the journal of biological chemistry vol. … · mpeb, which encode the a and subunits of pe 11,...

THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1993 by The American Society for Biochemistry and Molecular Biology, Inc

Vol. 268, No. 2, Issue of January 15, pp. 1236-1241,1993 Printed in U. S. A.

Rod Structure of a Phycoerythrin 11-containing Phycobilisome 11. COMPLETE SEQUENCE AND BILIN ATTACHMENT SITE OF A PHYCOERYTHRIN y SUBUNIT*

(Received for publication, September 18, 1992)

Sigurd M. WilbanksS and Alexander N. Glazer4 From the Division of Biochemistry and Molecular Biology, Department of Molecular and Cell Biology, University of California, Berkeley, California 94720

The major phycoerythrins of marine unicellular cyanobacteria Synechococcus spp. and those of red algae are isolated as stable complexes with the composition (a8)ey. The y subunits carry bilins and in this respect are unique among phycobilisome rod linker polypeptides. There has been no complete amino acid sequence data on any y subunit.

Synechococcus sp. WH8020 phycoerythrin I1 (PE 11) y subunit was isolated from purified P E I1 hexamers. Partial amino acid sequence determination showed that the y subunit was encoded by the mpeC gene, an open reading frame 276 base pairs 3‘ of mpeA and mpeB, which encode the a and subunits of P E 11, respectively (Wilbanks, S. M., and Glazer, A. N. (1993) J. Biol. Chem. 268, 1226-1235). A single phycourobilin is attached through a thioether bond to y-Cys-49. Derivatization with 4-vinylpyridine showed that the only other cysteinyl residue, y-Cys-64, is unsubsti- tuted.

MpeC encodes a polypeptide of 293 residues with a predicted molecular weight of 32,100 and a PI of 8.9. These properties are like those of non-bilin-bearing linker polypeptides associated with C-phycoerythrin and hence the y subunit is designated y (Liz). Alignment of the sequence of the PE 11-7 with those of the latter polypeptides shows that P E 11-7 has a 49-residue extension at the N terminus, that encompasses the phycourobilin attachment site, and is shorter by a similar number of residues at the C terminus. These differences in linker polypeptide structure offer a possible explanation for the observed much higher stability of P E I1 hexamers relative to those of C-phycoerythrins.

Phycobilisomes are large light-harvesting antenna complexes attached to the cytoplasmic surface of the thylakoid

* This research was supported in part by National Science Foun- dation Grants DMB-8816727 and MCB-9117221, National Institute of General Medical Sciences Grant GM28994, and the Lucille P. Markey Charitable Trust. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequencefs) reported in this paper has been submitted

M95288. to the GenBankTM/EMBL Data Bank with accession numberfs)

Recipient of a predoctoral fellowship from the Department of Health and Human Services (Training Grant 8T2 GM 08295). Pres- ent address: Dept. of Cell Biology, Stanford University, Stanford, CA 94305.

§To whom correspondence and reprint requests should be addressed Dept. of Molecular and Cell Biology: Stanley/Donner Ad- ministrative Services Unit, 229 Stanley Hall, University of California, Berkeley, CA 94720.

membrane in cyanobacteria and red algae (1, 2 ) . Phycoery- thrins form the distal portions of phycobilisome rods. These rods are stacks of three-to-four hexameric phycobiliprotein discs each of which has the composition (a@),LR, where LR is a rod linker polypeptide; in general, each disc in the rod has a distinctive linker polypeptide (for reviews, see Refs. 3-7).

Phycoerythrin is the most abundant phycobiliprotein in the phycobilisomes of red algae (8, 9) and of open-ocean strains of unicellular cyanobacteria Synechococcus spp. (10-12). The marine unicellular cyanobacteria have two phycoerythrins, P E I’ and PE I1 (13, 14). When red algal phycobilisomes or those of the marine unicellular cyanobacteria are exposed to low salt concentration at near-neutral pH, R-, B-, and PE I1 phycoerythrins are released as stable ( ( ~ @ ) ~ y complexes. In contrast, on dissociation of the phycobilisomes of freshwater cyanobacteria, C-phycoerythrins readily lose their linker polypeptides and are recovered as a mixture of complexes with the composition (a& = 1-6 (15). P E I behaves similarly to C-phycoerythrins (13).

The corresponding amino acid sequences of the (Y and /3 subunits of all phycoerythrins show a high degree of identity, particularly around bilin attachment sites. The difference in the stability of R-, B-, and PE I1 phycoerythrin complexes relative to those of C-phycoerythrins may be a consequence of differences in their linker polypeptides. The y subunits are the functional counterparts of the linker polypeptides of C- phycoerythrins. The y subunits of R-, B-, and PE I1 phycoerythrins all carry covalently attached bilins, whereas the linker polypeptides associated with C-phycoerythrins do not (16-

The genes encoding the non-bilin-bearing linker polypeptides associated with Calothrix PCC7601 C-phycoerythrin have been cloned and sequenced (20). No comparable infor- mation has been available on bilin-bearing phycoerythrin- associated linkers. All of the tryptic bilin peptides of Gastro- clonium coulteri R-phycoerythrin y subunit have been se- quetced (17). These peptides show no homology to any portion of the Calothrix linker polypeptide sequences.

The present study addresses two questions. In what manner does a particular phycoerythrin-associated linker polypeptide determine the stability of the multimeric protein? How do the bilins associated with linker polypeptides contribute to the energy transfer pathways within a phycoerythrin hexamer? These questions are answered in part in this report of the determination of the complete amino acid sequence of the y subunit of Synechococcus sp. WH8020 PE 11, and the characterization of its bilin attachment site.

19).

‘The abbreviations used are: PE, phycoerythrin; HPLC, high- performance liquid chromatography; PEB, phycoerythrobilin; PUB, phycourobilin; PAGE, polyacrylamide gel electrophoresis; TPCK, L- 1-tosylamido-2-phenylethyl chloromethyl ketone.

1236

Phycoerythrin 11 y Subuni t 1237

EXPERIMENTAL PROCEDURES

Culture Conditions-Synechococcus sp. WH8020 (11) was cultured in 500 ml of medium SN (21) in 2.8-liter Fernbach flasks a t -23 "C with daylight illumination a t -20 microeinsteins m-' s". The un- stirred cultures were fully resuspended by swirling at 2-4-day inter- vals and harvested at the end of about 5 weeks with cell yields ranging from 0.5 to 1.4 g wet weight per liter.

Preparation of Phycobilisomes-Phycobilisomes were prepared as described by Yamanaka et al. (22). Cells were harvested by centrifu- gation and resuspended to -0.12 g/ml in 0.75 M NaK phosphate, 1 mM EDTA, 0.1 mM phenylmethylsulfonyl fluoride a t pH 7.0 (0.75 M NaK-phosphate buffer). The cells were then broken by passage three times through an Aminco French pressure cell a t 20,000 p.s.i. Triton X-100 was added to the broken cell suspension to 1% (by volume); the mixture was stirred gently a t room temperature for 30 min and then centrifuged a t 31,000 X g for 45 min. Aliquots (5 ml) of the supernatant were applied to sucrose density-step gradients. The gradients consisted of 7.5 ml of 1.0 M sucrose, and 6.5-ml steps of 0.8, 0.6, 0.4, and 0.2 M sucrose, all in the 0.75 M NaK-phosphate buffer. The gradients were centrifuged for 14 h at 58,000 X g in a Beckman SW-28 rotor. Colored fractions were retrieved with a Pasteur pipet, diluted 2-fold with the 0.75 M NaK-phosphate buffer, and the proteins were precipitated overnight a t 4 "C by the addition of (NH4)2S04 to 65% of saturation.

Purification of Phycoerythrin II-Hexameric PE I1 (13) was purified from whole cells (or from phycobilisomes) by the procedure described above for phycobilisome preparation, with the following differences: all steps were carried out in 50 mM NaK-phosphate buffer (rather than 0.75 M), no detergent was used, and the sucrose density gradients were centrifuged for 30-36 h. Precipitated PE I1 was redis- solved in 50 mM NaK-phosphate buffer to a protein concentration of -1 mg/ml and stored a t 4 "C. SDS-PAGE-Electrophoresis and staining were performed as de-

scribed by Laemmli (23) using a 16% separating gel and a 10% stackinggel both with an acrylamide-cross-linker ratio of 37.5:l. Bilin peptides were detected as the fluorescent zinc complexes by soaking the unstained gel for 5 min in 100 mM ZnS04 and exciting with ultraviolet light (24). The gels were then stained with Coomassie Brilliant Blue R-250.

Tryptic Digestion and Reaction with 4-Vinylpyridine-PE I1 y subunit was purified by HPLC in aqueous trifluoroacetic acid/acetonitrile (25). Urea solution (0.5 ml of 9 M urea, pH 2.1) was added to the polypeptide (0.1-0.2 mg; 3.5-7 nmol) in aqueous trifluoroacetic acid/acetonitrile. The sample volume was reduced by rotary evaporation until urea crystals began to form. Trypsin (15 pg; 15 pl of a 1 mg/ml solution of Worthington TPCK-treated trypsin in 10 mM HCI) was added to the slurry which was then diluted with 1.0 M Tris- CI a t pH 8, to a final volume of about 3 ml and a final pH of 8.0. The solution was flushed with NP, sealed, and incubated for 2 h a t 37 "C in the dark. A further 15-pg aliquot of trypsin was then added, and the incubation was continued for 2 more hours. Digestion was terminated by addition of glacial acetic acid to a final concentration of 30%, by volume. The peptides were desalted on a Spice Cln sample preparation cartridge (Analtech). Peptides were eluted with 60% acetonitrile, 40% 100 mM Na-phosphate, v/v, pH 2.1, and the volume of the eluate was reduced by 60% in a Savant Speed-Vac concentrator.

Derivatization of the y subunit by reaction with 4-vinylpyridine to form the S-@-(4-pyridylethyl) derivative of cysteinyl residues was performed by a scaled down version of the procedure described by Friedman et al. (26). 8-Mercaptoethanol (2 pl) was added to a y subunit tryptic digest prepared exactly as described above. The mixture was flushed with N2, and reduction was allowed to proceed for 1 h a t room temperature in the dark; 4-vinylpyridine (6 pl) was then added, and the mixture was flushed again with NP and incubated for a further 3 h. The reaction was terminated by the addition of acetic acid, as described above, and the sample was reduced to a wet film by rotary evaporation and then mixed with 0.1 M Na phosphate, pH 2.1, before desalting on a Spice cartridge as above.

Amino Acid Analysis and Sequence Determination-For amino acid analyses, all hydrolysis and amino acid derivatization steps were performed using a Waters Pico-Tag amino acid analysis workstation as previously described (27). Amino acids were identified and quan- titated as their phenylthiocarbamyl derivatives.

Amino acid sequence determinations were performed with an automated gas-liquid phase sequenator (Applied Biosystems), and phenylthiohydantoin derivatives were identified by HPLC in a dedi- cated analyzer.

RESULTS

Characterization of Phycoerythrin 11-As previously observed (13), PE I1 purified either from whole cells or from phycobilisomes sedimented in sucrose density gradients as a hexamer. In 8 M urea at pH 1.9, the absorption spectra of different PE I1 preparations showed A4es nm of 1.20- 1.27, consistent with a ratio of phycourobilin (PUR): phycoerythrobilin (PEB) of 0.55-0.58 (for extinction coeffi- cients, see Ref. 17). On SDS-PAGE (Fig. 1, lane 2 ) the 18- kDa cy subunit and the 20-kDa /3 subunit were resolved as a closely spaced doublet from the 30-kDa y subunit. In presence of Zn2+ (24), the cy and /3 subunit bands fluoresced orange, whereas the y subunit showed a green fluorescence characteristic of PUB (13).

Separation of the Subunits of PE 11-The subunits of PE I1 were separated by reversed-phase HPLC (Fig. 2A) . Analysis by SDS-PAGE (Fig. 1, lanes 3-6) showed that the y subunit was the earliest eluting component, and that the second and third major components corresponded to the cy and /3 subunits, respectively. Spectroscopic analysis snowed that the cy and /3 subunits each carried 1 PUB and 2 PEB, as shown by Ong

Y

P , a'

FIG. 1. SDS-PAGE of Synechococcus sp. WH8020 phycobilisomes and their components. Samples are identified a t the top. PRS, purified phycobilisomes; PE I I , purified phycoerythrin 11; y , purified y subunit (Fig. 2, component eluting a t 22-23 rnin); cy,,,I~Vr

leading edge of the cy subunit peak (Fig. 2, component eluting a t 24- 24.5 rnin); 0, purified (Y subunit (Fig. 2, component eluting a t 24.5- 25.5 rnin); 0, purified 0 subunit (Fig. 2, component eluting a t 26.5- 27.5 min). Position of cy, 0, and y subunit bands are indicated a t left. Sample load was 1 pg, except for phycohilisomes, where the load was 5 Pg.

Elution Time (min)

""

Wavelength (nm)

FIG. 2. Purification of the subunits of phycoerythrin 11. A, HPLC separation of the subunits of PE I1 (-50 pg) on C4 reversed- phase resin monitored a t 495 nm (for details, see "Experimental Procedures"). R, the absorption spectra of each subunit at its elution peak in the profile in A: y, 22.5 min; cy, 25.00 min; 8, 26.9 min. The y subunit absorption is shown a t a 10-fold amplification.

1238 Phycoerythrin II y Subunit

and Glazer (13), and that the y subunit carried only PUB. Assuming 1 PUB per y subunit, integration of peak areas indicates a molar ratio of y:a:B of 1:12:12 as compared to a ratio of 1:66 indicated by protein staining of SDS-PAGE gels. SDS-PAGE analysis of the leading edge of the a subunit peak showed that additional 30-kDA 7 subunit was eluting in this position (Fig. 1, lane 4 ) . This component was not isolated, and, consequently, the difference between this material and the earlier-eluting y subunit is not known. All of the studies reported below were performed on the polypeptide indicated as y in Fig. 2A.

Characterization of the y Subunit-Fourteen steps of se- quential Edman degradation of the y subunit (Table I) yielded a sequence identical to that of the mpeC open reading frame (28) (Fig. 3). Amino acid analyses were performed on two independent preparations of the y subunit (Table 11). These compositions agreed reasonably well with that predicted from the sequence of mpeC. However, there were some discrepan- cies. In particular, the y subunit preparations showed a defi- ciency of proline and phenylalanine residues. Examination of the y subunit sequence predicted by mpeC suggested that the isolated y subunits may have been truncated by a proteolytic clip near the C terminus.

Purified y subunit was digested with trypsin and the tryptic digest fractionated by HPLC on Cls resin. The elution profile obtained for the bilin peptides in shown in Fig. 4. All of these peptides had spectra characteristic of PUB. The peptides in Peaks 11,111, and IV had very similar amino acid compositions (Table 11). Twelve steps of automated Edman degradation of the major PUB-peptide (Fig. 4, Peak 111) yielded the sequence Gln-X-X-Ala-Ala-Met-Gly-Ile-Gly-Ile-Gly-Pro, which corresponds to the sequence Gln-His-Cys-Ala-Ala-Met-Gly-Ile- Gly-Ile-Gly-Pro in mpeC (residues 47-58) which is flanked by -Lys-Arg on the N-terminal side and an Arg residue on the C-terminal side (Fig. 3). The PUB-peptide recovered from Peak IV (Fig. 4) was resistant to Edman degradation, sug- gesting that this component represents a peptide identical to that in Peak 111, but with the N-terminal glutamine residue cyclized to pyrrolidone carboxylic acid. The PUB peptide in Peak I1 gave the same sequence as that in Peak 111, except that it had an additional residue at the N terminus. This tryptic peptide is presumed to be Arg-Gln-His-Cys-Ala-Ala- Met-Gly-Ile-Gly-Ile-Gly-Pro-Arg and to arise from a tryptic cleavage between the Lys and Arg residues at positions 45

and 46 (28). The PUB is assumed to be attached by a thioether linkage to Cys-49.

The sequence of mpeC shows the presence of 2 cysteinyl residues, Cys-49 and Cys-64. To obtain independent confir- mation of the linkage of PUB to Cys-49, purified y subunit was treated with P-mercaptoethanol and then reacted with 4- vinylpyridine to convert cysteinyl residues to the pyridylethyl derivative. Chromatography of the tryptic digest of pyridyl- ethylated y subunit on reversed-phase Cls matrix gave the profile shown in Fig. 5. A single peptide containing pyridylethyl-cysteine was detected (identified by its characteristic absorbance at 254 nm, Fig. 523, Peak V). A number of smaller, but significant peaks are seen in the 254-nm trace. These are attributable to the ultraviolet absorbance of PUB peptides, which are easily identified from a 495-nm trace of the same chromatogram (Fig. 5C). Spectra of the three highest 254-nm peaks (I, 11, and V) are shown in Fig. 4, D-F. The spectra demonstrate that Peak V is unique in that there is no substantial contribution of PUB absorbance to the spectrum, and that it has a lower absorbance at 250 than at 254 nm, as expected from the Cys-pyridylethyl chromophore. Automated Edman degradation of Peak V (Table I) yielded the sequence Leu-Leu-Ser-Glu-X-Pro-Phe-Ala-Val-Thr-Phe-Asp which corresponds to the first 12 residues of the tryptic peptide Leu- Leu-Ser-Glu-Cys-Pro-Phe-Ala-Val-Thr-Phe-Asp-Arg (residues 60-72) predicted from the sequence of mpeC (28). These results show that Cys-64 is available for reaction with 4- vinylpyridine, but that Cys-49 does not react, consistent with the conclusion that Cys-49 is the PUB attachment site.

DISCUSSION

The y subunit purified from the major phycoerythrin (PE 11) of Synechococcus sp. WH8020 is encoded by the mpeC gene (28). This conclusion is based on the exact correspondence of the N-terminal sequence of the y subunit and the sequences of the tryptic peptides, which encompass residues 47 through 72, with the predicted sequence of MpeC (see Fig. 3). The amino acid composition of the y subunit shows the presence of about 5 fewer each of proline and phenylalanine residues, and about 4 fewer valine residues than expected from the sequence of mpeC. This observation and inspection of the mpeC sequence suggest that the isolated y subunit has been truncated by a proteolytic cleavage some 31 to 35 residues in from the C terminus. The sensitivity of linker poly-

TABLE I Automated Edman deeradation of PE XI Y subunit and certain of its tIYDtb2 DeDtides

y Subunit 11" 111" IV" V b Step

Res.' Yield' Res. Yield Res. Yield Res. Yield Res. Yield

1 M 30.1 NDd Q 102.6 Q 7 L 810 2 L 20.8 Q 20.0 ND ND L 800 3 G 14.2 ND ND ND S 31 4 A 19.0 ND A 149.5 A 15 E 400 5 E 10.7 A 115.9 A 77.4 A 14 ND 6 T 12.7 A 87.1 M 97.4 M 7 P 180 7 S 3.7 M 52.1 G 62.8 F 260 8 L 12.9 G 50.2 I 184.3 A 200 9 Q 5.0 I 115.8 G 97.1 V 300

10 A 10.1 G 41.8 I 205.3 T 72 11 L 12.9 I 109.8 G 15.2 F 210 12 T 6.2 G 38.5 P 59.5 D 50 13 S 7.5 P 54.1 14 A 7.4

-300 pmol from peak I1 (Fig. 4), -500 pmol from peak I11 (Fig. 4), and -150 pmol from peak IV (Fig. 4) were sequenced. * -800 pmol from peak V (Fig. 5) were sequenced. e Res., residue; yields at each step are expressed in picomoles.

ND, not determined (no residue could be identified at this step).

Phycoerythrin 11 y Subuni t 1239

mpec H L G A Z _ S i Q A L T S A T R T G P A A F S T I ( S K A G K N T V P R T V A G A I A ~ Y K R Q ~ C A A ~ G I G I G ? R L L cpeC EPFG CPClZ MPITS

286 289 85

FIG. 3. Alignment of linker polypeptide sequences. Alignment of the sequence of the mpeC gene product with those of linkers from Calothrix sp. PCC7601. The gene chosen as representative of phycocyanin-associated linkers was cpcI2 (29), C-phycoerythrin-associated linkers was cpeC (20), and of rod capping linkers was cpcD (29). Bold horizontal lines indicate the three regions which correspond to N- terminal and internal peptide sequences for the y subunit of P E 11; narrow sections of the line are positions for which no amino acid was determined. Gray arem identify agreement between two or more sequences. Dashes indicate gaps introduced to optimize alignment. The number of the last residue in each row is given a t right.

TABLE I1 Amino acid analyses of the y subunit and purified peptides and

comDarison with comDositions Dredicted from the seouence of mDeC

Amino y analyses” Predicted composition* Peptide analyses‘ acid A B mpeC Cys-49 Cys-64 I1 111 IV

D + N 32 32 29 0 1 0.7 0.1 0.4 E + Q 28 26 30 1 1 1.5 1.6 2.0 S 19 19 21 0 1 1.4 0.7 0.8 G 20 20 21 3 0 2.7 2.6 2.8 H 4 4 5 1 R

0 0.5 0.9 1.0 17 18 18 1 1 1.1 1.4 1.2

T 20 19 20 0 1 1.3 0.5 1.0 A 29 32 31 2 1 2.5 1.4 1.9 P 12 11 16 1 Y 9 9 1 0 0

1 1.0 1.0 1.0

V 0 0.4 0.0 0.4

16 16 20 0 1 1.0 0.4 0.5 M 7 9 7 1 0 0.8 0.8 1.0 I 10 10 12 2 L

0 1.4 1.4 1.8 21 21 22 0

F 2 0.7 0.6 0.8

10 11 16 0 K

2 0.3 0.6 0.0 12 12 13 0 0 0.2 0.3 0.4

PUBd 1.0 0.6 ND‘ ND ND “Two independent preparations (A and B) of the y subunit were

analyzed. In both cases the data were normalized to 21 leucine residues. A contaminant coeluting with the alanine derivative inter- fered with the quantitation of this residue.

‘The polypeptides for which composition was predicted are: MpeC, the product of mpeC from Synechococcus sp. strain WH8020; Cys-49, the predicted tryptic peptide (residues 47-59 of the mpeC gene product) containing Cys-49; and Cys-64, the predicted tryptic peptide (residues 60-72 of the mpeC gene product) containing Cys-64.

‘Tryptic peptides (Fig. 2, peaks 11, 111, and IV) of the y subunit were analyzed, and the data were normalized to 1 proline residue.

PUB content was determined with an internal norleucine stand- ard. Norleucine, a t a 201 ratio to phycourobilin (determined from absorbance a t 495 nm in acid urea), was added to samples before hydrolysis.

ND, not determined.

peptides to proteolysis during purification is well documented (30-33) and is discussed further below.

The genes encoding the non-bilin-bearing linker polypeptides associated with the C-phycoerythrin of the freshwater cyanobacterium Calothrix sp. PCC7601 have been cloned and sequenced (20). Comparison of these sequences with that of the PE I1 y subunit shows that the latter is of approximately the same length as the non-bilin-bearing linkers and shares a 220-residue region of partial homology with them. However, the region of homology is offset giving the y subunit a 50- residue N-terminal extension relative to the other sequences (Fig. 3). The single bilin (PUB) attachment site at Cys-49 lies

0.4

0.3 0 C m 3 0.2 0 v) n

0.1

0 14 16 18 20 22 24

Elution Time (min)

FIG. 4. Purification of y subunit tryptic bilin peptides. HPLC separation of the tryptic digest of the y subunit (200 pg) on CIS reversed-phase resin monitored a t 495 nm (for details, see “Ex- perimental Procedures”). PUB peptides, labeled I-IV, correspond to peptides designated in the same manner in Fig. 5C.

within this extension. To conform with the accepted nomen- clature for rod linker polypeptides (4), and yet distinguish between bilin-bearing from non-bilin bearing linkers, the y subunit is henceforth designated y (

The sequence about the bilin attachment site on y (Li2) bears no obvious resemblance to those about the six bilin attachment sites on the a and /3 subunits (13). The y subunits of red algal R- (17), and B-phycoerythrins (18, 34) carry multiple bilins. All of the tryptic bilin peptides from the y subunit of the R-phycoerythrin of the red alga Gastroclonium coulteri have been sequenced (17). None of these show an obvious sequence relationship to the PUB-bearing region of the PE I1 y subunit.

This study addressed two questions. In what manner does a particular phycoerythrin-associated linker polypeptide contribute to the unusual stability of the hexameric protein? How do the bilins associated with the linker polypeptide contribute to the energy transfer pathways within a phycoerythrin hexamer? To approach the answers to these questions, the results presented here need to be considered in the context of current knowledge of the three-dimensional structure of the phycobiliproteins and of the details of the interaction of these proteins with linker polypeptides.

Phycobilisome rods are made up of stacks of hexameric phycobiliprotein complexes with the composition (a/3)eL~, where LR is a rod linker polypeptide (3-7). The products of partial dissociation of rod substructures at low salt concentration vary from one phycobilisome to another. The products observed with the phycobilisomes of Anabaena sp. (31) are shown in Fig. 6. A different pattern of dissociation is seen

1240 Phycoerythrin II y Subunit

1.00

0.50

0 0.08

e 0.06 0.04

m

Y) 4 0.02

0.15 0

Elution Time (min)

W.".lmgfh 1 2 4 0 - 5 9 0 n T l

FIG. 5. Purification of they subunit tryptic peptide containing the S-@-(4-pyridylethyl)-cysteinyl derivative. A, HPLC separation of the tryptic digest of the pyridylethyl-derivative of the y subunit (200 pg) on Cla reversed-phase resin monitored at 210 nm (for details, see "Experimental Procedures"). B and C, the 254- and 495-nm traces, respectively, of the separation shown in A. Light gray shading under peaks, peaks with substantial contribution from PUB to the 254-nm absorbance; dark gray shading under peak, peak with no significant PUB contribution to the 254-nm absorbance. Four peptide elution peaks are labeled to correspond with peptides identified in Fig. 4. D, spectrum of peak I, acquired at 17.9 min; E, spectrum of peak 111, acquired at 19.9 min; F, spectrum of peak V, acquired at 20.6 min.

with the phycobilisome of the freshwater cyanobacterium Synechocystis sp. PCC6701. In this phycobilisome the core- distal half of the rods consists of two phycoerythrin hexamers, (afl)gLg.', and (ap)6Lp6 (19). Dissociation of phycobilisomes takes place in low salt, and these hexamers break down to smaller complexes including a& (cYB)L~.~, and (aB)Lp5 (35). This contrasts with the dissociation behavior of the rods of marine Synechococcu.s sp. WH8020 phycobilisomes, where an intact PE (11) hexamer is obtained (13). A consistent finding is that the rod linker polypeptides are resistant to proteolysis in the intact rods (22, 31), but that their C-terminal portion becomes very sensitive to proteolysis on dissociation of the rods (31-33).

Comparison of the the sequence of the y (LE) polypeptide with that of the product of Calothrix sp. PCC7601 cpeC gene, LF5, the isofunctional non-bilin-bearing, phycoerythrin-associated linker polypeptide, shows a region of partial homology between residues 51-249 of y (LE) and residues 1-199 of LP'. However, y (LE) has a %)-residue N-terminal extension relative to L?' (Fig. 3).

As shown in Fig. 3, the C-terminal region of rod linker polypeptides such as LF5 is homologous to a short polypeptide (LW'), present in phycobilisomes that contain phycocyanin as the sole rod phycobiliprotein, that functions as a "capping" polypeptide which terminates rod elongation (29). The C terminus of y (LE) is about 45 residues shorter at the C terminus than LW'.

These sequence comparison lead to a proposed structure for the phycoerythrin hexamer shown in Fig. 6B. The unusual stability of the hexamer is attributed to the insertion of the 50-amino acid N-terminal extension of y (LE) into the central channel of the core-distal trimer. This new interaction "sta- ples" the two trimers and leads to a stable hexamer.

"TYPICAL" PHYCOBILIPROTEIN HEXAMERIC BUILDING BLOCK OF PHYCOBILISOME RODS

A.

PHYCOERYTHRIN I1 HEXAMER

B. smE vmw

C. TOP VIEW

n

W 0 w9- A B*=

FIG. 6. Proposed models for the disposition of non-bilin- bearing linker polypeptides (LR) in phycobiliprotein hexamers and for the bilin-bearing y (Lga) in the PE I1 hexamer. A, this diagram is based on data for the building blocks of the rods of Anabaena sp. phycobilisomes in which the linker polypeptides do not carry bilins (31). Different phycobilisomes give different patterns of dissociation products (see "Discussion"). B , location of various seg- ments of the y (Lk') in the PE I1 hexamer. C, diagrammatic represen- tation of the spatial relationship of the proposed location of the y-49 PUB chromophore to the terminal energy acceptors within the hexamer, the 8-84 PEBs, which project into the central channel within each trimer of the hexamer (36-39).

Insertion of the C terminus of the rod linker polypeptide into the central channel of the core-distal trimer of the adjoining hexamer is required for addition of discs to the phycobilisome rod. In the case of the PE I1 y (LE), to compensate for the presence of 50 residues of the N-terminal extension within this space in the core-distal trimer, the C terminus, y (Lg) is shorter by some 43 residues (Fig. 6).

The three-dimensional structures of three phycocyanins (36-38), a phycoerythrocyanin (39), and a B-phycoerythrin (40) have been shown to be very similar by x-ray crystallog- raphy. The central location of LR shown in Fig. 6 is fully compatible with the crystallographic data. Data from studies of bilin locations in phycobiliproteins varying in bilin composition (13, 41, 42), crystallographic studies (36-38), and spectroscopy (43,44), indicate that the bilin at 8-84 functions as the terminal energy acceptor in phycobiliproteins. In a phycobiliprotein trimer, (a@)3, the three 8-84 bilins lie at the periphery of a channel in the center of the phycobiliprotein disc. Comparison of the spectrum of an LR-containing trimer ((Y@)3LR with that of ( L Y P ) ~ shows unambiguously that the 8- 84 bilin is the only bilin whose spectroscopic properties are strongly affected by the interaction of LR with (a8)3 (see, for example, such a comparison for R-phycocyanin I1 in Fig. 10 in Ref. 12).

The sequence alignment shown in Fig. 3 predicts that the sole bilin attached to y (LE), at 7-49, will lie in the center of the hexamer at or near the trimer-trimer interface. This PUB is closely surrounded by the three acceptor PEBs which project into the channel in each the core-proximal and core-

Phycoerythrin 11 y Subunit 1241.

distal trimers (Fig. 6C). Consequently, the PUB at 7-49 will transfer energy efficiently to the terminal acceptors. Since the absorption of PUB lies at 495 nm and that of PEB a t about 555 nm, the bilin at 7-49 will act solely as an energy donor.

In summary, comparison of the sequence of y (Liz) with those of rod linker polypeptides which do not carry bilins leads to a plausible explanation of the stability of the PE I1 hexamer and provides an indication of the location of the additional bilins carried by phycoerythrin-associated y subunits.

Acknowledgments-We are indebted to Dr. John Waterbury for supplying us with Synechococcus sp. strain WH8020 and t o Dr. Michael Moore of the Cancer Research Laboratory, University of California, Berkeley, for access to the protein sequencer.

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