Saccharomyces Cerevisiae (Plant Photoreceptor/In Vivo Reconstitution/Linear Tetrapyrrole) LIMING LI and J
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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,