subunit of a cryptomonad phycoerythrin

THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1989 by The American Society for Biochemistry and Molecuiar Biology, Inc. Vol. 264, No. 30, Issue of October 25, pp. 17860-17867,1989

Printed in U. S A .

Posttranslational Modifications of the @ Subunit of a Cryptomonad Phycoerythrin SITES OF BILIN ATTACHMENT AND ASPARAGINE METHYLATION*

(Received for publication, April 25, 1989)

Determination of the partial amino acid sequence of the 6 subunit of cryptomonad strain CBD phycoerythrin 566 established the nature, locations, and modes of attachment of the three bilin prosthetic groups and revealed a site of posttranslational methylation. Isola- tion of peptides cross-linked by a phycobiliviolin led to an unambiguous assignment of two thioether linkages, from residues 8-Cys-50 and @-Cys-61 to this bilin. Two bilins were attached through single thioether linkages, a phycobiliviolin at 6-Cys-158 and a phycoerythrobilin at 6-Cys-82 (the residue numbering is that for B-phycoerythrin; Sidler, W., Kumpf, B., Suter, F., Morisset, W., Wehrmeyer, W., and Zuber, H. (1985) Biol. Chem. Hoppe-Seyler 366, 233-244). The partial sequences (99 residues) established for phycoerythrin 566 @ subunit showed a 79% identity with that of the red algal Porphyridium cruentum B-phycoerythrin 6 subunit. A particularly remarkable finding is that the unique methylasparagine residue at position 6-72, highly conserved in cyanobacteriai and red algal phycobiliproteins (Klotz, A. V., and Glazer, A. N. (1987) J. Biol. Chern. 262, 17350-17355), is ais0 present at 6-72 in the cryptomonad phycoerythrin.

Comparison of the locations of donor and acceptor bilins in cryptomonad phycoerythrin with those found for cyanobacterial and red algal phycobiliproteins showed different favored pathways of energy migration in the cryptomonad protein.

Cryptomonads are a small group of unicellular biflagellated algae, the majority of which are phototrophs. The cryptomonad photosynthetic apparatus is unusual in that the light- harvesting components include both chlorophyll a/cz (or c,) light-harvesting proteins and a phycobiliprotein. Although several other algal classes contain chlorophyll a/c light-harvesting proteins, none of them contain phycobiliproteins. Aside from the cryptomonads, the only other organisms containing phycobiliproteins are the prokaryotic cyanobacteria and the red algae, all of which contain several phycobiliproteins but not chlorophyll c (1-3). The similarities and differ- ences between the cryptomonad phycobiliproteins and those of cyanobacteria and red algae are presented in a condensed

* This research was supported in part by the National Institute of General Medical Sciences Grant GM 28994 and National Science Foundation Grant DMB 8816727. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accord- ance with 18 U.S.C. Section 1734 solely to indicate this fact.

5 Recipient of a predoctoral fellowship from the Dept. of Health and Human Services Training Grant 5 T32 GM 07232-12.

form in Table I. This information suggests that the p subunit of the cryptomonad phycobiliproteins is very closely related to the corresponding subunit of red algal phycoerythrins. The origins of the a subunits, however, are more enigmatic since these do not appear to be divergent versions of the a subunits of red algal phycobiliproteins (11).

In common with the phycobiliproteins of cyanobacteria and red algae, those of the cryptomonads function as antenna pigments for photosystem I1 (13). In view of the correspond- ence in function, the distinctive location of the cryptomonad phycobiliproteins is particularly intriguing. In cyanobacteria and red algae, phycobiliproteins, assembled into phycobilisomes, are attached to the stromal side of the thylakoid membranes; in the cryptomonads, they are on the lumenal side ( 5 , 12, 17). However, the transmembrane orientation of the integral photosystem I and I1 complexes is the same in all three groups of organisms (18).

Gibbs and her co-workers (12; see also Ref. 19) have provided persuasive data to support the hypothesis that the progenitors of these photosynthetic zooflagellates phagocy- tosed a red alga-like cell and that with time the red algal partner in the resulting eukaryote-eukaryote symbiosis was reduced to a chloroplast and its surrounding structures. What- ever the evolutionary history of the cryptomonad photosynthetic apparatus might be, it is evident that the present day organism has retained one polypeptide component of the phycobilisome in a markedly different and differently assembled light-harvesting antenna (see Table I). No other struc- tural component of the phycobilisome appears to be present in the cryptomonads. Even the quaternary structure of the cryptomonad phycobiliprotein, which includes the retained f l subunit, has no counterpart among those present in cyanobacterial or red algal phycobilisomes (20, 21).

The limited data available show that the amino acid sequences of the p subunits of cryptomonad phycobiliproteins are remarkably homologous to that of the p subunit of red algal phycoerythrin (11, 16, 20, 22). We show here that the homology does not extend to conservation of the relative positions occupied by the donor and acceptor bilins on the 0 subunit which determine the preferred pathways of energy transfer. The distribution of donor and acceptor bilins on the cryptomonad phycoerythrin p subunit mandates a different preferred pathway of energy transfer than that seen univer- sally in cyanobacterial and red algal phycobiliproteins. This suggests that the functional properties of the red algal phycoerythrin 0 subunit required modification to optimize the contribution of this component in the “foreign” context of the cryptomonad light-harvesting assembly. A highly conserved methylasparagine residue, of unknown function, occupies position p-72 of cyanobacterial and red algal phycobiliproteins

17860

Posttranslational Modifications of Cryptomonad Phycoerythrin 17861 TABLE I

Phycobiliprotein structure and organization in cyanobacteria, red algae, and cryptomonads (see Refs. 1-11 for details)

Characteristic Cyanobacteria Red algae Cryptomonads

No. of major phycobiliproteins 2-3; allophycocyanin and 2-3; allophycocyanin and 1; phycocyanin or phyco- phycocyanin, sometimes phycocyanin; very fre- erythrin phycoerythrin or phyco- quently phycoerythrin as erythrocyanin as well well

Supramolecular organization of Phycobilisomes Phycobilisomes Rods (?)”

Subunit composition of major f fP a v p * phycobiliproteins

phycobiliprotein building blocks

Subunit size (residues) a subunit 164b 164‘ a’ 70; a* 80d /3 subunit 181* 177‘ 177d

Bilin groups per subunit Phycocyanin

a subunit 1 B subunit 2

Phycoerythrin a subunit @ subunit

2 or 3 3

1 3

1 3

Amino acid sequence homology between corresponding cyanobacterial, red algal, and cryptomonad phycobiliproteins

a subunit High High Very low P subunit High High High

Recent immunoelectron microscopy studies suggest that the cryptomonad phycobiliproteins are associated

Data for the a and 0 subunits of Fremyella diplosiphon (Calothrix sp. PCC7601) C-phycoerythrin (14). Data for the N and 0 subunits of P. cruentun B-phycoerythrin (15).

with the lumenal surface of the thylakoid membrane and organized into rod-like structures (12; see also Ref. 13).

dData for Chroomonas sp. phycocyanin-645 (11, 16)-

(23). We find that y-N-methylasparagine also occupies the homologous position in the cryptomonad phycobiliproteins. Given that numerous other phycobilisome components are apparently not conserved in the cryptomonads, the persist- ence of the unique posttranslational methylation of the asparagine residue at p-72 (and consequently of the specific protein methylase) is a remarkable finding.

EXPERIMENTAL PROCEDURES’

RESULTS

Properties of Cryptomonad Phycoerythrin 566-The native protein showed an absorption maximum at 566 nm with a pronounced shoulder at about 595 nm and a fluorescence emission maximum at 618 nm (Fig. 3). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of purified phycoerythrin 566 showed a purple bilin-bearing polypeptide of apparent M, about 20,000 ( p subunit) and a closely spaced quartet of bilin-bearing polypeptides ( a subunits) in the apparent M , 10,000-12,000 region (Fig. 3, inset). Other investi- gators have reported the presence of two or more separable a subunits in cryptomonad biliproteins (16, 21, 29-31).

Purification and Characterization of the p Subunit of Phy- coerythrin 566-Gel filtration on Bio-Gel P-150 in 3 M urea, 1 mM dithiothreitol, pH 2.0, permitted a clean separation of the /3 subunit from the a subunits (Fig. 2). Under these conditions, the subunit was purple, and the CY subunits were

Portions of this paper (including “Experimental Procedures,” Figs. 1-5, 7, and 8, and Tables 11-X) are presented in miniprint at the end of this paper. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are included in the microfilm edition of the Journal that is available from Waverly Press.

greenish blue. The absorption spectra of phycoerythrin 566 and of the purified 6 subunit, both in 3 M urea, 1 mM dithiothreitol, pH 2.0, and adjusted to the same absorbance at the maximum, are shown in Fig. 4. It is evident that the bilins on the a subunits contribute to the absorption of phycoerythrin denatured with acid urea primarily at the red edge. The pooled CY subunit peak (Fig. 2, 112-130 ml) in 3 M urea, 1 mM dithiothreitol, pH 2.0, showed an absorption peak at 592 nm with no sign of contamination by the p subunit (X,,, 571 nm under these conditions). Sodium dodecyl sulfate- polyacrylamide gel electrophoresis of the a subunit fraction showed the presence only of the apparent M , 10,000-12,000 components.

The number and nature of the bilins on the p subunit were investigated by fractionation of a tryptic digest of this polypeptide by HPLC’ on a reverse-phase CIS column. The elution profile (Fig. 5) showed four major peaks absorbing at 580 nm ( I , IIZ, I V , and V I ) and two minor peaks (IZ and V ) . Spectro- scopic studies and amino acid analyses indicated that peaks I11 and IV represented different forms of a single bilin peptide. The basis for the HPLC separation of this pair of chromopep- tides was not determined, but such behavior of bilin peptides has been noted previously (32). The 300-600-nm diode array absorption spectra at the peaks of fractions I, 111, IV, and VI showed a peak at 555 nm for I, a peak at 583 nm for both I11 and IV, and a peak at 590 nm for VI (Fig. 5). This indicated that the peptide-linked chromophore is a phycoerythrobilin in I (33) and phycobiliviolin (26) in the other three compo-

The abbreviations used are: HPLC, high performance liquid chro- matography; DTT, dithiothreitol; PTH, phenylthiohydantoin; TFA, trifluoroacetic acid; TPCK, L-1-tosylamido-2-phenylethyl chloro- methyl ketone.

17862 Posttranslational Modifications of Cryptomonad Phycoerythrin

nents. The peptides in peaks I, 111, IV, and VI were purified further by HPLC under isocratic conditions and then sub- jected to sequence analysis.

Sequence of Peptides @-1, p-2, and p-3"Sequence analysis was performed on peptides I, 111, and VI. These peptides were designated /I-1, P-2, and p-3 in order of their elution from the HPLC reverse-phase column.

The sequences for peptides /3-1 and p-2, determined from amino acid analysis data and sequential Edman degradation (Tables 111-V), are shown in Fig. 6 (aligned with residues 79- 84 and 151-171, respectively). The comparison of these sequences with that of the P subunit of B-phycoerythrin, using the residue numbering for B-phycoerythrin ( E ) , shows that p-1 corresponds to residues 79-84 with the phycoerythrobilin linked to Cys-82. Peptide p-2 corresponds to residues 151- 171 with the phycobiliviolin linked to Cys-158.

The partial sequence determined for peptide p-3 (Fig. 6, residues 38-74; Figs. 7 and 8) was based on sequential Edman degradation of peptide p-3 (Table VI), as well as on amino acid composition and sequence studies on three thermolysin peptides, p-3-Th-1, fi-3-Th-2, and p-3-Th-4 (Tables 111, VII, and VIII), derived from peptide p-3. Peptide p-3-Th-4 was characterized and shown to consist of a pair of peptides bridged by phycobiliviolin linked to 2 cysteinyl residues (Table VI11 and Fig. 7). Amino acid analysis provided support for the linkage to 2 cysteinyl residues (Table 111). Hydrolysis of peptide p-3-Th-4 in 6 N HCl containing 0.2 M dimethyl sulfoxide (27) yielded 2 residues of cysteic acid, and sequence determination detected no S-carboxymethylcysteine. The positions assigned to the 2 cysteine residues in the sequence of peptide 8-3 correspond precisely to the locations of cysteine residues which our earlier studies showed to be the attachment sites for doubly linked phycoerythrobilin (33, 34) in Porphyr- idiurn cruenturn B-phycoerythrin and for doubly linked phycourobilin (35,36) in Gastroclonium coulteri R-phycoerythrin. Amino acid analyses of peptides p-3-Th-3, Th-5, and Th-6 indicated that they are related to p-3-Th-4 by cleavage at alternate thermolysin sites.

Amino acid and sequence analyses on peptide p-3 and on peptides P-3-Th-1 and P-3-Th-2 revealed the presence of a single residue of y-N-methylasparagine at a position corre-

CBD p-PE 0-PC 645

p-B-PE

p-B-PE CBD p-PE p-PC 645


p-B-PE CBD 0-PE p-PC 645

P-B-PE

p-PC 645 CBD p-PE


FIG. 6. Comparison of the partial amino acid sequence of phycoerythrin 566 p subunit with the sequences of the corresponding subunits of P. cruentum B-phycoerythrin ( 1 5 ) and Chroomonas sp. phycocyanin 645 (16, 20). The residue numbering used is that for P. cruenturn B-phycoerythrin (15); the sequences of the cryptomonad phycobiliproteins have been aligned with this sequence to maximize homology. The bilin-linked cysteinyl residues and the 7-N-methylasparagine residue are outlined. PE, phycoerythrin; PC, phycocyanin.

sponding to p-72 (Tables 111, VI, and VII; Fig. 7). On acid hydrolysis, this derivative yields methylamine and aspartic acid. One residue equivalent of methylamine was found in the hydrolysates of peptides P-3-Th-1 and /3-3-Th-2 (Table 111), and it was also detected in the hydrolysate of peptide p-3. The PTH-derivative of authentic y-N-methylasparagine runs in the position of PTH-serine in the analytical system used here with a secondary peak in the region of PTH-glutamine, while serine shows a secondary peak in the region of PTH- arginine. The PTH-derivatives obtained on step 6 of the sequential Edman degradation of peptide P-3-Th-1 and on step 7 of the degradation of peptide p-3-Th-2 (Table VII) eluted at the positions expected for the PTH-derivative of y- N-methylasparagine.

Sequential Edman Degradation of Phycoerythrin 566 @ Sub- unit-Automated Edman degradation of the @ subunit allowed assignment (with two unassigned positions) of the first 44 residues of this polypeptide (Table IX). This sequence over- lapped with that determined for peptide p-3. Residue 38 of the amino-terminal sequence of the p subunit corresponded to the first residue of peptide p-3. The composite sequence is compared with that of B-phycoerythrin p subunit in Fig. 6.

Partial Characterization of Phycocyanin 645 Peptide 0-3- The amount of this peptide was too low to permit complete characterization. Amino acid analysis of this peptide showed the presence of methylamine and sequential Edman degradation (Table X) gave an amino-terminal sequence of Leu- Asp-Ala-Val-Asn-Ala-Ile-Val, identical to that of the tryptic peptide that includes residue p-72 in the Chroomonas sp. phycocyanin 645 sequenced by Sidler et al. (20).

DISCUSSION

In comparison to the extensive information available on the amino acid sequences of phycobiliproteins of cyanobacterial and red algal origins (8, 11, 37), the data on those of cryptomonads are very limited. The complete sequence of the @ subunit of only one cryptomonad phycobiliprotein, phycocyanin 645 (11, 16), and a partial sequence for the @ subunit of cryptomonad phycoerythrin 545 (16) is available. The partial sequence of the cryptomonad phycoerythrin 566 p subunit presented here is compared with that of the p subunit

1 M L D A F S R V V V N S D A K A A Y V G G S D L Q A L K S F

10 20 30

A L D A F S L V V T N A D O K A A Y V C C A D L E T L O K F M L D A F S R V V T G A D S K A A Y V G G A D L Q A L K K F

40 I A D G N K R L D A V N S I V S N A S C M V S D A V S G M I

50 60

I S E G N K R L D A V N S I V S N A S C I V S D A V S G M I V S E G N K R L D A V N A I V S N A S C I V S D A V S G M I

C E N P G L I S P C G N C Y T N R R M A A C L R D G E I I L 70 80 90

C E N P G L I S P S G M C Y M A A C L R C E N P S L I S P S G E C Y T N R R M A A C L R D A E I I L

110 120 100 R Y V S Y A L L A G D A S V L E D R C L N G L K E T Y I A L

R Y V S Y S L L S G D S S V L E D R C L S G L K E T Y A S L

G V P T N S S I R A V S I M K A Q A V A F I T N T A T E R K 130 140 150

G V P A A G N A R A V G I M K A T V V A F I N N T S N Q K K

M S F A A G D C T S L A S E V A S Y F D R V G A A I S 160 170 1 7 7

L S T P Q G D C S G L A S E V A G Y F D K L L T P S G D C S A L A S E A A C Y F D K V T S A . L A

Posttranslational Modifications of Cryptomonad Phycoerythrin 17863

FIG. 9. Bilin types and attachment sites in the @ subunits of cyanobacterial and red algal phycoerythrin and of cryptomonad phycobiliproteins. The abbreviations used are: PUB, phycourobilin; PEB, phycoerythrobilin; PXB, phycobiliviolin; PCB, phycocyanobilin. The numbers indicate approximate long wavelength absorption maxima for these prosthetic groups when present as components of native phycobiliproteins. Data from Refs. 11, 15, 16, 23, and 39.

B-PHYCOERYTHRIN C- o r

WH8103 o r R-PHYCOERYTHRIN

PEB 560 nm

Cryptomonad PHYCOERYTHRIN 566 8 11.'

7 f.Y f.11. f.3 0

PXB 568. 590 Cryptomonad Q Q Q 0 PHYCOCYANIN 1 1.11 f.:J .. _. _. .. .. .. .. .. .. 6 45

. . . . .. .. .

50 61 82 158 PCB 620 nm

nm

of phycocyanin 645 in Fig. 6. It should be noted that the former subunit carries one phycoerythrobilin and two phycobiliviolins, whereas the latter carries one phycobiliviolin and two phycocyanobilins (Fig. 9) (16, 38). This drastic difference in prosthetic group composition is not reflected in the amino acid sequence. In the 99 residues compared between the two sequences, 86 are identical. Zuber and his co-workers (11) concluded from a similar comparison of the sequence of phycocyanin 645 with the partial sequence of phycoerythrin 545 (which carries only phycoerythrobilin prosthetic groups) that both phycoerythrin and phycocyanin /3 subunit genes in the cryptomonads originated from a red algal phycoerythrin p subunit gene. The present results support this conclusion. The p subunit of phycocyanin 645 is 73% identical to that of P. cruentum B-phycoerythrin, and the partial sequence of the p subunit of phycoerythrin 566 shows a 79% identity (see Fig. 6).

Phycoerythrin 566 carries a doubly linked phycobiliviolin at p-Cys-50,61, a singly linked phycobiliviolin a t p-Cys-158, and a phycoerythrobilin a t p-Cys-82. The isolation of a frag- ment generated by thermolysin digestion which carried two peptides cross-linked through the phycobiliviolin has provided the first conclusive evidence for the existence of a doubly linked phycobiliviolin. Inferential evidence from sequence determination, suggesting the presence of a doubly linked phycobiliviolin, attached a t p-Cys-50 and p-Cys-61 in phycocyanin 645, was recently cited by Sidler and Zuber (11). Any mechanisms proposed for in vivo bilin addition will need to account for the generation of a doubly linked phycobiliviolin. Since singly linked phycoerythrobilin and phycourobilin each have a vinyl group at C-18' (34, 36), formation of doubly linked species can be rationalized by postulating thiol addition to this double bond. Singly linked phycobiliviolin has an ethyl group a t C-18' (26).

Fig. 9 compares the identity of the bilins at the three p subunit attachment sites of cyanobacterial and red algal phycoerythrins and cryptomonad phycoerythrin 566 and phycocyanin 645. Examination of all cyanobacterial and red algal phycobiliproteins reveals no bilin that absorbs at a lower energy (longer wavelength) than the bilin at p-82 (10, 39). From crystallographic studies and spectroscopic studies on C- phycocyanin, the bilin at p-82 has been assigned as the terminal energy acceptor (40-42). From extensive compari- sons of phycobiliproteins with varying bilin prosthetic group composition, it was concluded that the bilin at p-82 fulfilled the same role in both cyanobacterial and red algal phycoerythrins (39). As shown in Fig. 9, in C-, B-, and R-phycoerythrins and in phycoerythrins from marine cyanobacteria,

this bilin is a phycoerythrobilin, and no bilin absorbing at a longer wavelength is present on the p subunit. The same observation holds for phycocyanin 645, where the p-82 phycocyanobilin is one of the bilins absorbing at long wavelength. However, this generalization is broken by phycoerythrin 566 where the phycoerythrobilin at p-82 cannot serve as an acceptor for the energy absorbed by the two phycobiliviolins on the p subunit (Fig. 9). It is well established that the lowest energy acceptor bilins in cryptomonad phycobiliproteins are attached to the CY subunit (29,43-45). In the native phycocyanins, these acceptors have absorption maxima between 620 and 645 nm, depending on the protein. Consequently, energy transfer in the cryptomonad phycobiliproteins proceeds from the bilins on the p subunits to those on the CY, in the opposite direction to that seen in the component phycobiliproteins of phycobilisome rods (10). We conclude that these major alterations in the pathways of energy migration reflect the requirements for directional energy transfer imposed by the different spatial relationships of the cryptomonad phycobiliproteins with each other and/or with the chlorophyll-containing energy acceptor complexes within the thylakoid membrane.

The discovery that phycoerythrin 566 contains y-N-methylasparagine is of particular interest. y-N-Methylasparagine has been found only in components of the phycobilisome: phycoerythrin, phycocyanin, allophycocyanin, and the p's.2 polypeptide of the phycobilisome core. In all of these polypeptides, from both cyanobacteria and red algae, the methylation is found on P-Asn-72 in the highly conserved sequence Pro- Gly-Gly-Asn (23, 46). y-N-Methylasparagine also occupies position p-72 in phycoerythrin 566, but the sequence of residues 69-72 is Pro-Ser-Gly-Asn. In the sequence of Chroo- monas sp. phycocyanin 645, the /3 69-72 sequence was reported to be Pro-Ser-Gly-Glu (20). We have isolated the tryptic peptide encompassing residue p-72 from Chroomonas strain BL-78 phycocyanin 645 and have found that it releases an equivalent of methylamine on acid hydrolysis. This prelimi- nary result suggests that the methylation of residue 6-72 in cryptomonad phycobiliproteins may be of general occurrence.

In the face of the apparent loss of many genes encoding phycobilisome components from the putative red algal endo- symbiont ( la) , it is intriguing that the gene for the phycobiliprotein asparagine methylase (47) is retained in the cryptomonads. Its presence in the cryptomonads strengthens the argument for the red algal origin of the crptomonad chloroplast. The P-Asn-72 residue is within 3.5 A of the bilin at p- 82 (481, and it has been suggested that the function of the methylation is to modulate the absorption spectrum of this terminal acceptor bilin (46). The observation, discussed

17864 Posttranslational Modifications

above, that in phycoerythrin 566 the bilin at (3-72 is a donor rather than an acceptor, does not support such an explanation. The functional significance of this highly conserved posttranslational modification remains to be defined.

Acknowledgments-We are grateful to Dr. Paul Kugrens for pro- viding the cryptomonad strains for this study and to Dr. John West for his interest in this work and helpful discussions.

REFERENCES

1. Glazer, A. N. (1983) Annu. Reu. Biochem. 52, 125-157 2. Larkum, A. W. D., and Barrett, J. (1983) Adu. Bot. Res. 10, 3-

219 3. Wehrmeyer, W. (1988) in Photosynthetic Light Harvesting Sys-

tems. Structure and Function (Scheer, H., and Schneider, S., eds) pp. 35-47, Walter de Gruyter, Berlin

4. O’hEocha, C., O’Carra, P., and Mitchell, P. (1964) Proc. R. Zr. Acad. Sect. B Biol. Geol. Chem. Sci. 63, 191-200

5. Gantt, E. (1979) in Biochemistry and Physiology of Protozoa (Levandowsky, M., and Hutner, S. H., eds) 2nd Ed., Vol. I, pp. 121-137, Academic Press, New York

6. Gantt, E. (1980) Znt. Reu. Cytol. 66, 45-80 7. Glazer, A. N. (1981) in The Biochemistry of Plants (Hatch, M.

D., and Boardman, N. K., eds) Vol. 8, pp. 51-96, Academic Press, New York

8. Glazer, A. N. (1984) Biochim. Biophys. Acta 768, 29-51 9. MacColl, R., and Guard-Friar, D. (1987) Phycobiliproteins, pp. 1-

218, CRC Press, Inc., Boca Raton, FL 10. Glazer, A. N. (1989) J. Biol. Chem. 264, 1-4 11. Sidler, W., and Zuber, H. (1988) in Photosynthetic Light Har-

vesting Systems. Structure and Function (Scheer, H., and Schneider, S., eds) pp. 49-60, Walter de Gruyter, Berlin

12. Ludwig, M., and Gibbs, S. P. (1989) J. Cell Biol. 108 , 875-884 13. LichtlL, C., Duval, J. C., and Lemoine, Y. (1987) Biochim. Bio-

14. Sidler, W., Kumpf, B., Rudiger, W., and Zuber, H. (1986) Biol. Chem. Hoppe-Seyler 367, 627-641

15. Sidler, W., Kumpf, B., Suter, F., Klotz, A. V., Glazer, A. N., and Zuber, H. (1989) Bid. Chem. Hoppe-Seykr 370, 115-124

16. Sidler, W., Kumpf, B., Suter, F., Morisset, W., Wehrmeyer, W., and Zuber, H. (1985) Biol. Chem. Hoppe-Seyler 366,233-244

17. Spear-Bernstein, L., and Miller, K. R. (1987) in Progress in Photosynthesis Research (Biggins, J., ed) Vol. 2, pp. 309-312, Martinus Nijhoff Publishers, The Hague, Netherlands

18. Spear-Bernstein, L., and Miller, K. R. (1985) Protoplasma 129, 1-9

19. Whatley, J. M., and Whatley, F. R. (1981) New Phytol. 87, 233- 247

20. Sidler, W. A,, Nutt, H., Kumpf, B., Zuber, H., Wehrmeyer, W., and Brenzel, A. (1987) in Progress in Photosynthesis Research (Biggins, J., ed), Vol. 2, pp. 153-156, Martinus Nijhoff Publish- ers, The Hague, Netherlands

21. Morschel, E., and Wehrmeyer, W. (1975) Arch. Microbiol. 105,

phys. Acta 894, 76-90

153-158

of Cryptomonad Phycoerythrin 22. Glazer, A. N., and Apell, G. S. (1977) FEMS Microbiol. Lett. 1,

23. Klotz, A. V., and Glazer, A. N. (1987) J. Biol. Chem. 262, 17350-

24. Stein, J . (1958) Am. J. Bot. 45,664-672 25. Glazer, A. N., Cohen-Bazire, G., and Stanier, R. Y. (1971) Arch.

MicrobioE. 80, 1-18 26. Bishop, J. E., Rapoport, H., Klotz, A. V., Chan, C. F., Glazer, A.

N., Fuglistaller, P., and Zuber, H. (1987) J. Am. Chem. SOC.

27. Glazer, A. N., Hixson, C. S., and DeLange, R. J. (1979) Anal.

28. Hewick, R. M., Hunkapiller, M. W., Hood, L. E., and Dreyer, W.

29. Guard-Friar, D., and MacColl, R. (1986) Photochem. Photobiol.

30. Martin, C. D., and Hiller, R. G. (1987) Biochim. Biophys. Acta

31. Hiller, R. G., and Martin, C. D. (1987) Biochim. Biophys. Acta

32. Arciero, D. M., Bryant, D. A,, and Glazer, A. N. (1988) J. Biol.

33. Lundell, D. J., Glazer, A. N., DeLange, R. J., and Brown, D. M.

34. Schoenleber, R. W., Lundell, D. J., Glazer, A. N., and Rapoport,

35. Klotz, A. V., and Glazer, A. N. (1985) J. Biol. Chem. 260, 4856-

36. Nagy, J. O., Bishop, J. E., Klotz, A. V., Glazer, A. N., and

37. Zuber, H. (1987) in The Light Reactions (Barber, J., ed) pp. 197-

38. MacColl, R., and Guard-Friar, D. (1983) J. Biol. Chem. 258,

39. Ong, L. J., and Glazer, A. N. (1988) in Light Energy Transduction in Photosynthesis: Higher Plant and Bacterid Models (Stevens, S. E., and Bryant, D. A., eds) pp. 102-121, American Society of Plant Physiologists, Rockville, MD

40. Schirmer, T., Huber, R., Schneider, M., Bode, W., Miller, M., and Hackert, M. L. (1986) J. Mol. Biol. 188,651-676

41. Siebzehnrubl, S., Fischer, R., and Scheer, H. (1987) Z . Natur- forsch. Sect. C Biosci. 42, 258-262

42. Schirmer, T., and Vincent, M. G. (1987) Biochim. Biophys. Acta

43. Glazer, A. N., and Cohen-Bazire, G. (1975) Arch. Microbiol. 104 ,

44. Jung, J., Song, P.-S., Paxton, R. J., Edelstein, M. S., Swanson, R., and Hazen, E. E., Jr. (1980) Biochemistry 19,24-32

45. Hanzlik, C. A., Hancock, L. E., Knox, R. S., Guard-Friar, D., and MacColl, R. (1985) J. Lumin. 34,99-106

46. Rumbeli, R., Suter, F., Wirth, M., Sidler, W., and Zuber, H. (1987) Biol. Chem. Hoppe-Seyler 368 , 1401-1406

47. Glazer, A. N., Arciero, D. M., Klotz, A. V., and Swanson, R. (1988) FASEB J. 2, 2348 (abstr.)

48. Duerring, M., Huber, R., and Bode, W. (1988) FEES Lett. 236,

113-116

17355

109,875-881

Biochem. 92,489-496

J . (1981) J. Biol. Chem. 256, 7990-7997

43,81-85

923,88-97

923,98-102

Chem. 263,18343-18349

(1984) J. Biol. Chem. 259, 5472-5480

H. (1984) J. Biol. Chem. 259,5481-5484

4863

Rapoport, H. (1985) J. Biol. Chem. 260,4864-4868

259, Elsevier Science Publishers, Amsterdam

14327-14329

893,379-385

29-32

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