Considerable progress has been made in recent years on the biosynthesis of both apophytochrome and phytochromo- bilin (POB), the linear tetrapyrrole ...
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)
(10-12). In vitro holophytochrome assembly has been particularly useful for biochemical characterization of the structural features of both bilin chromophore and apophytochrome, which are necessary for pigment attachment and photoreversibility (10, 13). While the expression of recombinant apophytochrome and the in vitro assembly of holophytochrome is an invaluable research tool, the ability to reconstitute functionally active recombinant phytochrome in living cells will facilitate application of genetic methodologies to the analysis of photoreceptor structure and function. In this regard, the yeast Saccharomyces cerevisiae has proven particularly useful for genetic dissection of the structure and function of the mammalian steroid hormone receptor family (14-19) as well as the mammalian heterotrimeric G-protein coupled P2-adrenergic receptor (20). Like phytochrome, neither of these mammalian hormone receptor families occurs naturally in yeast, and their agonists are not known to regulate endogenous processes in this organism. The present studies were undertaken as the first step to develop an in vivo assay for phytochrome in yeast. Our studies show that yeast cultures expressing apophytochrome can assimilate exogenous PFB and phycocyanobilin (PCB), a chemically-related analog from algae, and incorporate either pigment into a photoactive holophytochrome in living cells. The in vivo spectrophotometric properties of these holophytochromes as well as their stability in yeast cells are also examined. These studies indicate that genetic approaches to the analysis of phytochrome structure and function in yeast are now practicable.
The biological activity of the plant photoreABSTRACT ceptor phytochrome requires the specific association of a linear tetrapyrrole prosthetic group with a large apoprotein. As an initial step to develop an in vivo assay system for structurefunction analysis of the phytochrome photoreceptor, we undertook experiments to reconstitute holophytochrome in the yeast Saccharomyces cerevisiae. Here we show that yeast cells expressing recombinant oat apophytochrome A can take up exogenous linear tetrapyrroles, and, in a time-dependent manner, these pigments combine with the apoprotein to form photoactive holophytochrome in situ. Cell viability measurements indicate that holophytochrome assembly occurs in living cells. Unlike phytochrome A in higher plant tissue, which is rapidly degraded upon photoactivation, the reconstituted photoreceptor appears to be light stable in yeast. Reconstitution of photoactive phytochrome in yeast cells should enable us to exploit the power of yeast genetics for structure-function dissection of this important plant photoreceptor.
Sensing and adapting to the light environment are essential for optimal plant growth and development. Plants therefore possess a number of photoreceptors that enable the perception of the direction, intensity, and/or spectral quality of light (1). These include photoreceptors for UV-B, UV-A/blue, and red/far-red regions of the light spectrum. The phytochrome photoreceptor mediates numerous photomorphogenetic responses to red and far-red light (2). Through its ability to reversibly photointerconvert between red- and far-red-light-absorbing forms, Pr and Pfr, phytochrome influences nearly every stage of plant development, from germination to floral induction and ultimately to senescence. To function as a receptor of visible light, the phytochrome molecule contains a linear tetrapyrrole pigment that is covalently bound to a large polypeptide via a thioether linkage (3). Light absorption by the phytochrome chromophore effects a protein conformational change that initiates a signaltransduction pathway(s) in the plant, the details of which are poorly understood at present. Considerable progress has been made in recent years on the biosynthesis of both apophytochrome and phytochromobilin (POB), the linear tetrapyrrole precursor of the phytochrome chromophore (reviewed in ref. 4). The observation that apophytochrome could be isolated from chromophoredeficient plants indicates that the synthesis of both components is not coupled in vivo (5-7). Subsequent in vitro studies have revealed that the assembly of photoactive holophytochrome occurs spontaneously when POB and apophytochrome are coincubated (8, 9). This feature of phytochrome biosynthesis has permitted many investigators to reconstitute holophytochromes in vitro using recombinant apophytochrome and linear tetrapyrrole chromophore precursors
Apophytochrome Expression in Yeast. The oat phytochrome A3 expression plasmid, pMphyA3, which contains a leu2 selection marker, was expressed in the yeast S. cerevisiae strain 29A (MATa leu2-3 leu2-112 his3-1 adel-101 trpl289) as described previously with the following modifications (11). Several individual colonies of yeast cells containing pMphyA3 were inoculated into 12 ml of synthetic raffinose (SR) medium supplemented with adenine, histidine, and tryptophan (each at 40 mg/liter) and grown overnight at 30°C with shaking at 300 rpm. The composition of SR is the same as synthetic dextrose (SD) medium except that glucose is replaced with raffinose (21). SR and SD both represent selective media for pMphyA3 since leucine is omitted. The 12-ml overnight cultures were used to inoculate 1 liter of SR medium in a 2-liter Fembach flask. This culture was incubated at 30°C for 10-12 h with shaking at 300 rpm. When the OD580 of the culture reached 0.3-0.6, 1% (wt/vol) galactose was added to induce apophytochrome expression. After a 24-h induction period, cells were utilized for in vivo holophytochrome assembly (see the next section) or for apophy-
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Abbreviations: PCB, phycocyanobilin; P4'B, phytochromobilin; Pr, red-light-absorbing form of phytochrome; Pfr, far-red-light-absorbing form of phytochrome; DMSO, dimethyl sulfoxide.
MATERIALS AND METHODS
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tochrome extraction according to the protocol of Wahleithner et al. (11). In Vivo Holophytochrome Assembly. Yeast cultures grown and induced as described above were collected by centrifugation for 5 min at 3000 rpm in an SS34 rotor. The resulting cell pellet was resuspended in fresh SR medium at a ratio of 0.5 g of fresh weight cells per ml. PCB or P(¶B [as 1 mM stock solutions in dimethyl sulfoxide (DMSO)] was then added to the cell suspension under a green safelight to give the desired final pigment concentration (see Results and Discussion). For in vivo spectrophotometric analysis and in vivo phytochrome stability studies, cell incubation mixtures were gently shaken for 5 h under a green safelight at room temperature or at 30°C. For the time course experiments of in vivo phytochrome assembly, a 280-,ul aliquot of each cell incubation mixture was removed at various times and transferred into a 50-ml polypropylene Falcon tube containing 30 ml of ice-cold wash buffer (50 mM NaCl/150 mM NaCl/1 mM EDTA/1 mM EGTA/1% DMSO). The resulting cell suspension was vortexed, and cells were collected by centrifugation at 3000 rpm for 5 min. After four such washes to remove free pigment, whole-cell SDS protein extracts and (NH4)2SO4fractionated protein extracts were prepared as described (11). The amount of holophytochrome that had assembled during the in vivo incubation period was determined using both spectrophotometric and zinc blot assays (see the following sections). Spectrophotometric Phytochrome Assays. In vivo spectrophotometric assay of holophytochrome in yeast cells was performed at 4°C using an Aviv/Cary 14DS UV/visible spectrophotometer equipped with a light-scattering sample compartment. Saturating red and far-red light irradiations (15 min for each) of stirred cell suspensions in 5-ml cuvettes were provided by a custom-made actinic light source [250-W, 24-V, Osram (Berlin) Xenophot HLX lamp] and red interference filters with a 10-nm band pass (650 nm for PCBapophytochrome adduct and 660 nm for P4B-apophytochrome adduct; Ditric Optics, Hudson, MA) or a far-red plastic filter (FRS 720 Plexiglas, Rohm & Haas dye no. 58015, 0.125 inch thick), respectively. The following parameters were employed for in vivo spectra: 2-nm wavelength increments, 10-nm band width, and 4.0-sec dwell time per reading. Holophytochrome concentrations in soluble protein extracts were estimated with a HP8450A UV/visible spectrophotometer using the absorbance difference assay described (13). Stability of Recombinant Holophytochrome in Yeast. These experiments were performed with S. cerevisiae strains 29A and Y14 (MATa adel his3 leu2-3 leu 2-112 trp 1-la), which had been transformed with the plasmid pMphyA3 (11). A 200-ml yeast culture was grown in SR medium and induced with galactose as described above in Apophytochrome expression in yeast. At the end of the induction period, cells were collected by centrifugation for S min at 5000 x g and then resuspended into 3 ml of sterile SR medium. The resulting cell suspension was divided into three equal parts, which were each transferred to a sterile 15-ml polypropylene Falcon tube. PCB (as a 1 mM stock solution in DMSO) was added to two of the three tubes to give a final PCB concentration of 45 ,uM and a final DMSO concentration of 4.5% (vol/vol). As a control, the same amount of DMSO was added to the third tube. All three cell suspensions were incubated at 30°C for S h with gentle shaking (100 rpm). After the incubation period, cells were washed four times with sterile SD medium by vortexing and centrifugation to remove free PCB. Washed cell pellets were then resuspended in 1 ml of sterile SR medium and used to inoculate 100 ml of fresh SR medium in a sterile 500-ml Erlenmeyer flask. One of the PCB-treated cultures was continuously irradiated with farred light (i.e., Pr culture), while the other was continuously irradiated with red light (i.e., Pfr culture). The third flask
Proc. Natl. Acad Sci. USA 91
containing DMSO-treated control cells (i.e., apophytochrome control) was irradiated with continuous red light. The far-red light source used for actinic irradiation consisted of two Sylvania F48T12/660 nm/VHO fluorescent tubes filtered with 0.125-inch-thick far-red FRS700 Plexiglas (Rohm & Haas dye no. 58015), whereas the red light source consisted of six Sylvania (Electric Products, Fall River, MA) F20T12 cool white fluorescent tubes, which were filtered through 0.125-inch-thick red Plexiglas (Rohm & Haas dye no. 2423). For these experiments, flasks were placed 6 inches (15.2 cm) from the light source on a rotary shaker and incubated at room temperature with a shaking speed of 200 rpm. After various incubation periods, 3-ml aliquots were removed from each culture for total protein isolation, and 10-ml aliquots were removed for preparation of (NH4)2SO4fractionated protein extracts as described by Wahleithner et al. (11). Whole-Cell Protein Isolation from Yeast Cells. Yeast suspension cultures (3 ml) were transferred into a 13 x 100 mm glass test tube and centrifuged for 5 min at 1000 x g. After removing the supernatant, 100 ,ul of 2x SDS sample buffer [125 mM Tris-HCl, pH 6.8/5.6% SDS/15% glycerol/5% 2-mercaptoethanol] and an equal volume of 0.5-mm acidwashed glass beads were added to the cell pellet. After the addition of 1 ,l4 of 200 mM phenylmethylsulfonyl fluoride in ethanol, the cell mixture was vortexed three times for 20 sec each. Between each 20-sec homogenization pulse, test tubes were kept on ice for at least 20 sec. After the final 20-sec vortexing step, an additional 100-,l aliquot of 2x SDS sample buffer was added to the mixture. After a brief mixing, homogenates were centrifuged for 2 min at 1000 x g, and the supernatants were removed and stored at -20°C prior to SDS/PAGE analysis. SDS/PAGE, Zinc Blot, Immunoblot, and Coomassie Blue Analyses. Yeast protein extracts were analyzed by SDS/ PAGE using 10% polyacrylamide minigels according to Laemmli (22). After electrophoresis, proteins were electrophoretically transferred to poly(vinylidene difluoride) membranes (Immobilon P; Millipore) for 1 h at 100 V. After transblotting, the same membrane was used for zinc blot, immunoblot, and Coomassie blue staining analyses as described (11).
RESULTS AND DISCUSSION Photoreversible Holophytochrome Adduct Assembly in Yeast. It is well established that yeast cells deficient in heme biosynthesis can grow normally in medium supplemented with heme (23, 24). Based upon these observations, it appeared reasonable that yeast cells would also be able to take up the structurally related linear tetrapyrrole (bilin) pigments. To test this hypothesis, we conducted experiments to determine whether yeast cells expressing recombinant oat apophytochrome A could assimilate exogenous bilins and produce photoactive holophytochrome intracellularly. The two bilin pigments, P4B and PCB, were chosen for these studies since both compounds have been previously shown to assemble with apophytochrome in vitro to form photoactive holophytochromes (11, 13). After a 5-h incubation period with 45 ,AM bilin and extensive washing, an in vivo spectrophotometric difference assay was performed. Fig. 1 Upper shows that the in vivo difference spectra for both PFB- and PCB-treated cultures are characteristic of the phytochrome photoreceptor. For the PFB-treated cultures, the absorbance difference maximum and minimum occurred at 660 nm and 730 nm, respectively. These values are very similar to those of native oat phytochrome A preparations (25). By comparison, the absorbance difference maximum and minimum of the PCB-treated cells were blue-shifted by 10 nm, occurring at 650 nm and 720 nm, values that are indistinguishable from
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Proc. Natl. Acad. Sci. USA 91 (1994)
0 0.5 1.0 2.5 4.0 5.5 8.5 (h)
0.02 0.01 U)
0U) -0.01a) -0.02-
0 .0 co
wavelength (nm) FIG. 1. Spectrophotometric analysis of recombinant holophytochrome 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 whole-cell suspensions were obtained (Upper). After the in vivo difference spectrophotometric measurements, (NH4)2504-fractionated protein extracts were prepared and spectrophotometrically Spectra obtained from PCB-treated yeast assayed (Lower). spectra obtained from P4DB-treated yeast cells; * *, cells; spectra obtained from the DMSO-only control cells. In this experiment, the amount of P4.B-apophytochrome adduct formed was -67% of that of the PCB-apophytochrome adduct. The difference spectra of the PFB-apophytochrome adduct in Upper and Lower are normalized to the scale of the PCB-apophytochrome spectra. ,
those of the PCB-apophytochrome A adduct produced in vitro (8, 11, 13). Control yeast cultures treated with DMSO alone failed to afford phytochrome photoactivity (Fig. 1 Upper), and yeast cells containing an antisense apophytochrome plasmid did not yield photoactive phytochrome after incubation with PCB (data not shown). For comparative purposes, phytochrome difference spectra were also obtained on soluble protein extracts prepared from both PCBand PNB-treated yeast cultures (Fig. 1 Lower). These difference spectra are indistinguishable from the in vivo difference spectra. Taken together, these investigations indicate that yeast cells are able to take up both bilins and to incorporate these pigments into functional holophytochromes. Time Course of Phytochrome Assembly for Yeast Cells. A time course experiment was performed to optimize conditions for in vivo assembly of phytochrome. Since purified PIB is difficult to obtain in large quantities, PCB was utilized for these experiments. Our experimental protocol entailed incubation of apophytochrome-expressing cells with 45 ,uM PCB at 300C followed by removal of a culture aliquot, protein extraction, and holophytochrome assay using a zinc blot procedure, The zinc blot procedure, which was originally designed for fluorescence detection of bilin-linked polypeptides in SDS gels (26), has been modified to permit quantitation of bilin attachment to apophytochrome after SDS/ PAGE and blotting to a poly(vinylidene difluoride) membrane (13). Fig. 2A shows the zinc blot analysis, which reveals the time dependence of the formation of a fluorescent polypeptide at 124 kDa. The fluorescence intensity of this band was only partially detectable after 30 min and continued to increase up to 4 h of incubation with PCB. Immunostaining of the same zinc blot membrane with a polyclonal phytochrome antibody indicated that this 124-kDa band corresponds to phytochrome and that an equivalent amount of
FIG. 2. Zinc-blot time course of phytochrome assembly in yeast. Yeast cells induced to express apophytochrome were treated with 45 ,uM PCB as described in Materials and Methods. At each time indicated, aliquots of the cell suspension were removed and protein extracts were prepared and fractionated with (NH4)2504. These extracts were resolved by SDS/PAGE, electroblotted to a poly(vinylidene difluoride) membrane, and sequentially analyzed with a zinc blot (A), immunostaining (B), and Coomassie blue-staining (C) procedures. Each lane contains 80 ,ug of total protein. The arrows on the left indicate the phytochrome polypeptide that migrates at 124 kDa. The dots on the right represent protein standards with molecular masses of 221, 115, 63, and 46 kDa.
apophytochrome was present in all sample lanes (Fig. 2B). These analyses also demonstrate that the phytochrome polypeptide remained full-length throughout the PCB incubation, thereby indicating that phytochrome does not undergo significant proteolysis under these experimental conditions. Coomassie blue staining of this membrane also confirms that equal amounts of total protein are present (Fig. 2C). Based on the above results, we conclude that the time-dependent change in the zinc-mediated fluorescence intensity of the phytochrome polypeptide represents quantitative differences in holophytochrome assembly. To corroborate the results of the zinc blot analysis, spectrophotometric phytochrome assays were performed on protein extracts from PCB-treated yeast cells. In this experiment, the percentage of in vivo holophytochrome assembly was estimated from the ratio of photoactive phytochrome measured in the initial protein extract to that determined in the same extract after subsequent incubation with PCB. Fig. 3 illustrates the percentage of in vivo holophytochrome assembly versus incubation time determined by this experiment. Consistent with the zinc blot analysis, this investigation shows that in vivo holophytochrome assembly was complete within a 4-h incubation period. The rate of phytochrome assembly does, however, vary from experiment to experiment, with saturation occurring between 2 and 6 h under our experimental conditions. As might be expected, the rate of in vivo assembly is affected by incubation temperature, bilin concentration, and cell growth conditions (unpublished observations). Similar time course experiments were also performed using two other compatible yeast strains. In all strains tested, photoactive holophytochrome could be reconstituted in vivo (data not shown). These results indicate that bilins are readily assimilated by yeast cells.
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Proc. Natl. Acad. Sci. USA 91
time (h) FIG. 3. Spectrophotometric time course of phytochrome assembly in yeast. Yeast cells induced to express apophytochrome were incubated with 45 uM PCB, and aliquots were removed after various incubation times as described in Materials and Methods. At each time point, soluble protein extracts were prepared and assayed for phytochrome spectrophotometrically. Each extract was then treated with 4 ,uM PCB, incubated for 1 h at 28°C, and then reassayed for phytochrome spectrophotometrically. The ratio of the amount of photoactive phytochrome before and after this in vitro PCB incubation x 100%o is plotted as a function of the length of the whole-cell incubation with PCB. Each point represents the average of the values from two independent experiments.
Photoreversible Holophytochrome Adducts Are Formed in Viable Yeast Cells. The ability to reconstitute photoreversible holophytochrome in yeast cells is expected to be a useful tool for the analysis of phytochrome structure and function. However, the application of yeast genetic methodology for this purpose requires that the formation of photoreversible holophytochrome adduct occurs within viable cells. Since it is possible that the above results reflect holophytochrome assembly in dying cells, additional experiments were performed to confirm whether holophytochrome-containing cells are viable. In the first experiment, cell aliquots were removed at different times throughout the PCB incubation treatment and then plated on both yeast extract/peptone/ dextrose (YPD) and SD (supplemented with adenine, histidine, and tryptophan) media. YPD medium was used to confirm the total number of viable yeast cells, whereas SD medium provided an estimate of plasmid-containing colonies. Table 1 shows that the total cell viability does not change during the first 2-h incubation with PCB. After a 4-h incuTable 1. The effect of PCB incubation on cell viability and apophytochrome expression Viable cells, % PC expression, % + PCB - PCB + PCB Time, h 0 100 ± 1.7 100 ± 1.7 100 1 98 ± 9.4 103 ± 4.9 100 2 107 ± 3.0 100 ± 4.5 100 4 94 ± 6.3 88 ± 5.6 100 6 86 ± 6.0 89 ± 6.9 100 Yeast cells expressing apophytochrome were incubated with or without PCB as described in Materials and Methods. After various incubation times, an aliquot of cells was removed, serially diluted, and plated on SD (supplemented with adenine, histidine, and tryptophan) medium. Colony numbers were recorded after 2 days of growth at 30°C and were normalized to the number of colonies found at the beginning of the incubation period (i.e., 0 h). Data for two independent experiments with three replicates each are represented. The percent phytochrome (PC) expression was determined by testing eight random colonies from each SD plate for their ability to express the phytochrome polypeptide as measured immunochemically (see Materials and Methods for details).
bation period, when holophytochrome assembly was nearly complete, >90% of the 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 numbers 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 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 time point and analyzed for apophytochrome expression. This involved inoculation of each individual colony into SR liquid medium, galactose induction, preparation of SDS whole-cell extracts, and SDS/PAGE and Western blot analysis as described in Materials and Methods. This experiment revealed that 100% of the colonies at every time point examined up to 6 h were able to express full-length apophytochrome (Table 1). Taken together with the evidence that nearly all of ligation-competent apophytochrome assembles in vivo within 4 h (Fig. 3), these experiments indicate that holophytochrome assembly occurs in viable yeast cells. Recombinant Holophytochrome Stability in Yeast. It is well established that phytochrome A is light labile in plants and that the protein half-life ofthe Pfr form is considerably shorter than that of the Pr form (reviewed in ref. 27). In cucumber seedlings for example, the half-life of PrA is 100 h compared with 1 h for PfrA (28). While the differential turnover of phytochrome A clearly plays an important role in the regulation of phytochrome A function and appears to be ubiquitin-mediated (29), the specific factors that mediate this differential turnover process are presently unknown. Since yeast and plants share many common eukaryotic cellular features, including ubiquitin-dependent protein turnover, it was of some interest to determine whether phytochrome A is light-labile in yeast. For this purpose, yeast cells expressing apophytochrome were treated with PCB, washed with SD medium, and grown in SR medium under continuous red or far-red light. An apophytochrome control culture that had not been treated with PCB was analyzed similarly. Total protein extracts were prepared from all three cultures and then analyzed for phytochrome protein using an immunochemical assay. Since SD medium contains glucose, which represses the transcription of apophytochrome in our expression plasmid pMphyA3, and SR medium lacks galactose, these growth conditions ensure that only the turnover of preexisting phyOh
Apo Pr Pfr
Apo Pr Pfr
Apo Pr Pfr
PC FIG. 4. Holophytochrome stability in yeast. Yeast cultures (200 ml) expressing apophytochrome were harvested, resuspended in 2 ml of fresh SR medium, and divided into three aliquots. Two aliquots were incubated with 45 ,uM PCB for 5 h as described in Materials and Methods, whereas the third aliquot was incubated with DMSO only as an apophytochrome control. After extensive washing with fresh SD medium, one of the PCB-treated cell suspensions was -placed under continuous far-red light (Pr) while the other was placed under continuous red light (Pfr). The apophytochrome control flask (Apo) was irradiated with continuous red light. All flasks were kept at room temperature and shaken at 200 rpm. At the indicated time, aliquots were removed from each flask, and total whole-cell SDS protein extracts were prepared. Immunoblots of SDS/polyacrylamide gels using a polyclonal phytochrome (PC) antibody are shown. Sample loadings represent equivalent volumes of the original cell culture.
Plant Biology: Li and Lagarias tochrome molecules was being estimated. As shown in Fig. 4, the immunoblot analysis reveals that the phytochrome polypeptide is quite stable in yeast and that no detectable difference is evident in the stability of apophytochrome, Pr, or Pfr, even up to 80 h of incubation with PCB. This experiment also demonstrates that the phytochrome polypeptide remained full-length throughout the incubation period. To test whether this phytochrome stability is strain specific, we also transformed pMphyA3 into yeast strain Y14. Qualitatively identical results were obtained in this strain with no detectable difference observed between the stability of the Pr and Pfr forms (data not shown). These results indicate that yeast cells lack the turnover machinery that supports the light-dependent turnover of phytochrome A. Applications of in Vivo Holophytochrome Assembly in Yeast. The ability to reconsitute photoactive phytochrome in living yeast cells should facilitate identification of plant factors that interact with phytochrome in a light-dependent manner (e.g., components of the phytochrome signal transduction pathway, molecules that participate in the light-dependent turnover of phytochrome, etc.). Depending on the complexity of phytochrome-regulated transcriptional regulation pathways, eventually it may be possible to reconstitute phytochromeregulated gene expression in yeast. With suitable reporter gene constructions, we anticipate that the yeast S. cerevisiae will prove as useful for the analysis of phytochrome structure and function as it has for mammalian receptors (14-20). This work was funded by National Science Foundation Grant MCB92-06110. 1. Kendrick, R. E. & Kronenberg, G. H. M. (1994) Photomorphogenesis in Plants (Nijhoff, Dordrecht, The Netherlands), 2nd Ed., p. 828. 2. Quail, P. H. (1991) Annu. Rev. Genet. 25, 389-409. 3. Lagarias, J. C. & Rapoport, H. (1980) J. Am. Chem. Soc. 102, 4821-4828. 4. Terry, M. J., Wahleithner, J. A. & Lagarias, J. C. (1993) Arch. Biochem. Biophys. 306, 1-15. 5. Jones, A. M., Allen, C. D., Gardner, G. & Quail, P. H. (1986) Plant Physiol. 81, 1014-1016.
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6. Konomi, K. & Furuya, M. (1986) Plant Cell Physiol. 27, 1507-1512. 7. Elich, T. D. & Lagarias, J. C. (1987) Plant Physiol. 84, 304310. 8. Elich, T. D. & Lagarias, J. C. (1989) J. Biol. Chem. 264, 12902-12908. 9. Lagarias, J. C. & Lagarias, D. M. (1989) Proc. Natl. Acad. Sci. USA 86, 5778-5780. 10. Deforce, L., Tomizawa, K., Ito, N., Farrens, D., Song, P.-S. & Furuya, M. (1991) Proc. Natl. Acad. Sci. USA 88, 1039210396. 11. Wahleithner, J. A., Li, L. & Lagarias, J. C. (1991) Proc. Natl. Acad. Sci. USA 88, 10387-10391. 12. Kunkel, T., Tomizawa, K., Kern, R., Furuya, M., Chua, N.-H. & Schaefer, E. (1993) Eur. J. Biochem. 215, 587-594. 13. Li, L. & Lagarias, J. C. (1992)J. Biol. Chem. 267, 19204-19210. 14. Metzger, D., White, J. H. & Chambon, P. (1988) Nature (London) 334, 31-35. 15. Schena, M. & Yamamoto, K. R. (1988) Science 241, 965-967. 16. Mak, P., McDonnell, D. P., Weigel, N. L., Schrader, W. T. & O'Malley, B. W. (1989) J. Biol. Chem. 264, 21613-21618. 17. Privalsky, M. L., Sharif, M. & Yamamoto, K. (1990) Cell 63, 1277-1286. 18. Purvis, I. J., Chotai, D., Dykes, C. W., Lubahn, D. B., French, F. S., Wilson, E. M. & Hobden, A. N. (1991) Gene 106, 35-42. 19. Pierrat, B., Heery, D. M., Lemoine, Y. & Losson, R. (1992) Gene 119, 237-245. 20. King, K., Dohlman, H. G., Thorner, J., Caron, M. & Lefkowitz, R. J. (1990) Science 250, 121-123. 21. Sherman, F. (1991) Methods Enzymol. 194, 3-21. 22. Laemmli, U. K. (1970) Nature (London) 227, 680-685. 23. Gollub, E. G., Liu, K.-P., Dayan, J., Adlersberg, M. & Sprinson, D. B. (1977) J. Biol. Chem. 252, 2846-2854. 24. Guarente, L. & Mason, T. (1983) Cell 32, 1279-1286. 25. Lagarias, J. C., Kelly, J. M., Cyr, K. L. & Smith, W. O., Jr. (1987) Photochem. Photobiol. 46, 5-13. 26. Berkelman, T. R. & Lagarias, J. C. (1986) Anal. Biochem. 156, 194-201. 27. Vierstra, R. D. (1994) in Photomorphogenesis in Plants, eds. Kendrick, R. E. & Kronenberg, G. H. M. (Nijhoff, Dordrecht, The Netherlands), 2nd Ed., pp. 141-162. 28. Quail, P. H., Schaefer, E. & Marme, D. (1973) Plant Physiol. 52, 128-131. 29. Shanklin, J., Jabben, M. & Vierstra, R. D. (1987) Proc. Natl. Acad. Sci. USA 84, 359-363.