Formation of Hybrid Phycobilisomes by Association Of
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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 inter- generic 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 ab- sorbance 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