Proc. Nat. Acad. Sci. USA Vol. 78, No. 2, pp. 428-431, February 1976 Biochemistry of and Rhodophyta: Homologous family of photosynthetic accessory pigments (/amino-terminal sequences/evolution/structure-function relationships) ALEXANDER N. GLAZER, GERALD S. APELL, CRAIG S. HIXSON, DONALD A. BRYANT, SARA RIMON*, AND DOUGLAS M. BROWN Department of Biological Chemistry, UCLA School of Medicine, and the Molecular Biology Institute, University of California, Los Angeles, Calif. 90024 Communicated by Emil L. Smith, December 1, 1975

ABSTRACT Amino-terminal sequence determinations carrying (s) (see Table 1). The molecular are re orted of the subunits of biliproteins of prokaryotic weights of the a and f3 subunits differ among the bilipro- unicel ular and filamentous cyanobacteria and of eukaryotic teins, and depend on the source organism, but, strikingly, all unicellular red . The biliproteins examined, allophyco- cyanin, -, R-phycocyanin, b-, and fall in the approximate range of 14,000-20,000 (2). The phycoerythrocyanin, vary with respect to the chemical na- prosthetic groups are invariably open chain (2). ture and the number and distribution of the chromo- These observations lead to the postulation of a common phores between the two dissimilar subunits. The amino-ter- ancestral gene for this group of (10). This hypothe- minal sequences fall into two classes, "a-type" and ",-type", sis is supported by the sequence data presented here. with a high degree of homology within each class. In those biliproteins where the number of bilin chromo- phores on the two subunits is unequal, the subunit with the MATERIALS AND METHODS greater number of has the j#-type amino-acid The unicellular blue-green alga Microcystis aeruginosa sequence. ATCC 22663 was obtained from the American Type Cul- Extensive homology also exists between a- and #-type se- quences, strongly supporting the view that these arose by ture Collection, Rockville, Md., and maintained in medium gene duplication to give rise to the ancestral a- and a-type BG 11, as described by Stanier et al. (11). The cells were genes early in the evolution of the biliproteins. The subse- grown to a density of approximately 1.5 g/liter and harvest- quent generation of the various classes of biliproteins ap- ed by centrifugation, and the pellets were stored at -20°. pears to be the result of further gene duplication of the a- The source of the organisms and the preparation of the and a-type genes, ultimately to give rise to families of poly- fractions from Anabaena variabilis and Porphy- chains of similar sequence, but varying in the num- cruentum B been 3). Al- ber of chromophore attachment sites and the structure of the ridium has described previously (1, chromophores. lophycocyanins and C- were purified by the general procedures described previously (4, 9). R-Phyco- The phycobiliproteins are the major light-harvesting pig- cyanin was purified by the procedure of Glazer and Hixson ments of photosystem II of cyanobacteria and Rhodophyta. (1). The details of the purification of phycoerythrocyanin (3) Three major classes of biliproteins have been recognized for and b-phycoerythrin (12) will be described elsewhere. a number of years: the allophycocyanins (Xmax 650 nm), and The subunits of C-phycocyanin, R-phycocyanin, and phy- C-phycocyanins (Xmax about 620 nm), both of which carry coerythrocyanin were separated by the method of Glazer covalently bound (PCB) prosthetic groups, and Fang (3), by stepwise elution from Bio-Rex 70 at pH 3.0 and (Xmax about 565 nm) with covalently at- with solutions of increasing urea concentration. Allophyco- tached (PEB) groups. R-Phycocyanins, cyanin subunits were separated by chromatography on found in certain , carry both PCB and PEB DEAE-Sephadex A-50 at pH 8.0 in 8 M urea, by the proce- moieties. The nomenclature and properties of biliproteins dure of Gysi and Zuber (13), but with stepwise rather than have been reviewed recently (1, 2). gradient elution. The fl subunit was eluted with 8 M urea- There are two other types of cyanobacterial biliproteins: B (Xmax about 670 nm) (3), and phycoer- ythrocyanin (Xmax 568 nm) (ref. 3, and unpublished observa- Table 1. Type and number of bilin chromophores on the tions). subunits of various biliproteins Within a given class of biliproteins, e.g., C-phycocyanins, there is a remarkable conservation of physical and immuno- ai-type logical properties (2, 4-6), primary structure (7), and chro- subunit (3-type subunit Ref. mophore content (8). Indeed, hybrid proteins have been suc- cessfully reconstituted from the subunits of C-phycocyanins Allophycocyanin 1 PCB 1 PCB 8 derived from unrelated organisms (9). C-Phycocyanin 1 PCB 2 PCB 8 The various classes of biliproteins also exhibit several com- R-Phycocyanin 1 PCB 1 PCB and 1 1 PEB mon characteristics. The monomer species is in each in- Phycoerythrocyanin PXB* 2 PCB * stance made up of two dissimilar subunits, a and (,B each b-Phycoerythrin 2 PEB 3-4 PEB t Abbreviations: PCB, phycocyanobilin; PEB, phycoerythrobilin; * PXB is a phycoerythrobilin-type chromophore (Xmax 330 and 590 PXB, a phycoerythrobilin-type chromophore of undetermined nm, in 8 M urea at pH 3.0) of as yet undetermined structure (D. A. structure. Bryant, F. E. Eiserling, and A. N. Glazer, unpublished observa- * Present address: Biochemistry Department, Tel-Aviv University, tions). Israel. t A. N. Glazer and C. S. Hixson, unpublished observations. 428 Downloaded by guest on September 28, 2021 Biochemistry: Glazer et al. Proc. Nat. Acad. Sci. USA 73 (1976) 429

Table 2. Percentage yields of certain phenylthiohydantoin derivatives obtained on sequential Edman degradation of biliprotein subunits Residue number - mol Source Protein 1 2 3 4 5 6 7 8 9 10 11 12 14 15 16 17 18 19 used 45 23 21 56 22 17 24 21 0.27 sp. 6301 Allophycocyanin (3 M. aeruginosa Allophycocyanin a 20 14 19 12 9 9 13 12 5 0.43 M. aeruginosa Allophycocyanin , 35 10 20 15 12 10 0.10 A. variabilis Allophycocyanin a 33 44 19 15 26 22 13 7 13 0.73 A. variabilis Allophycocyanin 3 52 52 26 16 35 26 52 26 31 4 8 0.62 P. cruentum Allophycocyanin a 19 6 6 12 6 3 8 5 0.31 P. cruentum Allophycocyanin 41 47 41 47 23 23 17 17 0.35 P. cruentum R-Phycocyanin a 67 34 67 34 34 55 0.24 P. cruentum R-Phycocyanin 3 63 51 32 51 29 32 32 17 8 0.24 P. cruentum b-Phycoerythrin a 38 76 29 38 19 10 19 19 0.21 P. cruentum b-Phycoerythrinj3 51 51 32 32 32 10 11 11 22 13 0.31 A. variabilis Phycoerythrocyanin ct 6 6 8 10 8 8 8 0.48 A. variabilis Phycoerythrocyanin 3 41 17 17 22 17 11 0.36

0.01 M K-phosphate-0.01 M 2-mercaptoethanol-0.05 M acid, or heptafluorobutyric acid). An aliquot was withdrawn NaCI at pH 8.0, and the a with the same buffer containing for quantitative amino-acid analysis and the remainder was 0.18 M NaCl. The subunits of b-phycoerythrin were sepa- added to the spinning cup at high speed (1600 rpm at 55°). rated by chromatography on CM-cellulose at pH 5.0 in 8 M The sample container was washed with 0.1 ml of solvent and urea with a NaCl gradient (Glazer and Hixson, manuscript the washings were added to the cup. The modifications to in preparation). Beckman Program 122974 were as follows: double coupling The sequential Edman degradation was performed in a on the first cycle, and increase in the restricted vacuum dry- Beckman sequencer model 890-C using the Beckman Pro- down times at program steps 19, 25, 31, 41, and 48-400, 200, tein Quadrol Program 122974 ("In Sequence", Issue No. 8, 260, 300, and 260 sec, respectively. Nitrogen dry-down at Oct. 1975, Beckman Instruments, Inc.), and 0.2 M quadrol program step 30 was increased from 30 to 230 sec. (single cleavage). Lyophilized polypeptide (0.1-0.75 ,mol) The butyl chloride extracts containing the anilinothiazoli- was dissolved in about 0.3 ml of an appropriate solvent (30% nones were dried under nitrogen at 50°, and conversion to vol/vol acetic acid, 70% vol/vol formic acid, trifluoroacetic the phenylthiohydantoin derivatives was accomplished with

Table 3. Amino-terminal sequences of biliprotein subunits* a-type subunits 1 5 10 15 Synechococcus sp. 6301 C-Phycocyanin a Subunitt Ser-Lys-Thr-Pro-Leu-( )-Glu-Ala-Val-Ala-Ala-Ala-Asx-( )-( )-Gly Anabaena variabilis C-Phycocyanin a Subunit Val-Lys-Thr-Pro-fle-Thr-Glu-Ala-Be-Ala-( )-Ala-( )-Asp-Gln-Gly-Arg-Phe-Leu-Gly-Asn Mastigocladus laminosus C-Phycocyanin a Subunit § Val-Lys-Thr-Pro-Ile-Thr-Asp-Ala-le-Ala-Ala-Ala-Asp-Thr-Gln-Gly-Arg-Phe-Leu-Ser-Asn-Thr-Glu Cyanidium caldarium C-Phycocyanin a Subunit¶ Met-Lys-Thr-Pro-lle-Thr-Glu-Ala-lle-Ala-Ala-Ala-Asx(Ala)Arg-Gly Porphyridium cruentum R-Phycocyanin a Subunit Met-Lys-Thr-Pro-Ile-Thr-Glu-Ala-Ile-Ala-Thr-Ala-Asp-Asn-Gln-Gly-Arg-Phe-Leu-( )Asn Anabaena variabilis Phycoerythrocyanin a Subunit Met-Lys-Thr-Pro-Leu-Thr-Glu-Ala-Ile-Gly-Ala-Ala-Asp-Val-Arg-Gly-( )-Tyr-Leu-( )-Asn Porphyridium cruentum b-Phycoerythrin a Subunit Met-Lys-Ser-Val-Ile-( }Thr-Val-Val-( -Ala-Ala-Asp-Ala-Ala-Gly(Arg)Phe-Pro 1 5 10 15 (Ble/I 20 Microcystis aeruginosa Allophycocyanin a Subunit Ser-Ile-Val-Thr-Lys-( )fle-Val-Asn-Ala-Asp-Ala-Glu-Ala-Arg-Tyr-Leu\Leu/Pro-Gly-Glu Anabaena variabilis Allophycocyanin a Subunit ( )-lle-Val-( )-Lys-( )-lle-Val-Asn-Ala-Asp-Ala-Glu-Ala-( )-Tyr-Leu-Leu Porphyridium cruentum Allophycocyanin ca Subunit Ser-lle-Val-Thr-Lys-( )-fIle-Val-Asn-Ala-Asp-Ala-Glu-Ala-Arg-Tyr-Leu ,-type subunits Synechococcus sp. 6301 Allophycocyanin 1 Subunit Met-Gln-Asp-Ala-Ile-( )-AIa-Val-fle-Asn-Ala-( )Asp-Val-Gln-Gly-Lys-Tyr-Leu-Asp Anabaena variabilis Allophycocyanin ,B Subunit Ala-Gln-Asp-Ala-lle-Thr-Ala-Val-lle-Asn-( )-Ala-Asp-Val-Gln-Gly-( )Thr-Leu-Asp Microcystis aeruginosa Allophycocyanin 1 Subunit Met-Gln-Asp-Ala-Ile-( -( )-Val-Ile-Asn-( )-( )-Asp-Val-Gln-( )-Lys-Tyr-Leu-Asp Porphyridium cruentum Allophycocyanin 13 Subunit Met:Gln-Asp-Ala-Ile-Thr-( )-Val-lle-Asn-Ala-Ala-Asp-Val-Gln-Gly-Lys-Tyr-Leu-Asp 1 5 10 15 Synechococcus sp. 6301 C-Phycocyanin , Subunitt Thr-Phe-Asp-Ala-Phe-Thr-Lys-Val-Val-Ala-Gln-Ala-Asp-Ala-Arg-Gly-Glu-Phe-Leu-Ser Anabaena variabilis C-Phycocyanin 13 Subunit Gly-Leu-Asp-Val-Phe-( )Lys-Val-( -( )-Gln-Ala-Asp-( )-Arg-Gly-Glu-Phe-Leu Mastigocladus laminosus C-Phycocyanin 1 Subunit § Ala-Tyr-Asp-Val-Phe-Thr-Lys-Val-Val-Ser-Gln-Ala-Asp-Ser-Arg-Gly-Glu-Phe-Leu-Ser Cyanidium caldarium C-Phycocyanin 13 Subunit I Met-Leu-Asx-Ala-Phe-Ala-Lys-Val(Val)Ala-Ala-Ala-Asx-Ala-Arg-Gly-Glu-Phe-Lys Porphyridium cruentum R-Phycocyanin 1 Subunit Met-Leu-Asp-Ala-Phe-Ala-Lys-Val-Val-Ala-Gln-Ala-Asp-Ala-Arg-Gly-Glu-Phe-Leu Anabaena variabilis Phycoerythrocyanin 1 Subunit Met-Leu-Asp-Ala-Phe-( )Lys-Val-Val-Glu-Gln-Ala-Asp-Arg-Lys-Gly-Asn-Tyr-Leu Porphyridium cruentum b-Phycoerythrin 13 Subunit Met-Leu-Asp-Ala-Phe-Thr-Arg-Val-Val-Val-Asn-Ala-Asp-Ala-Lys-Ala-Ala-Tyr-Val * Empty parentheses indicate that no definite identification of the residue at that position was made. A residue shown in parentheses was identified unambiguously by only one procedure. t Data from ref. 7. t P. Freidenreich, G. Apell, and A. N. Glazer, unpublished results. § Data from ref. 17. The a and 13 subunits of M. laminosus C-phycocyanin have nearly identical molecular weights. The designations a and ,1 originally given to these subunits were based upon electrophoretic mobility in the presence of urea. The subunit designated "a" by Frank et al. (17) has the 13-type amino-terminal sequence, and that designated "13", the a-type sequence. ' Data from ref. 18. Residue 13 in the a-type subunit and residues 3 and 13 in the 13-type subunit of Cyanidium caldarium C-phycocyanin were identified by Troxler et al. (18) as Asn. However, the data presented (18) do not exclude the possibility that the residue at these positions is actually Asp. Downloaded by guest on September 28, 2021 430 Biochemistry: Glazer et al. Proc. Nat. Acad. Sci. USA 73 (1976) 1 M HC under N2at 800 for 10 min. When the presence of Sufficient homology has remained between the sequences seryl or threonyl derivatives was suspected, conversion was of the a- and ,8-type subunits (e.g., Ile/Val at position 9, Ala restricted to 3 min. at 12, Asp at 13, Ala/Gly at 16, Phe/Tyr at 18) to demon- The phenylthiohydantoin derivatives were extracted with strate that these have arisen from a common ancestral gene, ethyl acetate and were identified, in each instance, by gas presumably by gene duplication. Of the proteins examined, chromatography (14), by thin-layer chromatography (15), the allophycocyanins showed the most pronounced conser- and by amino-acid analysis after regeneration in 6 M HC1 at vation of sequence and the most complete homology be- 1500 for 22 hr (16). Aliquots of the aqueous phase from the tween a and fl subunits. ethyl acetate extraction were spotted on Whatman 3MM The identity in the residues occupying two positions (Ala- paper and examined for the presence of arginine with Saka- Asp at 12 and 13) in all of the diverse subunits examined guchi reagent. here, and the extremely conservative substitutions at other Table 2 presents the amount of each of the subunits used positions (e.g., Leu/Ile/Val/Phe at residue 5; Ala/Val at 8; for the degradation, as well as the percentage yields of cer- Ile/Val at 9; Phe/Tyr at 18) is striking. The sequences exam- tain of the phenylthiohydantoin derivatives, as determined ined represent approximately 12% of the total sequence in by gas chromatography and, in most instances, by regenera- each instance, and only tentative conclusions can be reached tion as well. from such data. However, the observed homology provides compelling support for the view that certain elements of three-dimensional structure are conserved among the bil- RESULTS AND DISCUSSION iprotein subunits independent of the nature and number of t Prior to a discussion of the significance of the results in the chromophores they carry. Table 3, it is necessary to define the convention which deter- The classification of the biliproteins is based on the chem- sub- ical nature of the chromophores and on the absorption spec- mines the nomenclature used here for the biliprotein tra of the native proteins (1, 2). Table 1 lists the type and units. The designations a and ( were originally assigned to the subunits of biliproteins from various cyanobacteria on number of chromophores on the subunits of the biliproteins the basis of relative molecular weight, i.e., the smaller sub- examined in this study. Comparison of the data in Tables 1 unit was named a in each instance (4). However, it was and 3 leads to an important conclusion. Amino-terminal se- noted that the subunits of certain biliproteins from other cy- quence homology does not correlate in a simple manner anobacteria and Rhodophyta were identical in molecular with either the number of chromophores carried by the vari- weight (e.g., 17, 19, 20). The size is, therefore, inapplicable ous polypeptide chains or with the chemical nature of these for naming the subunits in such instances. In the present re- chromophores. These limited data strongly support the hy- port, the relative molecular weights are disregarded, and an pothesis that the various classes of biliproteins were generat- "a-type" subunit is arbitrarily defined as one whose amino- ed by attaching chromophores differing both in structure terminal sequence is homologous to that of the a subunit of and number onto basically similar polypeptide chains. For Synechococcus sp. C-phycocyanin as described by Glazer the biliproteins examined here, the (-type sequence is char- and Cohen-Bazire (4) and Williams et al. (7), and the ,B sub- acteristic of the subunit carrying the greater number of unit is similarly defined as homologous to the (-subunit of chromophores (Table 1). Synechococcus sp. C-phycocyanin (4, 7). It has been shown that in C-phycocyanin the energy ab- The biliproteins examined in this study span the full range sorbed by the a-subunit chromophore is re-emitted as fluo- of spectroscopically distinct proteins of this class and the rescence, whereas that absorbed by the (-subunit chromo- source organisms are far apart on the evolutionary scale. The phores sensitizes the of the a-subunit chromo- proteins listed in Table 3 are derived from both of the major phore (23, 24). For several of the proteins listed in Table 1 classes of cyanobacteria-the unicellular (Synechococcus (allophycocyanin, C-phycocyanin, phycoerythrocyanin), it sp., Microcystis sp.), and filamentous organisms (Anabaena has been possible to renature the separate subunits. In two of as well as from eukaryotic these instances, allophycocyanin and C-phycocyanin, the a sp., Mastigocladus laminosus), subunit exhibits a longer wavelength absorption maximum algae (Cyanidium caldarium, Porphyridium cruentum). than the (3. Further, in R-phycocyanin the chromophore ab- Fifteen amino-terminal sequences of diverse biliprotein ( subunits were determined in this study. These, and six addi- sorbing at the shorter wavelength is on the subunit. It is tional such sequences of C-phycocyanin subunits from other tempting, therefore, to make the generalization that the a- in Table 3. Limited data on type sequence is associated with the subunit which exhibits studies, are presented sequence the longer wavelength absorption band in the native state. the biliproteins obtained by others (17, 18, 21, 22) are consis- Phycoerythrocyanin does not fit this generalization. In this tent with those accumulated in this study. It is evident that (3 these sequences fall into two classes-a-type and (3-type. protein, the subunit carries the chromophores with the Very high conservation of primary structure is seen within longer wavelength absorption maxima. each group. Alignment of the sequences did not require the We are indebted to Dorothy McNall for the amino-acid analyses. introduction of any deletions. The NH2-terminal sequences This research was supported in part by Grant GM 11061. C.S.H. of the a-type subunits of allophycocyanin provide the sole and D.A.B. were supported by predoctoral fellowships from Train- exceptions. The homology between these sequences and ing Grants GM 00364 and GM 1531, respectively, from the Nation- those of the corresponding subunits of other biliproteins in- al Institutes of Health, U.S. Public Health Service. dicates that the first residue in the allophycocyanin a-type 1. Glazer, A. N. & Hixson, C. S. (1975) J. Biol. Chem. 250, sequences corresponds to residue 3 in the others (see Table 5487-5495. 3). The homology within each class of sequences is strikingly t The sites of attachment of the chromophores to the a and , sub- greater than that between the two classes. Thus the diver- units of Synechococcus sp. 6301 C-phycocyanin are distant from gence between a- and (3-type subunits was a very early the amino-terminal ends of these polypeptides (V. P. Williams event in the evolutionary history of the biliproteins. and A. N. Glazer, unpublished results). Downloaded by guest on September 28, 2021 Biochemistry: Glazer et al. Proc. Nat. Acad. Sci. USA 73 (1976) 431

2. Bogorad, L. (1975) Annu. Rev. Plant Physiol. 26,369-401. 15. Inagami, T. & Murakami, K. (1972) Anal. Biochem. 47, 501- 3. Glazer, A. N. & Bryant, D. A. (1975) Arch. Microbiol. 104, 504. 15-22. 16. Van Orden, H. 0. & Carpenter, F. H. (1964) Biochem. Bio- 4. Glazer, A. N. & Cohen-Bazire, G. (1971) Proc. Nat. Acad. Sci. phys. Res. Commun. 14,399-40. USA 68, 1398-1401. 17. Frank, G., Zuber, H. & Lergier, W. (1975) Experientia 31, 5. Berns, D. S. (1967) Plant Physiol. 42, 1569-1587. 23-26. 6. Glazer, A. N., Cohen-Bazire, G. & Stanier, R. Y. (1971) Proc. 18. Troxler, R. F., Foster, J. A., Brown, A. S. & Franzblau, C. Nat. Acad. Sci. USA 68,3005-3008. (1975) Biochemistry 14,268-274. 7. Williams, V. P., Freidenreich, P. & Glazer, A. N. (1974) Bio- 19. Vaughan, M. H., Jr. (1964) Ph.D. Dissertation, Massachusetts chem. Biophys. Res. Commun. 59,462-466. Institute of Technology, Cambridge, Mass. 8. Glazer, A. N. & Fang, S. (1973) J. Biol. Chem. 248,659-662. 20. Bennett, A. & Bogorad, L. (1971) Biochemistry 10, 3625- 9. Glazer, A. N. & Fang, S. (1973) J. Biol. Chem. 248,663-671. 3634. 10. Glazer, A. N. (1976) Photochem. Photobiol. Revs. 1, in press. 11. Stanier, R. Y., Kunisawa, R., Mandel, M. & Cohen-Bazire, G. 21. Brown, A. S., Foster, J. A., Voynow, P. V., Franzblau, G. & (1971) Bacteriol. Rev. 35,171-205. Troxler, R. P. (1975) Biochemistry 14,3581-3588. 12. Gantt, E. & Lipschultz, C. A. (1974) Biochemistry 13, 2960- 22. Harris, J. U. & Berns, D. S. (1975) J. Mol. Evol. 5, 153-163. 2966. 23. Glazer, A. N., Fang, S. & Brown, D. M. (1973) J. Biol. Chem. 13. Gysi, J. & Zuber, H. (1974) FEBS Lett. 48,209-213. 248,5679-5685. 14. Pisano, J. J., Bronzert, T. J. & Brewer, H. B., Jr. (1972) Anal. 24. Teale, F. W. J. & Dale, R. E. (1970) Biochem. J. 116, 161- Biochem. 45,43-59. 169. Downloaded by guest on September 28, 2021