Planta

Ptanta (1989) 178:147 156

9 Springer-Verlag1989

Phytochrome structure: Peptide fragments from the amino-terminal domain involved in protein-chromophore interactions* Alan M . Jones 1 ** and Peter H. Quail 2 *** Department of Biology, University of North Carolina, Chapel Hill, NC 27599, and z Department of Botany, University of Wisconsin, Madison, Wl 53706, USA

Abstract. We have undertaken a study of the struc-

ture of the amino-terminal domain of the phytochrome polypeptide purified from Arena sativa L. Amino-acid sequencing was used to identify arginine 52 as the precise location of a conformationspecific cleavage of phytochrome by subtilisin. The location of the epitopes for a class of monoclonal antibodies designated type 2 has been shown to be located between approx. 10 and 20 kilodaltons (kDa) from the amino terminus. These two new spatial markers, in addition to the chromophore and another epitope recognized by type 1 monoclonal antibodies and located within 6 kDa from the amino terminus, have been used to map the locations of several new protease-accessible sites along the polypeptide. After extensive digestion of phytochrome with subtilisin, a stable spectrally-active group of peptides remains. Within this group is a 16-kDa chromopeptide which, either alone or as part of an assemblage of peptides, elutes from a size-exclusion column under nondenaturing conditions at a volume consistent with a molecular mass of 35-40 kDa. This group of peptides has an absorbance spectrum similar to the red-absorbing form of phytochrome (Pr) and is red/far-red photoreversible between this and a photobleached form. These data indicate that this group of peptides still retains the principal structural requisites for Pr-chromophore-protein interactions and for * This work was presented, in part, at the XVI Yamada Conference on Phytochrome and Plant Photomorphogenesis, Okazaki, Japan, October 1986 ** To whom correspondence should be addressed *** Present address." Plant Gene Expression Center, U.S. Department of Agriculture, Albany, CA 94710, USA Abbreviations." Da=dalton; Mr=relative molecular mass; Pr, Pfr = red- and far-red-absorbing forms of phytochrome, respectively; SDS-PAGE = sodium dodecyl sulfate polyacrylamide gel electrophoresis

photoreversibility, but not for Pfr (far-red-absorbing phytochrome)-chromophore-protein interactions. It is uncertain if these structural requisites reside exclusively on the 16-kDa chromopeptide or result from an assemblage of these peptides. However, we have excluded any role for an adjacent 14-kDa fragment (approximately residues 50 to 200) in the observed spectral properties since it can be selectively removed without any effect on the photoreversibility. Key words: Arena (phytochrome) - Phytochrome

(structure, photoreversibility)

Introduction

Phytochrome is an important photoreceptor in plants that serves as a molecular switch for many developmental events by its capacity for photoconversion between two forms, a red-light-absorbing form (Pr) and a far-red-light-absorbing form (Pfr). The molecule, a chromoprotein, contains a linear tetrapyrrole adjunct covalently attached by a thioether linkage at cysteine residue 321 (Lagarias and Rapoport 1980; Hershey etal. 1985). The structure of this chromophore has been determined (Lagarias and Rapoport 1980; Rfidiger et al. 1983) but little is known concerning the noncovalent chromophore-polypeptide interactions which stabilize the two spectral forms. A number of studies have been directed at identifying the molecular domains necessary for correct chromophore-protein interaction in the Pr, Pfr, and photointermediate forms of phytochrome. Earlier, we reported that a 74-kilodalton (kDa) fragment, retaining the intact amino terminus, is identical to the undegraded molecule with respect

148

to the spectral parameters tested (Jones et al. 1985). These data indicate that amino-acid residues interacting with the Pr and Pfr chromophores must reside exclusively in the amino-terminal 74 kDa of the polypeptide and that the remaining 55-kDa carboxyl half is unnecessary for these interactions. Additional data indicate that the amino-terminal 10 kDa interacts in some way with the Pfr chromophore since loss of this region from the 74-kDa amino-terminal half (Jones et al. 1985), as well as from the intact 124-kDa polypeptide (Vierstra and Quail 1982), results in an 8-nm shift in the absorbance maximum of the Pfr form. This region apparently does not interact with the Pr chromophore, however, since no change in Pr absorbance occurs upon loss of the amino-terminal fragment (Jones et al. 1985). Other laboratories have sought to define yet smaller domains which provide the necessary structure for proper protein-chromophore interactions. Yamamoto and Furuya (1983) isolated a 40-kDa tryptic peptide from the Pr form of phytochrome of Pisum, and found it to be photoreversible between a form which absorbs maximally at 657 nm and a photobleached form. They concluded that this domain contains structural requisites for photoreversible absorbance changes. Using 124-kDa phytochrome (Pfr) from Arena, Reiff et al. (1985) obtained similar results and extended the work by examining the effects of proteolysis on the photointermediates. They purified a 39-kDa fragment which contains amino acids involved in stable chromophore-protein interactions in the Pr form and two of three known intermediates in the pathway of Pr~Pfr, but lacking the region necessary for stable Pfr formation. The chromophore in the stable photobleached product was determined to be the 15E form lacking specific polypeptide interaction (Thiimmler and Rfidiger 1984). The exact location of the 39/40-kDa domain within the polypeptide was not determined in either study. Thus the range of uncertainty for the location of this domain spanned 8 0 k D a of polypeptide, 40 kDa on each side of the chromophore, a range equivalent to the entire globular NHz-terminal domain. We have extended these previous investigations by locating the approximate position of the 40kDa fragments within the polypeptide. We have identified and determined the location of a group of peptides containing a chromopeptide. This group has spectral properties like the previously reported 40-kDa fragments. In addition, we have fine-mapped a new set of protease-accessible cleavage sites in the amino-terminal portion of the polypeptide.

A.M. Jones and P.H. Quail: Arena phytoehrome structure

Materials and methods Plant material and chemicals. Oat seeds (Arena sativa L., cv. Garry), purchased from Olds Seed Co., Madison, Wis., USA were sown on wetted vermiculite and grown in darkness for 3.5 d at 26 ~ C. Shoots were harvested the day before they were used in phytochrome purification, and stored at 4 ~ C in darkness. Subtilisin-BPN', immobilized subtilisin, 2-amino-2-(hydroxymethyl)-l,3-propanediol (Tris), ethylenediaminetetraacetic acid (EDTA), and phenylmethylsulfonyl fluoride (PMSF) were purchased from Sigma Chemical Co., St. Louis, Mo., USA. After experiments were completed, Sigma Chemical Co. notified users of Sigma Subtilisin BPN' that this product, prepared for Sigma by the same supplier since 1962, could not be confirmed as subtilisin. Recent data indicate that this product is probably subtilisin Carlsberg (Russell and Fersht 1986). Acrylamide, bisacrylamide and sodium dodecyl sulfate (SDS) were purchased from Bio-Rad Chemical Co., Richmond, Cal., USA. Coomassie blue-linked standards and Immunoprecipitin were purchased from Bethesda Research Laboratories, Bethesda, Md., USA. Antimouse immunoglobulin (Ig), prepared in rabbit, and antirabbit immunoglobulin conjugated with alkaline phosphatase were purchased from Kirkegaard and Perry Laboratories, Gaitherburg, Md. Monoclonal and polyclonal antibodies directed against 124-kDa phytochrome were prepared as described in Daniels and Quail (1984) and Hunt and Pratt (1979).

Phytochrome purification; irradiations; spectral measurements. Phytochrome was purified from etiolated oat as described in Vierstra and Quail (1983) with modifications described by Jones and Quail (1986). The specific absorbance ratio was always at least 0.90. Phytochrome samples were irradiated with light from a Unitron microscope illuminator passed through a red interference filter (transmission maximum at 666 nm, 5 J. m - 1. s-1) or a far-red cut-off filter (cut-off wavelength = 750 nm, 900 J- m - 1. s- 1 obtained from Corion Corporation (Holliston, Mass., USA). Spectra were recorded as described in Vierstra and Quail (1982) except for the experiments shown in Fig. 7 where a Shimadzu UV 3000 spectrophotometer was used instead of a Perkin Elmer 557.

Phytochrome digestion. Purified, 124-kDa phytochrome was digested as Pr or Pfr with subtilisin in the dark with a mass ratio of approx. 1:25 subtilisin:phytochrome. The phytochrome concentration varied among experiments but was always within the range of 0.1-0.4 mg/ml. The buffer for both phytochrome and subtilisin was 100 mM Tris (pH 7.8), 1 mM EDTA, 25% ethylene glycol. Digestion was stopped by the addition of PMSF (4 raM). The decrease in absorbance over time of incubation with subtilisin directly corresponds to the extent of digestion as judged by immunoblots. Although the rate of digestion may differ with different batches of phytochrome and subtilisin, it is easily controlled by adjusting the incubation temperature within the range of 5-20 ~ C. However, regardless of the batch of phytochrome digested or the activity of the protease, the change in absorbance always strongly correlates with defined changes in the peptide pattern such that at any '~percent absorbance remaining" a unique peptide profile can be expected.

Sodium dodecylsulfate polyaetylamide gel electrophoresis ( SDSPAGE); immunoblot analysis. The SDS-PAGE and the immunoblot analysis were performed as outlined by Jones and Quail (1986) except the milk buffer used in the antibody incubations and washes was 0.2% evaporated milk (Carnation, Los

A.M. Jones and P.H. Quail: Arena phytochrome structure Angeles, Cal. ; USA) instead of the previously used 3%. Silver staining of polyacrylamide gels was performed by the method of Merrill et al. (1983). Approximately 100 500 ng of intact or digested phytochrome was added to each lane of the immunoblots probed with either polyclonal or monoclonal antibodies. The immunoblot in Fig. 8 contains approx. 100 ng per lane of phytochrome digested with immobilized subtilisin (Sigma). According to Sigma Chemical Co. this product is the same as the free enzyme except linked to agarose beads. Digestion with immobilized subtilisin was performed as previously described for trypsin at 4~ C for 1 h (Jones and Quail 1986). This method of digestion is much milder than the method utilizing free subtilisin described above.

Visualization of chromopeptides on nitrocellulose blots. Chromophore-eontaining fragments separated by SDS-PAGE were visualized on nitrocellulose blots by soaking these blots in 1.3 M zinc acetate for 30 min prior to excitation with ultraviolet (UV) light (302 nm). This method of visualizing chromopeptides yields zinc-chromophore complexes which are fluorescent when viewed with UV light (Siegelman et al. 1966; Berkelman and Lagarias 1986). The pink fluorescence of the chromophore was visible by eye down to 100 ng/lane of 124-kDa phytochrome. Our procedure differs from a published protocol for detecting biliverdin-linked peptides in SDS-PAGE (Berkelman and Lagarias 1986) in two ways: First, we visualize the fluorescence of the chromopeptides after electrotransfer from SDS-PAGE to nitrocellulose. Second, zinc acetate is not added to the gel or electrophoresis buffer. Incubation of the blots in zinc acetate was sufficient to form the zinc-chromophore salt. Approximately 10 lag of phytochrome per lane was used on blots incubated with zinc acetate (Figs. 3, 6). This high load of phytochrome was only needed to obtain blots that photograph well. High-performance liquid chromotography. High-performance liquid chromatography (HPLC) was performed on a Beckman Instruments (Arlington, Ii1., USA) System 341 HPLC as described in Jones and Quail (1986). Eluted samples were monitored simultaneously at 280 and 656 nm with a Beckman 165 scanning spectrophotometer. The size-exclusion column (TSK2000SW; Toya Soda, Tokyo, Japan) was calibrated with bovine serum albumin, ovalbumin, soybean trypsin inhibitor, and lysozyme. The buffer was 100 mM Tris (pH 7), 150 mM NaC1, i mM EDTA.

Immunoprecipitation of digested and undigestedphytochrome. To 0.5 ml of intact phytochrome (Pr, 0.1 mg/ml) or phytochrome digested for 24 h with subtilisin as described above ( A 656 = 0.1), was added PMSF (2 mM final), and type 2 or type 3 monoclonal antibodies. The concentration of antibody in each reaction was 240 lag/ml (192 lag/reaction). These samples were gently mixed for 2 h in the dark at 4~ C, then 0.1 ml of 10% formalinkilled Staphylococcus aureus cells (Irnmunoprecipitin; Bethesda Research Labs) was added and the samples were mixed for another 30 rain. The samples were centrifuged at 12000.g for 3 min and spectra of the supernatants were taken. We found that glycerol above 5% interfered with these reactions. These experiments were repeated six times using at least two members each of the two classes of antibodies (type 2 and 3).

Preparation of digestedphytochromefor amino-acidsequencing. Phytochrome, as Pr (1.5 ml, 400 gg), was digested for 1 h at 4~ on a column packed with 1 ml of immobilized subtilisin as previously described for trypsin digestion (Jones and Quail 1986). The reaction was stopped by centrifugation and by the addition of PMSF (4 mM). Peptides, separated by preparative SDS-PAGE, were visualized by staining with Coomassie Blue

149 and a 61-kDa band was excised from the gel and electroluted by the method of Hunkapiller et al. (1983b). Upon subsequent SDS-PAGE, two bands were observed with relative molecular masses (Mrs) of 61 kDa and 53 kDa. This additional 53-kDa band, containing the same amino terminus as the 61-kDa peptide, is thought to have resulted from partial hydrolysis at residues D 5 3 9 and P54o during the staining-destaining steps (Marcus 1985). Automated Edman degradation was performed on a gas-phase sequencer (Model 470A; Applied Biosystems, Foster City, Cal., USA) and PTH-amino acids were analyzed by C-18 reverse-phase HPLC (Smithes et al. 1971; Hunkapiller etal. 1983a). Both electroelution and amino-acid sequence analysis were performed as a service by the University o f Wisconsin Biotechnology Center under the direction of Dr. Ron Niece.

Results

Changes in spectral properties during subtilisin digestion. P h y t o c h r o m e w a s digested f o r 24 h either as P r o r P f r a n d the a b s o r b a n c e s p e c t r a were determ i n e d o v e r this time (Fig. 1). T h e a b s o r b a n c e a n d wavelength maxima for both forms decreased during digestion a l t h o u g h to a different extent for either f o r m (Fig. 1). A t 23 h, P r a b s o r b a n c e h a d d e c r e a s e d to a p p r o x . 5 0 % o f the initial value, w h e r e a s o n l y 1 0 % o f the P f r a b s o r b a n c e r e m a i n e d . T h e P r m a x i m u m shifted f r o m 668 nan at time zero to 656 n m b y 24 h, while the P f r p e a k m a x i m u m shifted f r o m 730 n m at time z e r o to 724 n m at 6 h w h e r e f u r t h e r d e t e r m i n a t i o n s w e r e n o l o n g e r possible b e c a u s e o f l a c k o f a b s o r b a n c e . T h e r e m a i n i n g a b s o r b a n c e at 657 n m in the P f r digest c a n be acc o u n t e d f o r b y 1 2 % P r in the s t a r t i n g reaction, the p h o t o s t a t i o n a r y state o f p h y t o c h r o m e after actinic red i r r a d i a t i o n (Kelly a n d L a g a r i a s 1985). P h y t o c h r o m e t h a t h a d b e e n digested either as Pr o r P f r f o r 24 h w a s tested f o r p h o t o r e v e r s i b i l i t y b y i r r a d i a t i o n with red a n d f a r - r e d light. F i g u r e 2 s h o w s the p h o t o r e v e r s i b i l i t y o f a digested P r s a m ple b e t w e e n a f o r m ( s ) a b s o r b i n g m a x i m a l l y at 656 n m a n d a p h o t o b l e a c h e d f o r m . T h e s a m e results w e r e o b t a i n e d f o r the s a m p l e digested as P f r (data not shown). Complete conversion from Pfr to P r r e q u i r e d 15 m i n o f f a r - r e d light, a p p r o x i m a t e l y six times l o n g e r t h a n the time n e e d e d f o r complete conversion of undigested phytochrome.

Characterization of peptides generated by subtilisin digestion. P h y t o c h r o m e , as P r a n d Pfr, w a s digested w i t h h i g h levels o f subtilisin f o r 24 h a n d a l i q u o t s o f the d i g e s t i o n r e a c t i o n t a k e n t h r o u g h o u t this p e r i o d were s u b j e c t e d to S D S - P A G E , i m m u n o b l o t analysis, a n d h i g h - p e r f o r m a n c e liquid c h r o m a t o g r a p h y . F i g u r e 3 c o n t a i n s d u p l i c a t e nitrocellulose blots o f the s a m e samples, o n e p r o b e d w i t h a p o l y c l o n a l a n t i s e r u m directed a g a i n s t 124-

150

A.M. Jones and P.H. Quail: Arena phytochrome structure '

0.100

-

I

I

I

'

I

'

i

I

I

~

I

i

I

~

I

669

A

i

I

e

I

I

I

i

I

i

[

~

F

i

I

657

0

0.090 0.5 0,080

co~

0.070

--IA

0

~ 0.010 A

0.060 12

0.050

25

I

0.040

I 520

(.P

0.020

~n ...CI