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The Plant Cell, Vol. 4, 161-171, February 1992 O 1992 American Society of Plant Physiologists Localization of Protein-Protein lnteractions between Subunits of Phytochrome Michael D. Edgerton and Alan M. Jones’ Department of Biology, University of North Carolina, Chapel Hill, North Carolina 27599-3280 We have used a nove1assay based on protein fusions with h repressor to identify two small regions within phytochrome’s carboxy-terminal domain that are capable of mediating dimerization. Using an in vivo assay, fusions between the DNA binding, amino-terminal domain of h repressor and fragments from oat PhyA phytochrome have been assayed for increased repressor activity, an indicator of dimerization. In this assay system, regions of oat phytochrome between amino acids V6234673 and N1049-Q1129 have been shown to increase repressor activity. These short spans are highly conserved between proteins belonging to the phytochrome PhyA family. Embedded within these sequences are four segments that could potentially form amphipathic a helices. Two of the segments are well conserved between PhyA phytochrome and phytochromes encoded by the phyB and phyC genes, suggesting that heterodimers might form by way of subunit interaction at these sites. INTRODUCTION Phytochrome is a plant photoreceptor involved in photomorphogenic events ranging from seed germination and deetiolation to the induction of flowering. Phytochrome initiates some of these diverse responses over a fluence range that spans at least seven orders of magnitude (e.g., Jones et al., 1991). In this regard, phytochrome is unique among known photoreceptors. Also, phytochrome is uniquely photoreversible between a red light-absorbing form (Pr) and a far-redlight-absorbing form (Pfr) using red or far-red light, respectively. The Pfr form is thought to be the physiologically active form of phytochrome. In the natural environment, photoreversibility enables plants to detect both lightldark transitions and changes in redlfar red light ratios that occur under conditions such as shading in a forest canopy (Smith, 1982). Phytochrome is a homodimer of two independently photoreversible subunits. Each subunit is a 120- to 127-kD polypeptide with a covalently linked chromophore. Limited proteolysis has demonstratedthat phytochrome is divided into two major domains. The protein is composed of (1) a 65-kD aminoterminal domain that includes the chromophore and all structural requisites necessary for photoreversibility (Jones et al., 1985) and (2) a 60-kD carboxy-terminal domain that contains structures required for dimerization (Jones and Quail, 1986) and sequences possibly involved in rapid, light-mediated To whom correspondence should be addressed at Department of Biology, 010A Coker Hall, CB# 3280, University of North Carolina, Chapel Hill, NC 27599-3280. degradation of the protein (Shanklin et al., 1989). Phytochrome quaternary structure has been studied using ultracentrifugation (Jones and Quail, 1986), electron microscopy (Jones and Erickson, 1989), and size exclusion chromatography (SEC) (Lagarias and Mercurio, 1985).These studies and others have produced a rough picture of phytochrome quaternary structure. Phytochrome is tripartite in structure, consisting of two interacting carboxy domains that dimerize to form a base from which extend the two globular amino-terminal domains. The amino- and carboxy-terminal domains of each subunit are joined by a region defined as the hinge (Jones and Erickson, 1989). Comparison of the known physical properties of phytochrome with kinetic descriptions of phytochrome responses has led to a dimeric model for phytochrome action (VanDerWoude, 1985,1987). In VanDerWoude’s model, cells are thought to preceive mixed dimers in which one subunit is in the Pr form and the second subunit in Pfr conformation at low light levels. A second phytochrome-responsivesystem would also be measuring total Pfr levels at higher light levels. This model provides a possible explanation for the extraordinary fluence sensitivity of this photoreceptor and emphasizes the importance of its dimeric structure in attaining this sensitivity. Also, the existente of two or more different types of phytochrome raises the possibility of heterodimer formation. Pratt et al. (1991) have recently provided biochemical evidence that such heterodimers do indeed form in solution. For these reasons, we have focused our efforts on identification and characterization of regions of the protein involved in dimerization. 162 The Plant Cell We have developed a nove1 assay based on gene fusions to the DNA binding domain of the h repressor (cl) that has enabled us to identify segments of phytochrome capable of mediating homotypic protein-protein interactions that may be involved in dimerization. RESULTS h Repressor Assay for Dimerization Covalent cross-linking and SEC have been used successfully to map the dimerization region of phytochrometo a 37-kDtryptic fragment derived from the carboxy-terminalhalf of phytochrome (Jones and Quail, 1986). However, these methods have been limited in their ability to more precisely localize the dimerization region because of (1) aggregation of smaller phytochrome fragments (Yamamoto and Tokutomi, 1989), (2) the low resolution to which most monoclonal antibody epitopes have been mapped (Cordonnier, 1989), and (3) the limitations of SEC in distinguishing between dimeric molecules and molecules with extended shapes. We have developed an assay for dimerization that allows fine mapping of dimerization regions or, more precisely, sites of homotypic protein-protein interaction, and avoids the problems mentioned above. The premise of our assay is to begin with a protein that must be a dimer to function, remove that protein's dimerization region, and replace it with segments of a second protein. If one of the segments of the second protein contains a region through which the new chimeric protein is able to properly self-associate, activity will be restored. We have used the h repressor (cl) as the first component in our assay system. The native h repressor is a homodimer of 26-kD (236 residues) subunits. The protein is divided into two domains: an amino-terminal, DNA binding domain (residues 1 to 92) and a carboxy-terminal domain (residues 132to 236), which includes almost all of the structures necessary for dimerization of the repressor. The native h repressor binds to its operator sequence (OR1),with a dissociation constant of -2 x 10-8 M. In contrast, h repressor's aminoterminal domain, which contains only a single interprotein contact site, binds to OR1with a dissociation constant of -6 x M (Sauer et al., 1990). In our h repressor fusion vector, pME10, we have placed a polylinker immediately following the sequence encoding repressor helix 5, which ends at V92. Helix 5 was included in this construct even though it is a weak dimerization region because it may be requiredfor proper positioning of two amino-terminal domains on their operator sequence (Hecht et al., 1983; Weiss et al., 1987). In our dimerization assay, illustrated schematically in Figure 1, h repressor fusions are expressed under the control of the lactose promoter in pME10. Levels of expression from this promoter are increased by the addition of isopropyl P-D-thiogalactopyranoside (IPTG), which reduces the affinity of Lacl for its operator sequence. To report repressor activity, f LacZ hPf7 Figure 1. Schematic Diagram of h Repressor Dimerization Assay. Expression of 1 repressor fusion proteins from pMElO constructs is controlled by a lac promoter. The addition of IPTG leads to increased levels of fusion protein expression through its interaction with Lacl. To report repressor activity, expression was carried out in a cell line that contains lacZ under the control of ~ P RUpon . dimerization, h repressor fusions bound to PR,decreasing p-galactosidasesynthesis. Thus, the amount of p-galactosidaseactivity observed ata given leve1of IPTG depended upon the ability of the h repressor fusion proteins to form active dimers. Amp', ampicillin resistant; aa, amino acid. expression is carried out in a strain that contains a lacZ gene under the control of the h rightward promoter (PR). Thus, in this system, addition of IFTG leads to a decrease in fi-galactosidase levels. The manner in which these levels decrease is dependent upon both the affinity with which the repressor binds to its operator and the amount of repressor present. As an initial test of this system, we replaced the carboxyterminal half of h repressor with a portion of a yeast regulatory protein, GCN4, known to contain a leucine zipper dimerization motif (Hope and Struhl, 1987). The plasmid expressing this protein, pMElO/GCN4, contains sequences encoding GCN4 amino acids L169-R281 inserted into the Smal site of pME10. The resulting protein was stable in Escherichia coli, accumulating to 1 to 2% of total protein when induced with 0.5 mM IPTG. Cells containing pMElOlGCN4 were resistant to h phage infection. PGalactosidase levels measured in cells lysogenic for h200 were similar to those expressing the MElO/GCN4 protein and those expressing full-length h Phytochrome Dimerization represser, as shown in Figure 2A. Furthermore, the protein could be seen to form dinners when assayed by SEC, as shown in Figure 2B. As estimated by size exclusion data, the ME10/ GCN4 fusion protein has a dimerization constant in the micromolar range (between 2.5 and 25 nM). Hu et al. (1990) have independently used a similar assay to further study the leucine zipper region of GCN4. Their assay, as did ours, had approximately a 10-fold range in sensitivity. PLASMID #pfu [IPTG]x10' 6 M [IPTGJx 10-6M 0 1.0 10 0 1.0 10 950 850 750 0 0 0 4700 4900 2500 132 122 102 450 250 200 0 0 0 FG750 ME10 (5-Galactosidase ME10/GCN4 B Fraction Number 5 10 15 20 -•-71 — 44 3 8) en -28 ME10/_ GCN4 (0 -18.3 -15.3 t t 67 45 t 29 Apparent Molecular Mass (kD) Figure 2. A ME10 Fusion Protein Containing a Leucine Zipper Behaves as a Dimer. (A) A fusion between the amino-terminal DNA binding domain of X represser and sequences including a leucine zipper dimerization motif from GCN4 (L169-R281) was able to prevent lytic growth of wild-type X phage and suppress p-galactosidase activity in a manner similar to full-length X represser (encoded by FG750) when induced with IPTG. (B) ME10/GCN4 fusion protein was analyzed by SEC and found to be in equilibrium between monomeric and dimeric forms. Bacterial extract (100 jiL) containing ~25 mM ME10/GCN4 was separated on a TSK2000 size exclusion column. Individual fractions were collected and analyzed by immunoblotting with anti-X represser antiserum NCOS. Elution points for carbonic anhydrase (29 kD), ovalbumin (45 kD), and BSA (67 kD) are indicated. Fusion protein can be seen to have eluted as two peaks, presumably representing monomeric and dimeric forms. Both peaks migrated with slightly larger molecular mass than would be expected if ME10/GCN4 (213 residues) were a simple globular protein (48 kD expected for the dimer, 24 kD for the monomer), pfu, plaque-forming units. 163 Potential Dimerization Regions Reside between Residues V623-S673 and N1049-Q1129 Our efforts to map potential dimerization region(s) in phytochrome have focused on the carboxy-terminal half of the protein that has previously been shown to behave as a dimer (Jones and Quail, 1986). We have defined the carboxy-terminal domain as beginning at approximately amino acid L600 of oat PhyA APS, based on sequence data derived from fragments of proteolytically digested phytochrome (Grimm et al., 1988). Throughout this report, phytochrome amino acid designations will be in reference to those encoded by the oat gene phyA X\P3 (Hershey et al., 1985), with the initiation methioninecodon encoding amino acid 1. In reporting the results from our in vivo assay, we have defined a new constant, relative repression (Rr), which is a unitless value representing the relative ability of each fusion to repress p-galactosidase production. This value is derived by dividing the concentration of IPTG yielding half-maximal repression for a given construct by the concentration of IPTG that yields half-maximal repression for pME10 with no insert. Because the dimerization constant for phytochrome has not been measurable using conventional dissociation techniques (e.g., Jones and Quail, 1986; Choi et al., 1990), we use the Rr value as a relative measure of each fusion protein's dimerization coefficient. Segments encoding pieces of the carboxy-terminal half of oat phytochrome were excised from pCIB315 (Thompson et al., 1989) and inserted into pME10. Initially, an Xmnl fragment encoding amino acids V623-E1048, nearly all of the carboxyterminal half, was ligated into the Smal site of pME10. As expected, this construct, pME10/PC623-1048, repressed (3-galactosidase activity as IPTG levels increased. pME10/ PC623-1048 showed half-maximal repression of p-galactosidase at 0.9 ± 0.4 uM IPTG (n = 4). pME10 showed halfmaximal repression at 5.4 ± 0.1 uM IPTG (n = 4), yielding an Rr value of 6 ± 0.5 for pME10/PC623-1048. Thus, the level of IPTG giving half-maximal repression was roughly sixfold lower than the truncated X represser, ME10, encoded by pME10, as shown in Figure 3A. Unlike ME10/GCN4, the protein encoded by this construct and most other ME10/phytochrome fusions did not accumulate to significant levels when induced with high levels of IPTG (0.5 mM). Smaller fragments of phytochrome coding sequences spanning the entire carboxy-terminal domain were assayed individually (Figure 3B). Only four of the fusions were found to have Rr values significantly higher than pME10. Three constructs, pME10/PC488-673, pME10/PC623-723, and pME10/ PC599-683, all have in common sequences encoding a 5.5-kD region located between residues V623 and S673. Other constructs lacking these sequences had represser activity approximately equal to that encoded by pME10 (Figure 3A). One other construction with a relatively high Rr value was pME10/PC914-1129. Because represser fusion proteins containing the phytochrome segment between M914 and E1048 do not have represser activity, we concluded that a 164 The Plant Cell To separate these two factors, we have measured levels of fusion protein produced upon induction with 100 \iM IPTG (the lowest level of IPTG needed to reliably detect the fusion protein in immunoblots of crude extracts) by five constructs and pME10 by immunoblotting with polyclonal antiserum, NCOS, raised against X. represser. As shown in Figure 4, protein concentration was found to vary between constructs when induced to this level; however, this variation did not correlate with Rr values. For example, plasmids encoding proteins with Rr values of >1.0 were seen to be expressed at both relatively high (pME10/PC488-673) and low (pME10/PC599-683) levels. Plasmids encoding fusion proteins with Rr values of approximately 1.0 were also seen to vary in abundance. Thus, the observed Rr values do not reflect simple changes in either protein abundance or represser strength alone. CO. 1.0 2.5 5.0 10 25 50 [IPTG] (uM) B Amino Domain • CO2H ME10/PC623-1048 623——————— ME10/PC623-723 623 iri-i——723 ME10/PC488-673 488- ME10/PC599-683 3 4 5 6 Rr -1048 6.0 -11048 0.7 45.2 4.3 ^673 ME10/PC849-982 ME10/PC488-557 2 2.6 H:;:>723- ME10/PC723-1048 1 Carboxy Domain 849- -982;i; 488——557;g 0.5 1 599-SS-683 ME10/PC685-815 (585———815 ME10/PC914-1129 ill 0.8 2.2 0.9 914- (0 CO 29.5 2.4 Figure 3. Phytochrome Dimerization Regions Are Located between Residues V623-S673 and N1049-Q1129. (A) Dose-response curves for ME10/phytochrome gene fusions. Gene fusions between X represser and phytochrome segments were expressed in cells lysogenic for X200 and assayed for 3-galactosidase activity as IPTG concentration was increased. ME10 (•), ME10/PC6231048 (A), ME10/PC623-723(O), ME10/PC685-815 (D), and FG750 (fulllength c/) (•). The abscissa is log scale. (B) Map of ME10/phytochrome fusion proteins and their relative repression values. Relative repression values (Rr) are defined as the concentration of IPTG necessary to half maximally repress 3-galactosidase synthesis by ME10 divided by the concentration of IPTG, which half-maximally represses p-galactosidase expression with the construct being assayed. R, values are the mean of at least three separate experiments. All amino acid numbers are in reference to the oat phyA AP3 gene (Hershey et al., 1985). dimerization structure in the protein ME10/PC914-1129 resides carboxyl to residue E1048. Therefore, these data indicated that potential dimerization regions are located between residues V623-S673 and N1049-Q1129. Relative repression values are a function of both the dimerization constant and protein concentration for each construct. JS 3 U « O 18.7 15.5 Rr 1.0 0.9 2.6 0.7 4.3 0.8 Figure 4. Expression Levels of ME10 Fusion Proteins. Soluble protein (100 |ig) from cells induced with 100 uM IPTG was assayed for fusion protein expression levels by immunoblotting with anti-X. represser antiserum NCOS. The relative repression value (Rr) for each fusion is displayed below each lane. Lane 1, ME10; lane 2, ME10/PC685815; lane 3,10/PC599-683; lane 4, 10/PC723-1048; lane 5, 10/PC488673; lane 6, ME10/PC849-982. Levels of individual fusion protein can be seen to vary when induced with 100 uM IPTG. There is no apparent correlation between protein concentration and R, values at this level of induction. In each lane, two nonspecific bands can be seen; these serve as serendipitous internal controls for total protein concentration. Phytochrome Dimerization To further address the relative contributions of protein concentration and DNA binding affinity to R, values, we have used a filter binding assay to measure the amount of active DNA binding protein present in extracts of cells grown in the concentrations of IPTG used in measurements of R, values. PGalactosidase level was also determined for each IPTG concentration by both immunoblotting of the crude extracts and by activity measurement. By using saturating levels of radiolabeled operator DNA, we are able to measure the total abundance of OR1binding protein, independent of binding affinity (Riggs et al., 1970). Figure 5 shows that at the low levels of IPTG required to repress lacZ, the amount of DNA binding activity in extracts expressing MElO/PC914-1129 fusion protein and ME10 protein is the same, indicating that the level of the phytochrome repressor fusion protein and MElO protein is the same at the IPTG concentrationsmeasured. However, the repressivity of ME101PC914-1129 protein, as measured through p-galactosidaselevels, is much greater than for ME10 protein. Therefore, the decrease in p-galactosidase activity reported in R, values for pMElO/PC914-1129 is due to an increase in the affinity of the fusion protein for operator sequences dueto an improved ability to dimerize and not due to a difference in protein levels. DlSCUSSlON We are interested in the protein structure that forms and maintains phytochrome as a dimer because this dimeric character 112. 3OW I t ' 1 """I ' ' ' ""'I -i 1.50 -1.25 7 I I -" - 0.75 2 a -0.50 5w t 0 9 Av ..,:: .-,.--_. . . ,., #. e-Ya 12:5' - 0.25 6 O" 165 influences phytochrome fluence sensitivity and possibly its range of different responses. We began our study by noting that phytochrome behaves as a dimer even at concentrations at the lower detection limits of analytical assays and that phytochrome secondary structure is more sensitive to denaturants than its quaternary structure, suggesting to us that standard equilibria methodologies would not work to characterize dimerization (A.M. Jones, unpublished data). lnitial experiments with phytochrome expression in nonfusion systems were not successful because nearly all phytochrome sequences we expressed were nonnative, although coexpression with chaperonins did help significantly (M.D. Edgerton and A.M. Jones, unpublished data). It was therefore necessary to develop a new method for probing phytochrome structure. The method we have developed relies on modifying a stable prokaryotic protein, hcl, such that its activity is dependent, in part, upon additional foreign protein sequences involved in dimerization. Using this technique, we have identified two regions of phytochrome that are involved in dimerization, one residing between residues V623-S673 and the other between N1049Q1129. Each region when fused to the truncated h repressor restored repressor activity. In the first region, V623-S673, severa1 overlapping clones were assayed together with flanking regions to delineate the dimerization region. We have used immunoblot analysis to show that while the concentrations of these proteins vary at high IPTG levels, the variation does not correlate with R, values. Furthermore, fusions expressed at identical levels (ME10, MElO/PC599-683, and MElO/PC685815; Figure 4) demonstrate that, at least in this one case, phytochrome sequences are mediating dimerization. The second region that we mapped, N1049-Ql129, never accumulated to high enough levels to reliably quantitate by immunoblotting. Therefore, an in vitro DNA binding assay was used to demonstrate that while the MElO/PC914-1129 and ME10 protein concentrations varied with IPTG concentration in nearly identical fashion, the clone containing phytochrome sequences was better able to repress lacZ synthesis. The possible structure of these regions and the significance of phytochrome dimerization are discussed below. Structure Prediction of the Dimerization Regions ' 2; ' ;a' tIPTG1 (WW I i5' ' A' ' $0 Figure 5. Levels of Active MElO Fusion Protein Assayed by Filter Binding. Cells containing either pMElO (filled symbols) or pMElO/PC914-1129 (opensymbols) were grown in increasingamounts of IPTG.Crudecellular lysates were preparedby freeze-thaw extraction and assayed for total amount of active repressor by filter binding in the presence of excess labeledoperator DNA (squares). PGalactosidaselevels for each extract were also determinedby quantitative immunoblotting(circles). Values shown are from a single experiment. Repeat experimentsyielded qualitatively similar results, but differed in absolute levels of operator bound (OR1),perhaps due to differences in extraction efficiency. A comparison of the oat phytochromedimerization sequences to PhyA protein sequences from five other plants in Figure 6 shows that the regions between amino acids V623-SW3 and N1049-Ql129 are well conserved. The sequences between V623 and S673 are particularly well conserved, with 72% amino acid identity between corn and pea, the least similar of the six PhyA proteins examined. In comparison,the entire carboxyterminal domains have only 58% amino acid identity between corn and pea. This indicatesthat these conserved regions may be important in phytochrome function. The region between V623 and S673 contains three stretches that could potentially form amphipathic a helices, shown as 166 The Plant Cell AA# helix c helix a AA# helix b AA# AEL M A ~ ~ F E E D NEK ADL L S ~ Y E D D N KE PTAMGQ PAK... PSAVGR NK. HKSRTT HKLKG. ... 1129 1128 1131 1122 1124 1125 Figure 6. Conservation of Phytochrome Dimerization Regions. Sequence for the dimerization regions between V623-S673, N1049-Q1129,and flanking sequences of six different PhyA proteins (Hershey et al., 1985; Sharrock et al., 1986; Sato, 1988; Christensen and Quail, 1989; Kay et al., 1989; Sharrock and Quail, 1989). Putative helices a, b, c, and d are boxed. Residues that are 100°!o identical between all six phytochrome sequences are highlighted. Gaps in the sequence are represented by dots. AA, amino acid; A.t., Arabidopsis. helical wheel plots in Figure 7. Although we recognizethe limitations of secondary structuralpredictions(e.g., Nishikawaand Noguchi, 1991),for brevity we will refer to these potential helices as helices throughout this communication. The helices have charged and hydrophobic residues alternating with a periodicity of three to four residues. Chou-Fassman and Garnier-Osguthorpe-Robosonpredictionsof secondary structure (Devereux et al., 1984) assign helix propensity for the region between V623 and S673, particularly helix c (Figure 7; predictions not shown). We have defined the ends of these helices by choosing the preferred N- and C-cap residues described by Richardson and Richardson (1988). The amphipathic character of these three helices is conserved in all of the PhyA protein sequences examined. Helices a and c (Figure 7) are also well conserved between protein sequences encoded by two other membersof the phytochromegene family, phyS and phyC. Amphipathic a helices are often involved in protein-protein interaction and both heterotypic and homotypic dimerization (Segrest et al., 1990). The free energy change upon dimerization can be estimated from the energy of water exclusion for each helix (Richards, 1977). Using this method, the free energy of dimerization is calculated as 8.85, 8.7, and 4.8 kcallmol for helices a, b, and c (Figure7). As a comparison, the free energy of dimerization for the 24-amino-acid-long helix c of catabolite gene activation protein is 7.6 kcallmol when calculated by this method (McKay et al., 1982). Two other points of interest on the structure of the region between V623 and S673 are that (1) it is likely to be buriedwithin the protein and (2) it is adjacent to areas that are probably differentially exposed to solvent in Pr and Pfr forms. Computer predictions of surface probability suggest that the region between roughly amino acids S620 and E690 are located within the interior of phytochrome. More convincingly, this region is not readily accessible to proteases (Grimm et al., 1988), and a monoclonal antibody, GO-1, specific to this region is only able to recognize phytochrome by immunoblotting, and not by ELISA (Pratt et al., 1991). Sequences on either side of V623S673 are modified by a variety of agents when phytochrome is in the Pfr conformation. For example, the regions between L559-V669 and V748-G831 are thought to be ubiquitinatedas Pfr (Shanklin et al., 1989), K753 is more accessible to proteases as Pfr than Pr (Grimm et al., 1988), and S599 is preferentially phosphorylated in the Pfr conformation (McMichael and Lagarias, 1990). Sequences between N1049 and Q1129 contain a second region identifiedas being involved in protein-protein interaction. Overall, this segment is no better conserved than the entire carboxy-terminal domain. There is 58% amino acid identity between pea and corn carboxy-terminaldomains, versus 56% for this region. However,sequences between N1094 and G1110 show 71% amino acid identity between pea and corn. These sequences also have the potential to form an amphipathic a helix, depicted in a helical wheel plot in Figure 8. As shown in Figure 8, helix d, like helices a and c (Figure 7), is well conserved between proteins encoded by phyA, phyS, and phyC. However, PhyC protein contains an additional residue in what would be the center of helix d (Figure 8). Helix d, defined using preferred N- and C-cap residues, has a predicted free energy of water exclusion of 8.15 kcal/mol. An analysis of helical hydrophobic moments for four phytochrome proteins has also suggested that the region around helix d (Partis and Grimm, Phytochrome Oimerization M622 A629 phyA con: S E M V R L y E T A T V P A.t. phyB: R E M V R L I E T A T V P A.t.phyC: N E M V R L I D T A A V P 167 1990) as well as the regions around helices a and c have the potential to form an amphipathic a helix (Parker and Song, 1990). The leucine zipper is a common dimerization motif involving amphipathic a helices. Attempts to model the phytochrome dimerization sequences as coiled coils using both the algorithm of Lupas et al. (1991) and molecular dynamics simulations based on the leucine zipper structure described by OShea et al. (1991) have failed to support a coiled coil structure for these regions. Jones and Quail (1986) found that an 80-kD tryptic phytochrome fragment containing an intact amino terminus appeared to behave in SEC more like a monomer, implying that the dimerization region should lie in the carboxy-terminal 40-kD region (approximately residues 750 to 1129). Keeping in mind that SEC has low resolution, the data of Jones and Quail(l986) taken together with this work suggest that the region identified between V623 and S673, although important in protein-protein interactions, may not be sufficient to maintain phytochrome as a dimer. Significance of the Dimerization Regions "642 A649 phyAcon: G L V N G W N 7K i A E LTGL A.t. phyB: G C I N G W N A K I A E L T G L A.t. phyC: G V I N G W N S K A A E V T G L Severa1 plant developmental responses are regulated by phytochrome over a wide fluence range. The dimeric model of phytochrome action proposed by VanDerWoude (1985) postulates that at very low fluence levels, 0.1 to 100 nmolh" (e.g., Jones et al., 1991), plants have the ability to detect mixed phytochrome dimers, i.e., dimers in which one member of a pair is in the Pr conformation and the second member in the Pfr conformation. At higher fluences, 0.01 to 1.0 mmol/m2, plants measure the total amount of Pfr-Pfr dimers present. Thus, in VanDeWoude's model, dimerization extends the range of fluence sensitivity by three or more orders of magnitude. Although VanDerWoude's model is generally accepted, it should be noted that an alternative model by Blaauw-Jansen (1983) has been proposed that is not based upon the dimeric nature of phytochrome. Blaauw-Jansen proposes a fluencesensitive activation and deactivation of specific Pfr proteases. Figure 7. Consensus Sequence and Helical Wheel Plots of Putative Amphipathic a Helices between S620 and S673. phyAcon: e A I G E H i L T L V E D D A.t. phyB: E A M G K S L V S v L I Y K A.t. phyC: Q A I G K P * V S A L V E D D Helices c, a, and b are plotted in a helical wheel format to demonstrate the amphipathic character. The sequence shown is from the oat phyA gene product. Hydrophobic amino acids are representedby stip pled circles. Charged amino acids are indicated by (+) and (-) signs. The consensus (con) sequence based upon the six phytochrome sequences shown in Figure 6 is shown below each wheel. Upper case letters represent five or six identical residues; lower case letters represent three or four identical residues; two residues sharing the same line indicate positions at which all monocots had one residue (top) and all dicots a second residue (bottom). The corresponding sequences of the Arabidopsis (A.t.) phyB and phyC gene products are shown beneath each consensus sequence. Dots represent gaps, and carets represent the position of a residue. 168 The Plant Cell METHODS Bacterial Strains and Cloning 1109 D1102 L1095 1106 7 phyAcon: g L 1 L M N G D V R 1 R E A G A.t.phyB:K I L K L M N G E V Q Y I R E S E A.t.phyC: K L V K L M E,G T L R Y L R E S E R Figure 8. Consensus Sequence and Helical Wheel Plot of Putative Amphipathic a Helix between N1049 and Q1129. Helix d is plotted in helical wheel format to illustrate the amphipathic character. The consensus sequence and the corresponding sequences of the phy6 and phyC gene products are shown. Symbol usage is as in Figure 7. Physiologicaldata (e.g., Takimoto and Saji, 1984), biochemical data (Pratt et al., 1991), and DNAsequence data (Sharrock and Quail, 1989) have shown that at least two different pools of phytochromeexist within plant cells. Pratt et al. (1991) have shown that heterodimers form between the phyA gene product and at least one of these other types of phytochrome, possibly phyB andlor phyC gene products. The mapping of potentialdimerizationsites within PhyA proteins allows a comparison of these protein sequences with similar sequences encoded by the other members of the phytochrome gene family. We have shown that these regions contain sequences with the potential to form amphipathic a helices. The helices are conserved among members of the PhyA protein family and between PhyA proteins and Arabidopsis PhyB and PhyC proteins. The formation of homodimersand heterodimers has been shown to play an important role in modifying the activity of severa1 regulatory proteins such as calmodulin (ONeill and DeGrado, 1990), NF-~6/p50(Urban et al., 1991), and cAMPdependent protein kinase (Carr et al., 1991). It is possible that the fluence range, magnitude, andlor nature of phytochrome responses are modulated through the formation of heterodimers comprised of PhyA protein and other types of phytochromes. The plasmid pMElO was constructed by inserting aT4 DNA polymerasetreated 272-bp EcoRI-Sphl fragment containing the lacUV5 promoter and sequences encoding h repressor amino acids 1 to92 from KHlOl (Hu et al., 1990) into EcoRV-digestedpSP72 (Promega, Madison, WI) using standard cloning techniques (Sambrook et al., 1989). Fusion proteins were generated by inserting fragments from pCIB315 (Thompson et al., 1989), which contains sequences from oat phyA AP3, into pME10. MElOlGCN4 was assembled by inserting an Xbal-EcoRI fragment from LexA/GCN4 (Hope and Struhl, 1987) encoding GCN4 residues L169-R281 into the Smal site of pMElO after the fragment was blunted by treatment with the Klenow fragment of DNA polymerase I.All fusions were verified by restriction mapping and immunoblot analysis using antirepressor and/or antiphytochrome antibodies. For immunoblot analysis of fusion protein size, proteins were expressed in insoluble form in XL-1 Blue (Stratagene, La Jolla, CA). Repressor activity was assayed in Escherichia coli X90K (ara Alacpro73 nalA arg€[am] rif thi-7 [F'lac pro laclq lacZ::kan]) lysogenic for h200. A200 contains a lacZ fusion under the control of the h rightward promoter, PR(Meyer et al., 1980). PGalactosidase Assay and Relative Repression p-Galactosidaseactivity was assayed as described by Miller (1972) using SDS and chloroform cell lysis. We have defined a coefficient of relative repression (R,) as being equal to the IPTG concentration at which a given repressor has half-maximalactivity divided by the IPTG concentration at which the protein encoded by pMElO has half maximal activity. This value was found to be a more reliable indicator of repressor function than p-galactosidase values taken at a single IPTG concentration. The curves of P-galactosidase activity dependence on IPTG induction were performed at least three times. In each experiment, pMElO expression was also measured as a control. SDS-PAGE and lmmunoblots Crude extracts for immunoblotting were produced by growing 50-mL cultures in Luria broth with antibioticsto an ODWoof 0.6, adding IPTG to 0.1 mM, and allowing growth to continue for 2 hr. The induced cells were collected by centrifugation, washed in 10 mL of protein resuspension buffer (50 mM Tris, pH 8.0, 100 mM NaCI, 5 mM EDTA, 140 mM 2-mercaptoethanol, 10% glycerol), and resuspended in 10 mL of protein resuspensionbuffer. Cells were lysed by passage through a Carver press. Unbroken cells and cell debris were removed by centrifugation at 27,0009 for 20 min. Soluble proteins were concentrated by precipitation with 75% ammonium sulfate and resuspended in a final volume of 500 pL of protein resuspensionbuffer. Protein concentrationswere estimated using micro-Bradford assays (Bradford, 1976). SDS-PAGE was carried out as described by Laemmli (1970) with slight modification. Proteins were transferred to nitrocellulosefor immunoblot analysis. The filters were blocked for 1 hr in blocking buffer (5% nonfat dry milk in 1 x TBS [I0 mM Tris, pH 7.45, 150 mM NaCI, 0.05% NaN,]) at room temperature. lncubation with primary antibody was carried out in incubation buffer (0.1% BSA in 1 x TBS) with 10% Phytochrome Dimerization crude E. coliextract overnight at 4%. The crude bacterial cell extract used in the incubation step was prepared by growing the appropriate E. coli strain to an ODmoof 1.0 with 0.5 mM IPTG. The cells were then harvested by centrifugation, resuspended in 1/50th volume protein resuspension buffer, and lysed by passage through a Carver press. The lysate was then cleared at 27,0009 for 20 min. Anti-X repressor antiserum was generated as described by Jones et al. (1991) using insoluble full-length 1repressor as an antigen. Antisera to phytochrome (4032 and 3032) were kindly provided by Peter Quail (University of California at Berkeley, Berkeley, CA). Size Exclusion Chromatography Crude extract from cells expressing the pMElO/GCN4 protein was prepared from cells as described above. The extract was further purified by the addition of 10% polyethyleneimine to a final concentration of 0.2% followed by centrifugation at 27,0009 for 20 min. Saturated ammonium sulfate was added to the supernatant to 75% saturation. Protein was precipitated by centrifugation at 27,000s for 20 min and resuspended in 200 WL of protein resuspension buffer. Fusion protein concentration was estimated by comparison of extract stained with Coomassie Brilliant Blue R 250 to known concentrations of carbonic anhydrase after separation by SDS-PAGE. Approximately 6.5 mg of crude extract was separated over a 0.75 x 30 cm Spherogel TSK 2000SW size exclusion column (Beckman, Berkeley, CA) at O5 mUmin in TSK buffer (50 mM Tris, pH 7.0,50 mM NaCI, 0.5 mM EDTA, 5 mM 2-mercaptoethanol, 10% glycerol). Fractions (0.25 mL) were collected and analyzed by SDS-PAGE, followed by immunoblot analysis. A rough estimate of a dimerizationconstant for the MElO/GCN4 fusion protein shown in Figure 2 was determined by finding a load concentrationwhere approximately half of the fusion protein appeared dimeric. This was not possible with the low leve1 of expression observed for the phytochrome fusion proteins. Filter Binding Filter binding assays were carried out in a manner similar to that described by Johnson et al. (1980). Cells were grown in media A (Miller, 1972) to an ODmoof 0.4 with varying amounts of IPTG. Cells (1.5 mL) were harvestedby centrifugation at 12,0009, washed once with 500 pL of protein resuspension buffer, repelleted, and resuspended in 30 pL of protein resuspension buffer. The cells were then frozen in liquid nitrogen and thawed on ice. The freeze-thaw cycle was repeated, and the lysate was centrifuged for 20 min at 28,0009. This supernatant was used for filter binding assays and also assayed for P-galactosidase content by quantitative immunoblot analysis. Binding reactions were carried out in 50 pL of 10 mM Pipes, pH 6.6, 50 mM KCI, 2 mM CaCI,, 0.1 mM EDTA, 100 pg/mL BSA, 5% DMSO to which had been added 8.5 pg of sheared calf thymus DNA, and 50 pmol of 32P-labeleddouble-stranded oligonucleotides containina an OR, ... bindina site (5’-GATCCTATCACCGCCAGAGGTAGGATC-3! specific activity 500 cpmlpmol). Ten microlitersof freeze thaw supernatant was assayed for repressor content. The reaction was incubated on ice for 1 hr, followed by filtration through nitrocellulose (0.45 pm, NitroME; Micron Separations Inc., Westboro, MA) in a slot blotting system (Minifold (I;Schleicher and Schuell, Keene, NH). Each reaction was washed three times with 0.3 mL of wash buffer (10 mM Tris, pH - I 169 7.0, 50 mM KCI, 2 mM CaCI2, 0.1 mM EDTA). The filter was then allowed to dry, and individual slots were cut out and counted. 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M D Edgerton and A M Jones Plant Cell 1992;4;161-171 DOI 10.1105/tpc.4.2.161 This information is current as of June 17, 2017 Permissions https://www.copyright.com/ccc/openurl.do?sid=pd_hw1532298X&issn=1532298X&WT.mc_id=pd_hw15322 98X eTOCs Sign up for eTOCs at: http://www.plantcell.org/cgi/alerts/ctmain CiteTrack Alerts Sign up for CiteTrack Alerts at: http://www.plantcell.org/cgi/alerts/ctmain Subscription Information Subscription Information for The Plant Cell and Plant Physiology is available at: http://www.aspb.org/publications/subscriptions.cfm © American Society of Plant Biologists ADVANCING THE SCIENCE OF PLANT BIOLOGY