24). Based upon these observations, it ap- 200-ml yeast culture was grown in SR medium and induced peared reasonable that yeast cells would also be able to take with galactose as described above in Apophytochrome ex- up the structurally related linear tetrapyrrole (bilin) pigments. pression in yeast. At the end of the induction period, cells To test this hypothesis, we conducted experiments to deter- were collected by centrifugation for S min at 5000 x g and mine whether yeast cells expressing recombinant oat apo- then resuspended into 3 ml of sterile SR medium. The phytochrome A could assimilate exogenous bilins and pro- resulting cell suspension was divided into three equal parts, duce photoactive holophytochrome intracellularly. The two which were each transferred to a sterile 15-ml polypropylene bilin pigments, P4B and PCB, were chosen for these studies Falcon tube. PCB (as a 1 mM stock solution in DMSO) was since both compounds have been previously shown to as- added to two of the three tubes to give a final PCB concen- semble with apophytochrome in vitro to form photoactive tration of 45 ,uM and a final DMSO concentration of 4.5% holophytochromes (11, 13). After a 5-h incubation period (vol/vol). As a control, the same amount of DMSO was with 45 ,AM bilin and extensive washing, an in vivo spectro- added to the third tube. All three cell suspensions were photometric difference assay was performed. Fig. 1 Upper incubated at 30°C for S h with gentle shaking (100 rpm). After shows that the in vivo difference spectra for both PFB- and the incubation period, cells were washed four times with PCB-treated cultures are characteristic of the phytochrome sterile SD medium by vortexing and centrifugation to remove photoreceptor. For the PFB-treated cultures, the absorbance free PCB. Washed cell pellets were then resuspended in 1 ml difference maximum and minimum occurred at 660 nm and of sterile SR medium and used to inoculate 100 ml of fresh SR 730 nm, respectively. These values are very similar to those medium in a sterile 500-ml Erlenmeyer flask. One of the of native oat phytochrome A preparations (25). By compar- PCB-treated cultures was continuously irradiated with far- ison, the absorbance difference maximum and minimum of red light (i.e., Pr culture), while the other was continuously the PCB-treated cells were blue-shifted by 10 nm, occurring irradiated with red light (i.e., Pfr culture). The third flask at 650 nm and 720 nm, values that are indistinguishable from Downloaded by guest on September 30, 2021 Plant Biology: Li and Lagarias Proc. Natl. Acad. Sci. USA 91 (1994) 12537 0.04 0 0.5 1.0 2.5 4.0 5.5 8.5 (h) 0.031 A 0.02 0.01 U) 0- /~~~~~~~~~/ U) -0.01- .~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ a) -0.02- 10 0 B C1 L.0 0.01. 0 0.005- -I-, ' .0 co 0...... D_ -0.005- /.A...... -0.01- C 550 600 650 700 750 800 wavelength (nm) FIG. 1. Spectrophotometric analysis of recombinant holophy- tochrome adducts in yeast. Yeast cultures (200 ml) expressing apophytochrome were divided into three aliquots and incubated with 45 AM PCB or P4B or DMSO (control) for 5 h at 30°C in SR medium as described in Materials and Methods. After extensive washing to remove free pigments, far-red minus red difference spectra on FIG. 2. Zinc-blot time course of phytochrome assembly in yeast. whole-cell suspensions were obtained (Upper). After the in vivo Yeast cells induced to express apophytochrome were treated with 45 difference spectrophotometric measurements, (NH4)2504-fraction- ,uM PCB as described in Materials and Methods. At each time ated protein extracts were prepared and spectrophotometrically indicated, aliquots of the cell suspension were removed and protein

assayed (Lower). , Spectra obtained from PCB-treated yeast extracts were prepared and fractionated with (NH4)2504. These

cells; -, spectra obtained from P4DB-treated yeast cells; * *, extracts were resolved by SDS/PAGE, electroblotted to a poly(vi- spectra obtained from the DMSO-only control cells. In this experi- nylidene difluoride) membrane, and sequentially analyzed with a zinc ment, the amount of P4.B-apophytochrome adduct formed was blot (A), immunostaining (B), and Coomassie blue-staining (C) -67% of that of the PCB-apophytochrome adduct. The difference procedures. Each lane contains 80 ,ug of total protein. The arrows on spectra of the PFB-apophytochrome adduct in Upper and Lower are the left indicate the phytochrome polypeptide that migrates at 124 normalized to the scale of the PCB-apophytochrome spectra. kDa. The dots on the right represent protein standards with molec- ular masses of 221, 115, 63, and 46 kDa. those of the PCB-apophytochrome A adduct produced in vitro (8, 11, 13). Control yeast cultures treated with DMSO apophytochrome was present in all sample lanes (Fig. 2B). alone failed to afford phytochrome photoactivity (Fig. 1 These analyses also demonstrate that the phytochrome poly- Upper), and yeast cells containing an antisense apophy- peptide remained full-length throughout the PCB incubation, tochrome plasmid did not yield photoactive phytochrome thereby indicating that phytochrome does not undergo sig- after incubation with PCB (data not shown). For comparative nificant proteolysis under these experimental conditions. purposes, phytochrome difference spectra were also ob- Coomassie blue staining of this membrane also confirms that tained on soluble protein extracts prepared from both PCB- equal amounts of total protein are present (Fig. 2C). Based and PNB-treated yeast cultures (Fig. 1 Lower). These differ- on the above results, we conclude that the time-dependent ence spectra are indistinguishable from the in vivo difference change in the zinc-mediated fluorescence intensity of the spectra. Taken together, these investigations indicate that phytochrome polypeptide represents quantitative differences yeast cells are able to take up both bilins and to incorporate in holophytochrome assembly. these pigments into functional holophytochromes. To corroborate the results of the zinc blot analysis, spec- Time Course of Phytochrome Assembly for Yeast Cells. A trophotometric phytochrome assays were performed on pro- time course experiment was performed to optimize condi- tein extracts from PCB-treated yeast cells. In this experi- tions for in vivo assembly of phytochrome. Since purified ment, the percentage of in vivo holophytochrome assembly PIB is difficult to obtain in large quantities, PCB was utilized was estimated from the ratio of photoactive phytochrome for these experiments. Our experimental protocol entailed measured in the initial protein extract to that determined in incubation of apophytochrome-expressing cells with 45 ,uM the same extract after subsequent incubation with PCB. Fig. PCB at 300C followed by removal of a culture aliquot, protein 3 illustrates the percentage of in vivo holophytochrome extraction, and holophytochrome assay using a zinc blot assembly versus incubation time determined by this experi- procedure, The zinc blot procedure, which was originally ment. Consistent with the zinc blot analysis, this investiga- designed for fluorescence detection of bilin-linked polypep- tion shows that in vivo holophytochrome assembly was tides in SDS gels (26), has been modified to permit quanti- complete within a 4-h incubation period. The rate of phy- tation of bilin attachment to apophytochrome after SDS/ tochrome assembly does, however, vary from experiment to PAGE and blotting to a poly(vinylidene difluoride) mem- experiment, with saturation occurring between 2 and 6 h brane (13). Fig. 2A shows the zinc blot analysis, which under our experimental conditions. As might be expected, reveals the time dependence ofthe formation of a fluorescent the rate of in vivo assembly is affected by incubation tem- polypeptide at 124 kDa. The fluorescence intensity of this perature, bilin concentration, and cell growth conditions band was only partially detectable after 30 min and continued (unpublished observations). Similar time course experiments to increase up to 4 h ofincubation with PCB. Immunostaining were also performed using two other compatible yeast of the same zinc blot membrane with a polyclonal phy- strains. In all strains tested, photoactive holophytochrome tochrome antibody indicated that this 124-kDa band corre- could be reconstituted in vivo (data not shown). These results sponds to phytochrome and that an equivalent amount of indicate that bilins are readily assimilated by yeast cells. Downloaded by guest on September 30, 2021 12538 Plant Biology: Li and Lagarias Proc. Natl. Acad. Sci. USA 91 (1994) bation period, when holophytochrome assembly was nearly complete, >90% ofthe cells remained viable. When PCB was omitted from the incubation medium as a control, the results were essentially the same (see Table 1). Throughout an extended 9-h incubation period, no significant difference was observed in both absolute number and ratio of colony num- 50 0 bers on YPD and SD plates, thus confirming that plasmid loss is insignificant during this incubation period. Since it was conceivable that the viable cells remaining at the end of the 25- PCB incubation period had lost their ability to express apophytochrome and that we were observing assembly only in a small subpopulation of dead cells, eight random colonies were selected from the SD plates for each PCB incubation IrII 0 2 4 6 time point and analyzed for apophytochrome expression. This involved inoculation of each individual colony into SR time (h) liquid medium, galactose induction, preparation of SDS whole-cell extracts, and SDS/PAGE and Western blot anal- FIG. 3. Spectrophotometric time course of phytochrome assem- bly in yeast. Yeast cells induced to express apophytochrome were ysis as described in Materials and Methods. This experiment incubated with 45 uM PCB, and aliquots were removed after various revealed that 100% of the colonies at every time point incubation times as described in Materials and Methods. At each examined up to 6 h were able to express full-length apophy- time point, soluble protein extracts were prepared and assayed for tochrome (Table 1). Taken together with the evidence that phytochrome spectrophotometrically. Each extract was then treated nearly all of ligation-competent apophytochrome assembles with 4 ,uM PCB, incubated for 1 h at 28°C, and then reassayed for in vivo within 4 h (Fig. 3), these experiments indicate that phytochrome spectrophotometrically. The ratio of the amount of holophytochrome assembly occurs in viable yeast cells. photoactive phytochrome before and after this in vitro PCB incuba- Recombinant Holophytochrome Stability in Yeast. It is well tion x 100%o is plotted as a function of the length of the whole-cell established that phytochrome A is light labile in plants and incubation with PCB. Each point represents the average ofthe values that the protein half-life ofthe Pfr form is considerably shorter from two independent experiments. than that of the Pr form (reviewed in ref. 27). In cucumber Photoreversible Holophytochrome Adducts Are Formed in seedlings for example, the half-life of PrA is 100 h compared Viable Yeast Cells. The ability to reconstitute photoreversible with 1 h for PfrA (28). While the differential turnover of holophytochrome in yeast cells is expected to be a useful tool phytochrome A clearly plays an important role in the regu- for the analysis of phytochrome structure and function. lation of phytochrome A function and appears to be ubiqui- However, the application of yeast genetic methodology for tin-mediated (29), the specific factors that mediate this dif- this that the ferential turnover process are presently unknown. Since purpose requires formation of photoreversible yeast and plants share many common eukaryotic cellular holophytochrome adduct occurs within viable cells. Since it features, including ubiquitin-dependent protein turnover, it is possible that the above results reflect holophytochrome was of some interest to determine whether phytochrome A is assembly in dying cells, additional experiments were per- light-labile in yeast. For this purpose, yeast cells expressing formed to confirm whether holophytochrome-containing apophytochrome were treated with PCB, washed with SD cells are viable. In the first experiment, cell aliquots were medium, and grown in SR medium under continuous red or removed at different times throughout the PCB incubation far-red light. An apophytochrome control culture that had not treatment and then plated on both yeast extract/peptone/ been treated with PCB was analyzed similarly. Total protein dextrose (YPD) and SD (supplemented with adenine, histi- extracts were prepared from all three cultures and then dine, and tryptophan) media. YPD medium was used to analyzed for phytochrome protein using an immunochemical confirm the total number of viable yeast cells, whereas SD assay. Since SD medium contains glucose, which represses medium provided an estimate ofplasmid-containing colonies. the transcription of apophytochrome in our expression plas- Table 1 shows that the total cell viability does not change mid pMphyA3, and SR medium lacks galactose, these growth during the first 2-h incubation with PCB. After a 4-h incu- conditions ensure that only the turnover of preexisting phy- Table 1. The effect of PCB incubation on cell viability and O h 4 h 18 h apophytochrome expression Viable cells, % PC expression, % Apo Pr Pfr Apo Pr Pfr Apo Pr Pfr Time, h - PCB + PCB + PCB 0 100 ± 1.7 100 ± 1.7 100 PC 1 98 ± 9.4 103 ± 4.9 100 2 107 ± 3.0 100 ± 4.5 100 4 88 ± 5.6 94 ± 6.3 100 FIG. 4. Holophytochrome stability in yeast. Yeast cultures (200 ml) expressing apophytochrome were harvested, resuspended in 2 ml 6 86 ± 6.0 89 ± 6.9 100 of fresh SR medium, and divided into three aliquots. Two aliquots Yeast cells expressing apophytochrome were incubated with or were incubated with 45 ,uM PCB for 5 h as described in Materials and without PCB as described in Materials and Methods. After various Methods, whereas the third aliquot was incubated with DMSO only incubation times, an aliquot of cells was removed, serially diluted, as an apophytochrome control. After extensive washing with fresh and plated on SD (supplemented with adenine, histidine, and tryp- SD medium, one of the PCB-treated cell suspensions was -placed tophan) medium. Colony numbers were recorded after 2 days of under continuous far-red light (Pr) while the other was placed under growth at 30°C and were normalized to the number ofcolonies found continuous red light (Pfr). The apophytochrome control flask (Apo) at the beginning of the incubation period (i.e., 0 h). Data for two was irradiated with continuous red light. All flasks were kept at room independent experiments with three replicates each are represented. temperature and shaken at 200 rpm. At the indicated time, aliquots The percent phytochrome (PC) expression was determined by testing were removed from each flask, and total whole-cell SDS protein eight random colonies from each SD plate for their ability to express extracts were prepared. Immunoblots of SDS/polyacrylamide gels the phytochrome polypeptide as measured immunochemically (see using a polyclonal phytochrome (PC) antibody are shown. Sample Materials and Methods for details). loadings represent equivalent volumes of the original cell culture. Downloaded by guest on September 30, 2021 Plant Biology: Li and Lagarias Proc. Natl. Acad. Sci. USA 91 (1994) 12539 tochrome molecules was being estimated. As shown in Fig. 6. Konomi, K. & Furuya, M. (1986) Plant Cell Physiol. 27, 4, the immunoblot analysis reveals that the phytochrome 1507-1512. polypeptide is quite stable in yeast and that no detectable 7. Elich, T. D. & Lagarias, J. C. (1987) Plant Physiol. 84, 304- difference is evident in the stability of apophytochrome, 310. Pr, 8. Elich, T. D. & Lagarias, J. C. (1989) J. Biol. Chem. 264, or Pfr, even up to 80 h of incubation with PCB. This 12902-12908. experiment also demonstrates that the phytochrome poly- 9. Lagarias, J. C. & Lagarias, D. M. (1989) Proc. Natl. Acad. Sci. peptide remained full-length throughout the incubation pe- USA 86, 5778-5780. riod. To test whether this phytochrome stability is strain 10. Deforce, L., Tomizawa, K., Ito, N., Farrens, D., Song, P.-S. specific, we also transformed pMphyA3 into yeast strain & Furuya, M. (1991) Proc. Natl. Acad. Sci. USA 88, 10392- Y14. Qualitatively identical results were obtained in this 10396. strain with no detectable difference observed between the 11. Wahleithner, J. A., Li, L. & Lagarias, J. C. (1991) Proc. Natl. stability of the Pr and Pfr forms (data not shown). These Acad. Sci. USA 88, 10387-10391. results indicate that yeast cells lack the turnover machinery 12. Kunkel, T., Tomizawa, K., Kern, R., Furuya, M., Chua, N.-H. & Schaefer, E. (1993) Eur. J. Biochem. 215, 587-594. that supports the light-dependent turnover of phytochrome 13. Li, L. & Lagarias, J. C. (1992)J. Biol. Chem. 267, 19204-19210. A. 14. Metzger, D., White, J. H. & Chambon, P. (1988) Nature Applications ofin Vivo Holophytochrome Assembly in Yeast. (London) 334, 31-35. The ability to reconsitute photoactive phytochrome in living 15. Schena, M. & Yamamoto, K. R. (1988) Science 241, 965-967. yeast cells should facilitate identification ofplant factors that 16. Mak, P., McDonnell, D. P., Weigel, N. L., Schrader, W. T. & interact with phytochrome in a light-dependent manner (e.g., O'Malley, B. W. (1989) J. Biol. Chem. 264, 21613-21618. components of the phytochrome signal transduction path- 17. Privalsky, M. L., Sharif, M. & Yamamoto, K. (1990) Cell 63, turn- 1277-1286. way, molecules that participate in the light-dependent 18. Purvis, I. J., Chotai, D., Dykes, C. W., Lubahn, D. B., over of phytochrome, etc.). Depending on the complexity of French, F. S., Wilson, E. M. & Hobden, A. N. (1991) Gene phytochrome-regulated transcriptional regulation pathways, 106, 35-42. eventually it may be possible to reconstitute phytochrome- 19. Pierrat, B., Heery, D. M., Lemoine, Y. & Losson, R. (1992) regulated gene expression in yeast. With suitable reporter Gene 119, 237-245. gene constructions, we anticipate that the yeast S. cerevisiae 20. King, K., Dohlman, H. G., Thorner, J., Caron, M. & Lefkow- will prove as useful for the analysis ofphytochrome structure itz, R. J. (1990) Science 250, 121-123. 21. Sherman, F. (1991) Methods Enzymol. 194, 3-21. and function as it has for mammalian receptors (14-20). 22. Laemmli, U. K. (1970) Nature (London) 227, 680-685. 23. Gollub, E. G., Liu, K.-P., Dayan, J., Adlersberg, M. & Sprin- This work was funded by National Science Foundation Grant son, D. B. (1977) J. Biol. Chem. 252, 2846-2854. MCB92-06110. 24. Guarente, L. & Mason, T. (1983) Cell 32, 1279-1286. 25. Lagarias, J. C., Kelly, J. M., Cyr, K. L. & Smith, W. O., Jr. 1. Kendrick, R. E. & Kronenberg, G. H. M. (1994) Photomor- (1987) Photochem. Photobiol. 46, 5-13. phogenesis in Plants (Nijhoff, Dordrecht, The Netherlands), 26. Berkelman, T. R. & Lagarias, J. C. (1986) Anal. Biochem. 156, 2nd Ed., p. 828. 194-201. 2. Quail, P. H. (1991) Annu. Rev. Genet. 25, 389-409. 27. Vierstra, R. D. (1994) in Photomorphogenesis in Plants, eds. 3. Lagarias, J. C. & Rapoport, H. (1980) J. Am. Chem. Soc. 102, Kendrick, R. E. & Kronenberg, G. H. M. (Nijhoff, Dordrecht, 4821-4828. The Netherlands), 2nd Ed., pp. 141-162. 4. Terry, M. J., Wahleithner, J. A. & Lagarias, J. C. (1993) Arch. 28. Quail, P. H., Schaefer, E. & Marme, D. (1973) Plant Physiol. Biochem. Biophys. 306, 1-15. 52, 128-131. 5. Jones, A. M., Allen, C. D., Gardner, G. & Quail, P. H. (1986) 29. Shanklin, J., Jabben, M. & Vierstra, R. D. (1987) Proc. Natl. Plant Physiol. 81, 1014-1016. Acad. Sci. USA 84, 359-363. Downloaded by guest on September 30, 2021