Formation of Hybrid Phycobilisomes by Association Of

Proc. NatL Acad. Sci. USA Vol. 79, pp. 5277-5281, September 1982 Botany

Formation of hybrid phycobilisomes by association of phycobiliproteins from Nostoc and Fremyella (energy transfer/blue-green algae/photosynthetic accessory pigment) ORA CANAANI* AND ELISABETH GANTT Radiation Biology Laboratory, Smithsonian Institution, 12441 Parklawn Drive, Rockville, Maryland 20852 Communicated by Richard C. Starr, June 2, 1982 ABSTRACT Formation of phycobilisomes has been accom- the reassociation of the various components into energetically plished in vitro from isolated phycobiliprotein fractions obtained functional phycobilisomes. Recently, in vitro intragenerict' from the same blue-green alga (intrageneric) and from different phycobilisome formation was reported, whereby separate frac- blue-green algae (intergeneric). Phycobilisomes, which are supra- tions of allophycocyanin and phycoerythrin-phycocyanin com- molecular complexes of phycobiliproteins, serve as major light- plexes from Nostoc sp. were recombined to give energetically harvesting antennae for photosynthesis in blue-green and red al- functional phycobilisomes (14). This has made it possible to gae. Intrageneric association into energetically functional phyco- begin testing the specificity ofthe phycobiliprotein association. bilisomes, previously reported to occur with Nostoc sp. allophy- The purpose of this investigation was to see whether interge- cocyanin and phycoerythrin-phycocyanin complexes [Canaani, neric (hybrid)1: phycobilisomes could be formedby reassociation O., Lipschultz, C. A. & Gantt, E. (1980) FEBS Lett 115, 225-229], has been obtained with FremyeUa diplosiphon. By their spectral offractions ofphycoerythrin-phycocyanin and allophycocyanin properties (absorption, fluorescence excitation, and emission) and derived from various sources. Our primary emphasis was on two electron microscopic images, the native and in vitro-associated filamentous blue-green algae-Nostoc sp. and Fremyella di- phycobilisomes were virtually indistinguishable. Intergeneric plosiphon. A briefpreliminary report (15) of this in vitro asso- phycobilisomes have been produced from allophycocyanin ofNos- ciation ofhybrid phycobilisomes from two blue-green algae has toc sp. strain Mac. and phycoerythrin-phycocyanin of F. diplosi- appeared elsewhere. phon, as well as from the reverse mixtures. The yield of intergeneric phycobilisomes, favored by higher phycobiliprotein content MATERIALS AND METHODS in 0.75 M phosphate, pH 7.0/2.0 M sucrose, was 40-60%. Energy transfer to the terminal long-wavelength-emitting allophycocy- The strain ofNostoc sp. strain Mac. was originally obtained from anin in the phycobilisomes was evident from the 670-675 nm flu- C. Van Baalen and F. diplosiphon was kindly supplied by L. orescence emission peaks. Furthermore, excitation spectra showed Bogorad and H. W. Siegelman. Cultures ofFremyella and Nos- the contribution ofthe respective phycoerythrins (Fremyella, Am. toc were grown in liquid media under continuous illumination 570; Nostoc, Am 573 and 553 nm), as well as that ofphycocyanin with daylight fluorescent lamps (ca. 1,500 tW/cm2) as de- and short-wavelength-absorbing allophycocyanin. Phycobilisomes scribed (16). Porphyridium sordidum was grown at 18°C in an of Nostoc and Fremyella, analyzed by NaDodSO4/polyacryla- artificial seawater medium with reduced salinity (13). Cells of mide gel electrophoresis, possessed a number ofpolypeptides hav- Phormidium persicinum were a gift from D. S. Berns and R. ing similar molecular weights: the usual a- and ,B-phycobilin-con- MacColl. Some F. diplosiphon cultures were also grown in red taining polypeptides of Mr 15,000-22,000, a faint band at Mr ca. light (>600 nm; ca. 1,000 ,uW/cm2) by using a red plastic filter 95,000, and a prominent band at Mr ca. 31,000. The Mr 31,000 (P-14, Gelatin Products, Glen Cove, NY). polypeptide is assumed to provide the recognition site for attach- Phycobilisomes were isolated by the procedure of Gantt et ment of the phycoerythrin-phycocyanin complexes with the allo- aL (1) with one modification. Instead of breaking the cells in a phycocyanin core. In vitro association was not obtained between French pressure cell, we suspended them in 0.75 M KPO4 buff- allophycocyanin from Nostoc and phycoerythrin-phycocyanin er, pH 7.0/3% Triton X-100 (vol/vol) and incubated the mix- complexes from Phormium persicinum orPorphyridium sordidum. ture for 1 to 2 hr as in Rigbi et aL (4). The isolation procedure Phycobilisomes are ordered pigment aggregates that function described in ref. 1 was then followed. as light harvesters for photosynthesis. From spectral analysis For separation of the phycobiliprotein components, phyco- ofisolated phycobilisomes, it is clear that the constitutive phy- bilisomes were suspended (2-4 mg of protein/ml) in 0.4 M cobiliproteins channel their energy to special allophycocyanins KPO4 buffer (pH 7.0) and dialyzed for 2 hr at 22°C against 0.1 (1-5). Blue-green algal phycobilisomes have been studied ex- M KPO4 buffer/0.1 M NaCl, pH 7.0. A 2-ml sample was lay- tensively recently and several important concepts on the phy- ered on 20 ml of a linear gradient of 0.25-1.0 M sucrose/0.4 cobilisome structure have emerged. It is generally accepted Abbreviations: KPO4, KH2PO4 titrated with K2HPO4 to pH 7.0. that the core of the phycobilisome contains the allophycocy- * Present Address: Department of Biochemistry, The Weizmann In- anins, while the phycocyanins and phycoerythrins are attached stitute of Science, Rehovot, Israel. as radial rods to the core (3, 6-8). Special polypeptides, which t Polypeptides thatbecome visible only after staining gels with aprotein are uncoloredt on NaDodSO4/polyacrylamide gels, are impli- stain are often designated as uncolored or colorless because the phy- cated as being responsible for the ordered phycobiliprotein ar- cobilin chromophore appears absent. Treatment ofphycobiliproteins ray in the phycobilisome (9-13). for NaDodSO4/polyacrylamide gel electrophoresis causes extensive fading; thus, it is possible that the uncolored polypeptides were col- Full elucidation of the phycobilisome structure necessitates ored in vivo. t Phycobilisomes obtained from phycobiliprotein fractions derived The publication costs ofthis article were defrayed in part by page charge from the same organism are referred to as intrageneric; those derived payment. This article must therefore be hereby marked "advertise- from fractions of different organisms are referred to as intergeneric ment" in accordance with 18 U. S. C. §1734 solely to indicate this fact. or hybrid. 5277 Downloaded by guest on September 27, 2021 5278 Botany: Canaani and Gantt Proc. Natl. Acad. Sci. USA 79 (1982) M KPO4, pH 7.0, over 2 M sucrose (4 ml) and sedimented at 200C, constant 10 mA), gels were stained with Coomassie blue 136,000 X g for 4 hr at 200C. An allophycocyanin-rich fraction R-250 and destained in 10% acetic acid. To determine the ap- was collected by syringe from the upper portion ofthe gradient parent molecular weights ofthe polypeptides, marker proteins and the fraction of phycoerythrin-phycocyanin (existing as a in the molecular weight range 14,400-200,000 (Bio-Rad) were complex) was collected from the middle (14). used. In Nostoc sp., the phycoerythrin-phycocyanin complex thus For electron microscopy, phycobilisomes in 0.75 M KPO4/ prepared had absorption maxima at 553-573 and 620 nm and 1 M sucrose, pH 7.0, were placed on carbon films on copper was energetically functionally coupled as evidenced by the flu- grids, fixed for 3 min in 0.3% glutaraldehyde/0.75 M KPO4, orescence emission at 655 nm (excitation, 545 nm) arising from rinsed with deionized water, and stained with 1% uranyl sulfate phycocyanin (14). Its phycoerythrin/phycocyanin ratio (mol/ (8) or 1% uranyl acetate. mol) was ca. 1.4: 1. The complex from F. diplosiphon had absorbance maxima at 570 and 620 nm, with the fluorescence RESULTS emission at 650 nm (excitation, 545 nm), and a phycoerythrin/ It has been reported previously that phycobilisomes from Nos- phycocyanin ratio (mol/mol) of 3: 1. In this species, some ap- toc sp. can be separated into two fractions, one containing phy- parently aggregated phycoerythrins (5-10%) sedimented in a coerythrin-phycocyanin in a complex and the other containing separate band and had a fluorescence emission maximum at 582 allophycocyanin. Under appropriate conditions, these two frac- nm. With P. persicinum, higher ionic strengths were required tions can be reassociated into functional phycobilisomes (14). for isolation of the phycoerythrin-phycocyanin complex; thus, Association into phycobilisomes was enhanced by high protein phycobilisomes were dialyzed in 0.4 M KPO4 (pH 7.0) for 3 hr concentration in 0.75 M KPO4/2.0 M sucrose. Except for some and then centrifuged on a sucrose step gradient (1). This com- improvements, the same methods were used here to obtain plex had a phycoerythrin/phycocyanin ratio (mol/mol) of 10: 1, energetically functional phycobilisomes. Criteria for intactness absorbance maxima at 545 nm and 620 nm, and an emission of in vitro-associated phycobilisomes were the same as for native maximum at 585 nm (excitation, 545 nm). The complex from P. ones-(i) functional competence as seen by energy transfer from sordidum isolated in 10 mM KPO4 (pH 7.0) on a sucrose gra- phycoerythrin (excitation, 545 nm) to allophycocyanin (emis- dient (13) had a phycoerythrin/phycocyanin ratio (mol/mol) of sion, ca. 675 nm), (ii) sedimentation into and recovery from the 1:1, absorbance maxima at 567 nm and 625 nm, and an emission 1 M sucrose layer of a phycobilisome isolation gradient, and maximum at 655 nm (excitation, 545 nm). (iii) morphological images consisting of short rods and a core. The allophycocyanin fractions prepared from Nostoc sp. and Only phycoerythrin-containing phycobilisomes of several from F. diplosiphon had absorbance maxima at ca. 650 nm and species were used for these experiments, because phycoery- emission maximaat 665 nm (excitation, 620 nm). Although these thrin has the advantage that it can be independently excited and fractions contained some phycoerythrin (ca. 5%), it was not en- thus energy transfer to phycocyanin and to allophycocyanin can ergetically coupled to allophycocyanin according to the emis- be readily ascertained. Phycobilisomes of Nostoc sp. and Fre- sion spectra and was easily removed on a brushite column with- myella were used primarily because, on dissociation, the phy- out affecting the association characteristics ofthe allophycocyanin cobiliproteins were separable into two main bands, allophyco- fraction. Similarly, some high molecular weight allophycocy- cyanin near the top of the gradient and phycoerythrin- anin (ca. 3% of total phycobiliprotein) was present in the phy- phycocyanin in the middle of the gradient. Phycobilisomes of coerythrin-phycocyanin fractions and was not energetically Porphyridium sordidum and Phormidium persicinum yielded coupled. multiple bands, from which the phycoerythrin-phycocyanin For in vitro association, various fractions were mixed (see fractions were selected. Table 1) and dialyzed at room temperature (ca. 22°C) for 6-8 The results (Table 1) show that the highest yields of in vitro- hr in 0.75 M KPO4/2.0 M sucrose, pH 7.0, and then for 3 hr associated phycobilisomes were obtained from Nostoc sp. (Exp. in 0.75 M KPO4; 2-ml samples were then layered on sucrose 1) and from Fremyella (Exp. 2). Of particular interest was the gradients as used for phycobilisome isolation. Phycobilisomes high yield of hybrid phycobilisomes obtained from mixtures of were recovered in the 1 M sucrose layer. Fremyella and Nostoc sp. (Exps. 3 and 4). This appears to be Spectra were determined at room temperature as described a specific and not a random interaction, because phycobilisomes (17). For fluorescence spectra, samples were diluted in KPO4 were not obtained with Nostoc allophycocyanin and phycoer- (pH 7.0) to a protein concentration ofca. 35 ,g/ml (A, ca. 0.1 ythrin-phycocyanin of either Phormidium (Exp. 5) or Porphyr- at the maximum of the predominant phycobiliprotein). Phy- idium (Exp. 6). Since the phycobiliprotein concentration for cobiliprotein concentrations were always determined by absor- these species was as great or greater than that in Exps. 1-4, bance of samples at low ionic strength (0.1 M KPO4, pH 7.0) inadequate protein concentration is improbable. Furthermore, by using the following molar extinction coefficients: C-phy- large aggregates, equivalent to phycobilisomes, did not occur coerythrin, 4.88 X 105 Mm-cm'-, 562 nm (18); C-phycocyanin, with the allophycocyanin fractions (Exps. 8 and 10) or with the 2.81 x 105 M'1 cm-1, 620 nm (19); allophycocyanin, 2.3 X 105 phycoerythrin-phycocyanin fractions (Exps. 7 and 9), also sug- Mmcm', 650 nm (20). The relative absorbance ofphycocyanin gesting that random association did not occur. at 650 nm/620 nm = 0.2 and of allophycocyanin at 620 nm/ Phycobilisomes, whether native or associated in vitro, were 650 nm = 0.47 in both Fremyella and Nostoc. Use of the ex- distinct for Fremyella and Nostoc; the phycoerythrin of Nostoc tinction coefficients determined by Bennett and Bogorad (21) had a double absorption maxima (at ca. 553 and 573 nm), while yielded essentially the same phycobiliprotein ratios. that of Fremyella had a single maximum (at ca. 570 nm). Ab- NaDodSO4/polyacrylamide gel electrophoresis was carried sorption of phycocyanin at ca. 620 nm and of allophycocyanin out essentially according to the method of Laemmli (22) on 1.5- ca. 650 nm was the same in both. Also, the phycobiliprotein or 3-mm-thick slab gels (15 cm x 30 cm) with a gradient of compositions calculated from native phycobilisomes of these 8-15% acrylamide (acrylamide/bisacrylamide, 30:0.8) contain- two organisms were different. For native phycobilisomes of ing 0.1% NaDodSO4. For electrophoresis, samples were dis- Nostoc, the relative phycoerythrin/phycocyanin/allophyco- solved in 1% NaDodSO4/10% 2-mercaptoethanol/5O mM cyanin ratio (mol/mol) was ca. 1.4:1.1:1.0, whereas the cor- Tris.HCI/0.002% bromophenol blue/10% glycerol (vol/vol) responding ratio for Fremyella was ca. 2.8:1.0:1.0. The ab- and heated to 100TC for 3 min. After electrophoresis (12 hr, sorption spectra of in vitro-associated Nostoc and Fremyella Downloaded by guest on September 27, 2021 Botany: Canaani and Gantt Proc. Nati. Acad. Sci. USA 79 (1982) 5279

Table 1. In vitro phycobilisome association from phycobiliprotein mixtures of various algal species Mixture Conc., Conc., Phycobilisome Exp. Component A mg/ml Component B mg/mi yield, % 1 Nostoc allophycocyanin 0.15-0.30 Nostoc phycoerythrin-phycocyanin 0.30-0.50 50-70 2 Fremyella allophycocyanin 0.06-0.12 Fremyella phycoerythrin-phycocyanin 0.24-0.46 50-70 3 Nostoc allophycocyanin 0.15-0.30 Fremyella phycoerythrin-phycocyanin 0.24-0.46 40-60 4 Fremyella allophycocyanin 0.12-0.24 Nostoc phycoerythrin-phycocyanin 0.35-0.50 40-60 5 Nostoc allophycocyanin 0.30 Phormidium* phycoerythrin-phycocyanin 1.0 0 6 Nostoc allophycocyanin 0.30 Porphyridiumt phycoerythrin-phycocyanin 1.0 0 7 Fremyella phycoerythrin-phycocyanin 0.24-0.46 Fremyella phycoerythrin-phycocyanin 0.24-0.46 0 8 Fremyella allophycocyanin 0.06-0.12 Fremyella allophycocyanin 0.24-0.46 0 9 Nostoc phycoerythrin-phycocyanin 0.35-0.50 Nostoc phycoerythrin-phycocyanin 0.35-0.50 0 10 Nostoc allophycocyanin 0.15-0.30 Nostoc allophycocyanin 0.15-0.30 0 Phycobilisome yields were determined as the percent phycobiliprotein recovered, in the 1 M sucrose layer of the phycobilisome isolation gradient, of the total initially layered on the gradient. * Phormidium persicinum. tPorphyridium sordidum. phycobilisomes (Fig. 1A) were almost identical to the corre- Intergeneric phycobilisomes recovered from the isolation sponding spectra offreshly isolated native phycobilisomes (not gradient (Fig. 1D) could be readily distinguished by the spectral shown), except for a slight decrease in the absorption at 650 nm characteristics of the phycoerythrins because of the single ab- (unpublished result). sorption maximum of Fremyella and the double one of Nostoc.

20 Uj z w 10 Z LU z I0U m 500 600 700 500 600 700 500 600 700 w 0co I) 0 4 60 3) LU 50 >

0 500 600 700 500 600 700 500 600 700 WAVELENGTH (nm) FIG. 1. Spectra of in vitro-associated phycobilisomes. (A and D) Absorption. (B and E) Excitation. (C and F) Emission. (A-C) Intrageneric phycobilisomes ofNostoc (-) andFremyella (---) (Table 1, Exps. 1 and 2, respectively). (D-F) Intergeneric phycobilisomes ofNostoc allophycocyanin and Fremyella phycoerythrin-phycocyanin (---; Table 1, Exp. 3) and of Fremyella allophycocyanin and Nostoc phycoerythrin-phycocyanin (-; Table 1, Exp. 4). Absorptionandexcitation were normalizedat650 nm. Forexcitation andemissionspectra,A570 = 0.08; theemissionwavelength was 680 am and the excitation wavelength was 545 nm in 0.75 M KPO4/1 M sucrose, pH 7.0, at 22°C. (Excitation and emission spectra were made in the quantum-corrected mode.) Downloaded by guest on September 27, 2021 5280 Botany: Canaani and Gantt Proc. Nad Acad. Sci. USA 79 (1982) A B C Mr 10-3 95

38 31

7,wl1 5

FIG. 3. Polypeptide bands of phycobilisomes resolved by Na- FIG. 2. Electron micrographs of in vitro-associated phycobili- DodS04/polyacrylamide gel electrophoresis. Lanes: A, intergeneric somes. (Main) Phycobilisomes of Nostoc allophycocyanin and Fre- phycobilisomes formed from Nostoc allophycocyanin and Fremyella myellaphycoerythrin-phycocyanin (Table 1, Exp. 3). (Insets) Phycobil- phycoerythrin-phycocyanin; B, intrageneric phycobilisomes from isomes ofFremyella phycoerythrin-phycocyanin and allophycocyanin Fremyella phycoerythrin-phycocyanin and allophycocyanin; C, intra- (Upper, Table 1, Exp. 2), Nostoc phycoerythrin-phycocyanin and al- generic phycobilisomes from Nostoc phycoerythrin-phycocyanin and lophycocyanin (Middle; Table 1, Exp. 1), and Nostoc phycoery- allophycocyanin. thrin-phycocyanin and Fremyella allophycocyanin (Lower, Table 1, Exp. 4). (One percent uranyl acetate; Main, x 124,600; Insets x 139,500.) that the smaller ones were derived from larger ones that were damaged during handling. It is worth noting that the absorbance ratio 570 nm/650 nm was ca. 4.6 in the intrageneric Fremyella phycobilisomes (Fig. 1A) The ready association between phycobiliproteins of Nostoc but ca. in and Frenyella raised the possibility that they had similar poly- only 3.0 the intergeneric phycobilisomes of Fremyella peptide patterns. Thus, the polypeptides were analyzed by phycoerythrin-phycocyanin and Nostoc allophycocyanin (Fig. NaDodSO4/polyacrylamide gel electrophoresis. The bilin-con- 1D), indicating loss of phycoerythrin, a larger allophycocyanin taining polypeptides ofphycobilisomes from Exps. 1-3 in core, or the to core. Table binding offewer rods the 1 were very similar (Fig. 3) and had Mr values of15,000-22,000. Migration of the intrageneric and intergeneric phycobili- An uncolored polypeptide having a of ca. 31,000 was somes into the 1 M sucrose that M, ob- layer indicated they had the served in the intraspecies phycobilisomes ofFremyella (Fig. 3, same sedimentation properties as native phycobilisomes. How- lane B) and in the lane ever, only by their fluorescence excitation 1 B and and interspecies phycobilisomes (Fig. 3, A). (Fig. E) A polypeptide with a similar molecular weight was present in emission (Fig. 1 C and F) spectra was it possible to demonstrate Nostoc phycobilisomes as well lane all that were (Fig. 3, C). Similarly, had the phycobilisomes functionally active and capable faint polypeptide bands to a Mr of of energy ex- corresponding ca. 95,000. transferring between the phycobiliproteins. The Additional uncolored polypeptide bands corresponding to Mr citation spectra (emission, 680 nm) indicated that the quantum values of ca. 34,500 and 36,500 and of ca. 34,000 and 38,000 contribution from allophycocyanin (650 nm) was higher than that expected from the absorption spectra. To the contrary, the were found in Fremyella and Nostoc, respectively. contribution from phycoerythrin (570 nm), although present in substantial amounts, was relatively low, especially in the 553- DISCUSSION nm region, which was much less in the excitation spectra. The We have demonstrated the in vitro association of phycobili- contribution from phycocyanin (ca. 620 nm) was somewhat somes in the blue-green alga Fremyella diplosiphon from allo- lower than that predicted from the absorption spectra, espe- phycocyanin and phycoerythrin-phycocyanin fractions. More cially in comparison with allophycocyanin. The variations be- importantly, we have also demonstrated that hybrid (interge- tween absorption and excitation spectra were similar in both neric) phycobilisomes can be obtained in high yield (Table 1) native (data not shown) and in vitro-associated phycobilisomes. from Fremyella and Nostoc phycobiliproteins. By sedimenta- Comparison of the emission spectra shows that all phycobili- tion and electron microscopic analysis, the in vitro-associated proteins were energetically tightly coupled because the emis- phycobilisomes had the size and shape of native phycobili- sion from allophycocyanin at 670-680 nm was high whereas, somes. They were functionally active; energy transfer from phy- from the predominant phycoerythrin, the emission (ca. 580 nm) coerythrin to allophycocyanin occurred, as evidenced by the was very low. Clearly, the intergeneric phycobilisomes had the fluorescence excitation and emission spectra (Fig. 1). same functional integrity as the intrageneric phycobilisomes. The association between allophycocyanin and phycoery- Structural integrity was also evident by electron microscopy. thrin-phycocyanin was judged to be specific and not random. The phycobilisomes recovered from the 1 M layer of the iso- Ifrandom association had occurred, it would have been favored lation gradient were found to be aggregates ofshort rods similar by high protein concentration and 0.75 M KPO4/2 M sucrose, to native phycobilisomes from blue-green algae (3, 7, 8). Ag- and aggregates should have resulted when allophycocyanin was gregated clusters composed of 4 or 5 rods were present (Fig. mixed with the phycoerythrin-phycocyanin from Porphyridi- 2) in all preparations, but many had fewer rods. It is probable um or Phormidium, and they did not. In this respect, it is worth Downloaded by guest on September 27, 2021 Botany: Canaani and Gantt Proc. Natl. Acad. Sci. USA 79 (1982) 5281 noting that dissociated phycoerythrin and phycocyanin com- for exploring phylogenetic relationships within and among red plexes of either Nostoc or Fremyella could be readily reasso- and blue-green algae. ciated in 0.75 M KPO4/2 M sucrose. These reassociated complexes also formed phycobilisomes when combined with an This work was supported in part by Contract AS05-76-ER 0430 from allophycocyanin fraction, providing that the Mr 31,000 poly- the U.S. Department of Energy. peptide(s) was present. In Nostoc, we have found that, when 1. Gantt, E., Lipschultz, C. A., Grabowski, J. & Zimmerman, B. K. phycoerythrin-phycocyanin fractions are no longer able to as- (1979) Plant Physiol. 63, 615-620. sociate with allophycocyanin, the Mr 31,000 polypeptide is ab- 2. Gantt, E. (1981) Annu. Rev. Plant Physiol 32, 327-347. sent but a new band at Mr ca. 27,000 is present (unpublished 3. Glazer, A. N., Williams, R. C., Yamanaka, G. & Schachman, H. results). The full significance of the Mr 30,000-31,000 poly- K. (1979) Proc. Natl Acad. Sci. USA 76, 6162-6166. 4. Rigbi, M., Rosinski, J., Siegelman, H. W. & Sutherland, J. C. peptide(s) is not known, but it is probable that it is involved in (1980) Proc. Natl Acad. Sci. USA 77, 1961-1965. the attachment of the phycoerythrin-phycocyanin complexes 5. Zilinskas, B. A., Zimmerman, B. K. & Gantt, E. (1978) Photo- to the allophycocyanin core. Analyses of phycobilisomes from chem. Photobiol 27, 587-595. various organisms have led to the conclusion that such poly- 6. Morschel, E., Koller, K.-P., Wehrmeyer, W. & Schneider, H. peptides are structurally and functionally highly conserved (1977) Cytobiologie 16, 118-129. (10-12). In fact, we have also found a Mr 31,000 polypeptide 7. Bryant, D. A., Guglielmi, G., Tandeau de Marsac, N., Castets, in Anacystis A. & Cohen-Bazire, G. (1979) Arch. Mikrobiol 123, 113-127. nidulans and in Tolypothrix tenuis grown under 8. Rosinski, J., Hainfeld, J. A., Rigbi, M. & Siegelman, H. W. light of various wavelengths or in darkness. Lundell et al. (10) (1980) Ann. Bot. (London) 47, 1-12. have shown that the presence of a Mr 30,000 polypeptide cor- 9. Tandeau de Marsac, N. & Cohen-Bazire, G. (1977) Proc. Natl relates with the formation ofphycocyanin into stacked phycobil- Acad. Sci. USA 74, 1635-1639. isome type rods while a Mr 27,000 polypeptide seems to be at 10. Lundell, D. J., Williams, R. C. & Glazer, A. N. (1981) J. Biol the end of the rods. The polypeptides of ca. 31,000 present Chem. 256, 3580-3597. Mr 11. Bryant, D. A. & Cohen-Bazire, G. (1981) Eur. J. Biochem. 119, in Fremyella and Nostoc may be functionally equivalent to the 415-424. Mr 27,000 polypeptide described by Lundell et al. (10). Not 12. Yamanaka, G. & Glazer, A. N. (1981) Arch. Microbiol. 130, 23- only was a Mr 31,000 polypeptide present in the phyco- 30. erythrin-phycocyanin complexes, itwas also present even when 13. Lipschultz, C. A. & Gantt, E. (1981) Biochemistry 20, 3371-3376. the phycoerythrin was reduced by growing cells in red light, 14. Canaani, O., Lipschultz, C. A. & Gantt, E. (1980) FEBS Lett. as observed by Tandeau de Marsac and Cohen-Bazire (9). The 115, 225-229. middle 15. Gantt, E., Canaani, 0., Lipschultz, C. A. & Redlinger, T. (1981) polypeptide bands (Mr 34,500 and 36,500) ofFremyella in Photosynthesis III. Structure and Molecular Organisation of are assumed to be linked with phycoerythrin because they were the Photosynthetic Apparatus, ed. Akoyunoglou, G. (Balaban In- absent when phycoerythrin was absent (e.g., in cells grown in ternational Science Services, Philadelphia, PA), pp. 143-153. red light). 16. Gray, B. H., Lipschultz, C. A. & Gantt, E. (1973) J. Bacteriol The presence of a Mr 95,000 polypeptide in both Fremyella 116, 471-478. 17. Grabowski, J. & Gantt, E. (1978) Photochem. Photobiol. 28, 39- and Nostoc as well as in Pseudanabaena (11) is noteworthy, es- 45. pecially since it may be similar to that found in the core fraction 18. Glazer, A. N. & Hixson, C. S. (1975) J. Biol Chem. 250, 5487- of Porphyridium cruentum phycobilisomes and may function 5495. as an anchor protein (23). The in vitro-association studies are 19. Glazer, A. N., Fang, S. & Brown, D. M. (1973) J. Biol Chem. only in their beginning stages, and it would be premature to 248, 5679-5685. draw any phylogenetic conclusions from these results. When 20. Cohen-Bazire, G., Beguin, S., Rimon, S., Glazer, A. N. & it becomes possible to fully interpret the roles of most Brown, D. M. (1977) Arch. Microbiol 111, 225-238. of the 21. Bennett, A. & Bogorad, L. (1973) J. Cell Biol 58, 419-435. polypeptides, interspecies crosses such as used here will be 22. Laemmli, U. K. (1970) Nature (London) 227, 680-685. useful for probing the phycobilisome structure and perhaps also 23. Redlinger, T. & Gantt, E. (1981) Plant Physiol 68, 1375-1379. Downloaded by guest on September 27, 2021