Further evidence for a phycobilisome model from selective dissociation, fluorescence emission, immunoprecipitation, and electron microscopy.

Phycobilisomes, isolated in 500 mM Sorensen's phosphate buffer pH 6.8 from the red alga, Porphyridium cruetum, were analyzed by selective dissociation at various phosphate concentrations. The results are consistent with a structural model consisting of an allophycocyanin core, surrounding by a hemispherical layer of R-phycocyanin, with phycoerythrin being on the periphery. Such a structure also allows maximum energy transfer. Intact phycobilisomes transfer excitation energy ultimately to a pigment with a fluorescence emission maximum at 675 nm. This pigment is presumed to be allophycocyanin in an aggreagated state. Uncoupling of energy transfer among the pigments, and physical release of the phycobiliproteins from the phycobilisome follow a parallel time-course; phycoerythrin is released first, followed by R-phycocyanin, and then allophycocyanin. In 55 mM phosphate buffer, the times at which 50% of each phycobiliprotein has dissociated are: phycoerythrin 40 min, R-phycocyanin 75 min, and allophycocyanin 140 min. The proposed arrangement of phycobiliproteins within phycobilisomes is also consistent with the results from precipitation reactions with monospecific antisera on intact and dissociated phycobilisomes. Anti-phycoertythrin reacts almost immediately with intact phycobilisomes, but reactivity with anti-R-phycocyanin and anti-allophycocyanin is considerably delayed, suggesting that the antigens are not accessible until a loosening of the phycobilsome structure occurs. Reaction wbilisomes, but is much more rapid in phycobilisomes of Nostoc sp. which contains 6-8 times more allophycocyanin. It is proposed that allophycocyanin is partially exposed on the base of isolated intact phycobilisomes of both algae, but that in P. cruentum there are too few accessible sites to permit a rapid formation of a precipitate with anti-allophyocyanin.     

Further evidence for a phycobilisome model from selective dissociation,fluorescence emission,immunoprecipitation, and electron microscopy

Phycobilisomes, isolated in 500 mM Sorensen's phosphate buffer pH 6.8 from the red alga, Porphyridium cruentum, were analyzed by selective dissociation at various phosphate concentrations. The results are consistent with a structural model consisting of an allophycocyanin core, surrounded by a hemispherical layer of R-phycocyanin, with phycoerythrin being on the periphery. Such a structure also allows maximum energy transfer.Intact phycobilisomes transfer excitation energy ultimately to a pigment with a fluorescence emission maximum at 675 nm. This pigment is presumed to be allophycocyanin in an aggregated state. Uncoupling of energy transfer among the pigments, and physical release of the phycobiliproteins from the phycobilisome follow a parallel time-course; phycoerythrin is released first, followed by R-phycocyanin, and then allophycocyanin. In 55 mM phosphate buffer, the times at which 50% of each phycobiliprotein has dissociated are: phycoerythrin 40 min, R-phycocyanin 75 min, and allophycocyanin 140 min.The proposed arrangement of phycobiliproteins within phycobilisomes is also consistent with the results from precipitation reactions with monospecific antisera on intact and dissociated phycobilisomes. Anti-phycoerythrin reacts almost immediately with intact phycobilisomes, but reactivity with anti-R-phycocyanin and anti-allophycocyanin is considerably delayed, suggesting that the antigens are not accessible until a loosening of the phycobilisome structure occurs. Reaction with anti-allophycocyanin is very slow in P. cruentum phycobilisomes, but is much more rapid in phycobilisomes of Nostoc sp. which contains 6–8 times more allophycocyanin. It is proposed that allophycocyanin is partially exposed on the base of isolated intact phycobilisomes of both algae, but that in P. cruentum there are too few accessible sites to permit a rapid formation of a precipitate with anti-allophyocyanin.Phycobilisome dissociation is inversely proportional to phosphate concentration (500 mM to 2 mM), and is essentially unaffected by protein concentration in the range used (30–200 μg/ml). Phycobiliprotein release occurs in the same order (phycoerythrin > R-phycocyanin > allophycocyanin) in the pH range 5.4–8.0.     

Molecular architecture of a light-harvesting antenna. Comparison of wild type and mutant Synechococcus 6301 phycobilisomes

Phycobilisomes of the cyanobacterium Synechococcus 6301 contain C-phycocyanin and allophycocyanin in a molar ratio of approximately 3.8:1, a minor biliprotein, allophycocyanin B, and nonpigmented polypeptides of 75, 33, 30, and 27 kilodaltons. A nitrosoguanidine-induced mutant AN112 produces altered phycobilisomes with the molar ratio of C-phycocyanin to allophycocyanin reduced to approximately 1.4:1 and without any of the 33- and 30-kilodalton polypeptides. The mutant and wild type phycobilisomes contain the same molar amount of the 75- and 27-kilodalton polypeptides relative to allophycocyanin. As seen by electron microscopy, the allophycocyanin-containing core of the mutant and of the wild type phycobilisomes appears the same. In some views of the core, each of the two core units in the mutant particles can be seen to consist of four discs approximately 3 nm thick. In wild type phycobilisomes five or six rods, made up of two to six stacked discs (11.5 X 6 nm) are attached to the core. In the mutant, no such rods are seen; rather, single disc-shaped elements, ranging from two to six in number, are found attached. Spectroscopic measurements show that the assembly form of phycocyanin in the mutant phycobilisomes differs from that in the wild type particles but reveal no difference in the organization of the core elements. These results indicate that the portions of the rod substructures of wild type phycobilisomes, beyond the disc proximal to the core, are made up of phycocyanin and the 33- and 30-kilodalton polypeptides. Emission from phycocyanin is a significant component in the fluorescence from isolated Synechococcus 6301 phycobilisomes and indicates an upper limit of 94% for the efficiency of energy transfer from phycocyanin to allophycocyanin and allophycocyanin B in these particles.     

Allophycocyanin B (λmax 671, 618 nm)

A hitherto undescribed red fluorescent phycobiliprotein (maximum emission at ∼ 680 nm), characterized by long wavelength absorption maxima in the visible region at 671 nm (ε=172000 M⁻¹·cm⁻¹ per monomer of mol. wt. 30600) and 618 nm, has been purified to homogeneity from a unicellular cyanobacterium, Synechococcus sp., and from a filamentous cyanobacterium, Anabaena variabilis. The name allophycocyanin B has been proposed for the new protein. A. variabilis allophycocyanin B is characterized by a native molecular weight of 89000 ± 5000 (in 0.05 M phosphate at pH 7.2), an isoelectric point of 5.09, and a subunit molecular weight, based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, of 15300. The protein contains one phycocyanobilin chromophore per subunit. In common with allophycocyanin from the same organism, allophycocyanin B does not contain either histidine or tryptophan. In other respects, the amino acid compositions of the two proteins are significantly different. Synechococcus sp. (Anacystis nidulans) allophycocyanin B gives two components of 16000 and 17000 mol. wt., of equal staining intensity, on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Allophycocyanins B from both organisms cross-react with rabbit antisera directed against either Synechococcus sp. or Anabaena sp. allophycocyanin, but not with antisera against the phycocyanins of the same organisms. It is suggested that allophycocyanin B occupies a position between allophycocyanin and chlorophyll a in the energy transfer path from the accessory pigments to species of chlorophyll a with absorption maxima at λ>670 nm.     

Fluorescent tandem phycobiliprotein conjugates. Emission wavelength shifting by energy transfer.

A fluorescent tandem phycobiliprotein conjugate with a large Stokes shift was prepared by the covalent attachment of phycoerythrin to allophycocyanin. The efficiency of energy transfer from phycoerythrin to allophycocyanin in this disulfide-linked conjugate was 90%. A distinctive feature of this phycocyanin conjugate is the wide separation between the intense absorption maximum of phycoerythrin (epsilon = 2.4 x 10(6) cm-1 M-1 at 545 nm) and the fluorescence emission maximum of allophycocyanin (660 nm). Energy transfer from a donor to a covalently attached acceptor can be used to adjust the magnitude of the Stokes shift. Tandem phycobiliprotein conjugates can be used to advantage in fluorescence-activated cell sorting, fluorescence microscopy, and fluorescence immunoassay analyses.     

Isolation and Function of Allophycocyanin B of Porphyridium cruentum

Allophycocyanin B was purified to homogeneity from the eukaryotic red alga Porphyridium cruentum. This biliprotein is distinct from the allophycocyanin of P. cruentum with respect to subunit molecular weights, and spectroscopic and immunological properties. The purified allophycocyanin B has a long wavelength absorption maximum at 669 nm at room temperature and at 675 nm at −196 C while the fluorescence emission maximum is at 673 nm at room temperature and 679 nm at −196 C. The emission spectrum of allophycocyanin shifted only 1 nm, from 659 to 660 nm, on cooling to −196 C, and was the same with allophycocyanin crystals as it was with pure solutions of the pigment. Phycobilisomes from P. cruentum have a major fluorescence emission band at 680 nm at −196 C which emanates from the small amount of allophycocyanin B present in the phycobilisomes. Light energy absorbed by the bulk of the biliprotein pigments is transferred to allophycocyanin B with high efficiency.     

Algal phycocyanins promote growth of human cells in culture

Summary The growth-promoting substances in a non-dialyzable extract of Synechococcus elongatus var. on RPMI 8226 cells (a human myeloma cell line) were separated by gel filtration and ion exchange chromatography. By gel filtration with Sepharose 4B, the dialyzate was separated into two fractions. One fraction was green-colored (P-1) and the other was blue-colored (P-2). The P-2 fraction had a higher growth-promoting activity than P-1. By ion exchange chromatography, the P-2 fraction was separated into two blue-colored fractions of phycocyanin and allophycocyanin. Both biliproteins promoted the growth of RPMI 8226 cells; however, allophycocyanin was more active than phycocyanin. Editor's statement This reporrt that phycobiliprotein from algae is capable of stimulating animal cell growth is unique, and raises the possiblity that related compounds such as biliverdin might also be similarly active. Some puzzling aspects, such as the lack of increase in specific activity during purification, as well as the possibility that the activity might be due to a contaminant, remain to be resolved.     

Allophycocyanin I and the 95 Kilodalton Polypeptide : The Bridge between Phycobilisomes and Membranes

Allophycocyanin was isolated from dissociated phycobilisomes from Nostoc sp. and was separated into allophycocyanin I, II, III, and B as described elsewhere. If the separation of the proteins following phycobilisome isolation is done in the presence of the protease inhibitor, phenylmethylsulfonylfluoride, associated with allophycocyanin I are two colored polypeptides of 95 kilodalton (kD) and 80 kD, belonging to the class of Group I polypeptides as defined by Tandeau de Marsac and Cohen-Bazire (Proc Natl Acad Sci USA 1977 74: 1635-1639). Allophycocyanin I has a fluorescence maximum of 680 nanometers as do intact phycobilisomes and has thus been suggested to be the final emitter of excitation energy in phycobilisomes. Thylakoid membranes washed in low ionic strength buffer containing phenylmethylsulfonylfluoride lose all biliproteins, but retain the 95 kD and 80 kD polypeptides. As suggested by Tandeau de Marsac and Cohen-Bazire, these are likely to be the polypeptides involved in binding the phycobilisome to the membrane. As these polypeptides are isolated with allophycocyanin I, structural evidence is provided for placing allophycocyanin I as the bridge between the phycobilisome and the membrane. These Group I polypeptides and the 29 kD polypeptide (involved in rod attachment to the APC core) are particularly susceptible to proteolytic breakdown. It is thought that in vivo the active protease may be selectively attacking these polypeptides to detach the phycobilisome from the membrane and release the phycoerythrin and phycocyanin containing rods from the allophycocyanin core for greater susceptibility of the biliproteins to protease attack.     

Properties of allophycocyanin II and its α- and β-subunits from the thermophilic blue–green alga Mastigocladus laminosus

Purified allophycocyanin II and its subunits have been examined with respect to spectroscopic properties, sedimentation, reconstitution and isoelectric behaviour. In 0.02m-potassium phosphate buffer, pH8.0, and at a concentration of 0.25mg/ml, allophycocyanin II and its alpha- and beta-subunits show visible absorption maxima at 650, 615 and 615nm respectively, whereas the fluorescence emission maxima were determined to be at 662, 640 and 630nm respectively. The absorption difference spectrum (dilution difference) of allophycocyanin II displays maxima at 650 and 590nm with a minimum at 610nm. The c.d. spectrum of allophycocyanin II showed only one positive-ellipticity band at 635nm, and a major negative-ellipticity band at 340nm. Oxidation of allophycocyanin II, low- and high-pH solutions (pH3.0 and 11.0), various ethanol concentrations as well as dialysis against distilled water induce a spectral change leading to phycocyanin-like characteristics. In most cases these shifts are reversible. Allophycocyanin II is thermostable over a period of 60min at temperatures up to 60 degrees C. The isoelectric points of allophycocyanin II and its alpha- and beta-subunits are 4.65, 4.64 and 4.82 respectively. Estimated molecular weights from sedimentation-equilibrium analyses were 102500 for allophycocycanin II, 16000 for the alpha- and 31500 for the beta-subunit. Recombination of alpha- and beta-subunits leads to allophycocyanin II, which is indistinguishable from native allophycocyanin with respect to its spectral form, to its gel-filtration and to its electrophoretic behaviour.     

Phycobiliprotein synthesis in protoplasts of the unicellular cyanophyte, Anacystis nidulans.

Stable and metabolically active protoplasts were prepared from the unicellular cyanophyte, Anacystis nidulans, by enzymatic digestion of the cell wall with 0.1% lysozyme. The yield of protoplasts from intact algal cells was approx. 50%. Incorporation of L-[U-14C]leucine into cold trichloroacetic acid-insoluble material from protoplasts preparations was linear for 1.5 h and continued for an additional 2.5 h. Incorporation of radiolabeled leucine into hot trichloroacetic acid-insoluble material from protoplast preparations demonstrated protein synthesis in protoplasts in vitro. Phycocyanin is the principal phycobiliprotein and allophycocyanin is a minor phycobiliprotein in A. nidulans cells. The light-absorbing chromophore of both of these phycobiliproteins is the linear tetrapyrrole (bile pigment), phycocyanobilin. Radiolabeled phycocyanin and allophycocyanin were isolated from protoplast preparations which had been incubated with L-[U-14]leucine or delta-amino[4-14C] levulinic acid (a precursor of phycocyanobilin). The radio-labeled phycobiliproteins were purified by ammonium sulfate fractionation and ion-exchange chromatography on brushite columns. The specific radioactivity of phycocyanin and allophycocyanin in brushite column eluates (protoplasts incubated with radiolabeled leucine) was 106 000 and 82 000 dpm/mg, respectively. The specific radioactivity of phycocyanin and allophycocyanin in brushite column eluates (protoplasts incubated with radiolabeled delta-aminolevulinic acid) was 33 000 and 38 000 dpm/mg, respectively. Phycobiliproteins from protoplasts incubated with radiolabeled leucine were examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. 25% of the incorporated radioactivity in protoplast lysates and approx. 60% of the incorporated radioactivity in protoplast lysates and approx. 60% of the incorporated ratioactivity in phycocyanin and allophycocyanin (in brushite column eluates) comigrated with the subunits of these phycobiliproteins on sodium dodecyl sulfate-polyacrylamide gels. Chromic acid degradation of phycobiliproteins from protoplast preparations incubated with delta-amino[4-14C] levulinic acid yielded radiolabeled imides which were derived from the phycocyanobilin chromophore. Imides from radiolabeled phycobiliproteins isolated from protoplast preparations incubated with L-[U-14C]leucine did not contain radioactivity. These results show that both the apoprotein and tetrapyrrolic moieties of phycocyanin and allophycocyanin were synthesized in A. nidulans protoplasts in vitro.     

C-phycocyanin and allophycocyanin in two species of blue-green algae

1. The biliproteins C-phycocyanin and allophycocyanin were purified from the blue-green alga Anabaena variabilis by ammonium sulphate fractionation and gel filtration. 2. An assay procedure that enabled the proportion of the two pigments, present as a mixture, to be determined was devised by using the data provided by spectrophotometric analysis of the purified biliproteins. 3. The degree of association and relative proportions of the two pigments were analysed by the application of this procedure to the components separated by thin-layer gel filtration. 4. The C-phycocyanin/allophycocyanin ratio remained essentially constant in algal extracts prepared at various stages throughout the growth cycle or after growth under conditions of reduced illumination. 5. The behaviour of the C-phycocyanin aggregate species from Anacystis nidulans suggested that they were of appreciably lower molecular weight than those observed in extracts of Anabaena variabilis.     

Dynamic aspects of phycobilisome structure

In exponentially growing cells of Synechococcus sp. 6301, over 95% of the phycobiliproteins are located in phycobilisomes, and the remainder is present in the form of low molecular weight aggregates. In addition to the subunits of the phycobiliproteins (C-phycocyanin, allophycocyanin, allophycocyanin B), the phycobilisomes of this unicellular cyanobacterium contain five non-pigmented polypeptides. During the initial phase of starvation (24 h after removal of combined nitrogen from the growth medium), the phycobiliproteins in the low molecular weight fraction largely disappeared. Phycocyanin was lost more rapidly from this fraction than allophycocyanin. Simultaneous changes in the phycobilisome were (1) a decrease in sedimentation coefficient, (2) a decrease in phycocyanin: allophycocyanin ratio, (3) a shift in the fluorescence emission maximum from 673 to 676 nm, and (4) a selective complete loss of a 30,000 dalton non-pigmented polypeptide. Upon extensive nitrogen starvation (72 h), the intracellular level of phycocyanin decreased by over 30-fold. These results indicate that in the early stage of nitrogen starvation, the free phycobiliproteins of the cell are degraded, as well as a significant proportion of the phycocyanin from the periphery of the phycobilisome. However, the structures partially depleted of phycocyanin still function efficiently in energy transfer. On extended starvation, total degradation of residual phycobilisomes takes place, possibly in conjunction with the detachment of these structures from the thylakoids.None of the effects of the absence of combined nitrogen were seen when cells were starved in the presence of chloramphenicol, or in a methionine auxotroph starved for methionine.Abbreviations Used NaK-PO₄ NaH₂PO₄ titrated with K₂HPO₄ to a given pH - SDS sodium dodecyl sulfate - Tris Tris(hydroxymethyl)aminomethane     

Light Intensity Adaptation and Phycobilisome Composition of Microcystis aeruginosa

Phycobilisomes isolated from Microcystis aeruginosa grown to midlog at high light (270 microeinsteins per square meter per second) or at low light intensities (40 microeinsteins per square meter per second) were found to be identical. Electron micrographs established that they have a triangular central core apparently consisting of three allophycocyanin trimers surrounded by six rods, each composed of two hexameric phycocyanin molecules. The apparent mass of a phycobilisome obtained by gel filtration is 2.96 × 10⁶ daltons. The molar ratio of the phycobiliproteins per phycobilisome is 12 phycocyanin hexamers:9 allophycocyanin trimers. The electron microscopic observations combined with the phycobilisome apparent mass and the phycobiliprotein stoichiometry data indicate that M. aeruginosa phycobilisomes are composed of a triangular central core of three stacks of three allophycocyanin trimers and six rods each containing two phycocyanin hexamers. Adaptation of M. aeruginosa to high light intensity results in a decrease in the number of phycobilisomes per cell with no alteration in phycobilisome composition or structure.     

Protein aggregation. Studies of larger aggregates of C-phycocyanin

Aggregates of phycocyanin sedimenting at 17s, 22s and 27s are demonstrated to constitute more than 40% of crude blue-green-algal extracts, pH6.0 and I0.1, and are retained in highly purified preparations. Sedimentation-velocity studies of the large aggregates as a function of pH are reported. Sucrose-density-gradient experiments performed as a function of time of sedimentation indicate that: (1) with increasing time of sedimentation, the largest aggregates are dissipated at the leading protein boundary and the several phycocyanin species present are not completely resolved; (2) phycocyanin fractions with the largest aggregates exhibit the highest E(620)/E(280) ratio and the largest relative fluorescence efficiency. Gel-filtration experiments with Sephadex G-200 do not resolve the species completely, and reapplication of phycocyanin gel-filtration fractions to the column results in an elution pattern similar to the original, except that there is an enhancement of the allophycocyanin fraction and the amount of denatured protein. Increasing the sedimentation times in a sucrose density gradient also enhances the allophycocyanin fraction. Fluorescence results demonstrate that there are possibly three excitation maxima, one corresponding to the monomer (approx. 600mmu), one for higher aggregates (625-630mmu) and one for the allophycocyanin fraction (approx. 650mmu). Only a single fluorescence-emission band is detected, which is fairly symmetrical and which has a red shift with higher aggregation and with the appearance of allophycocyanin. The appearance of allophycocyanin may be correlated with the irreversible disaggregation of the largest phycocyanin species. It is suggested that the largest protein aggregates are in the size range of the biliprotein aggregates reported in electron microscopy of algal cells.     

Spectral analysis of allophycocyanin I, II, III and B from Nostoc sp. phycobilisomes

Low temperature (-196C) and room temperature (25C) absorption spectra of a family of allophycocyanin spectral forms isolated from Nostoc sp. phycobilisomes as well as of the phycobilisomes themselves have been analyzed by Gaussian curve-fitting. Allophycocyanin I and B share long wavelength components at 668 and 679 nm, bands that are absent from allophycocyanin II and III. These long wavelength absorption components are apparently responsible for the 20 nm difference between the 680 nm fluorescence emission maximum of allophycocyanin I and B and the 660 nm maximum of II and III. This indicates that allophycocyanin I and B are the final acceptors of excitation energy in the phycobilisome and the excitation energy transfer bridge linking the phycobilisome with the chlorophyll-containing thylakoid membranes. These Gaussian components are also found in resolved spectra of phycobilisomes, are arguing against this family of allophycocyanin molecules being artifactual products of protein purification procedures.     

Chromophores of allophycocyanin and R-phycocyanin

The biliprotein allophycocyanin was purified from Phormidium luridum, Anabaena variabilis and Plectonema boryanum. R-phycocyanin was purified from Rhodymenia palmata. The chromophores were cleaved from the denatured protein by methanol hydrolysis. They were purified and crystallized as the dimethyl esters. Chromatographic and absorption-spectral (visible–ultraviolet and infrared) comparisons with reference material have established phycocyanobilin as the chromophore of allophycocyanin. Phycocyanobilin and phycoerythrobilin were shown to be the chromophores of R-phycocyanin.     

Spectroscopic properties of the C-phycocyanin-allophycocyanin conjugate and the isolated phycobilisomes from Spirulina platensis

C-phycocyanin (CPC) and allophycocyanin (APC) were purified from Spirulina platensis, then the CPC was attached covalently to the APC by reacting their ∈-amino groups. The excitation energy could be transferred from the CPC to the APC in the CPC-APC conjugate. Intact phycobilisomes (PBS), consisting of CPC, APC, colourless linker polypeptides, and APC B or Lcm, were isolated from S. platensis. Spectroscopic properties of the isolated PBSs kept at 20 °C for various times showed that the connection between the APC and the APC B or Lcm was looser than that between the CPC and the APC in the isolated PBSs. The CPC-APC conjugate was more stable than the isolated PBSs, and the linker polypeptides had a minor influence on the excitation energy transfer characteristic between different phycobiliproteins in the PBS.     

Growth and Chromatic Adaptation of Nostoc sp. Strain MAC and the Pigment Mutant R-MAC

A spontaneous, stable, pigmentation mutant of Nostoc sp. strain MAC was isolated. Under various growth conditions, this mutant, R-MAC, had similar phycoerythrin contents (relative to allophycocyanin) but significantly lower phycocyanin contents (relative to allophycocyanin) than the parent strain. In saturating white light, the mutant grew more slowly than the parent strain. In nonsaturating red light, MAC grew with a shorter generation time than the mutant; however, R-MAC grew more quickly in nonsaturating green light.

When the parental and mutant strains were grown in green light, the phycoerythrin contents, relative to allophycocyanin, were significantly higher than the phycoerythrin contents of cells grown in red light. For both strains, the relative phycocyanin contents were only slightly higher for cells grown in red light than for cells grown in green light. These changes characterize both MAC and R-MAC as belonging to chromatic adaptation group II: phycoerythrin synthesis alone photocontrolled.

A comparative analysis of the phycobilisomes, isolated from cultures of MAC and R-MAC grown in both red and green light, was performed by polyacrylamide gel electrophoresis in the presence of 8.0 molar urea or sodium dodecyl sulfate. Consistent with the assignment of MAC and R-MAC to chromatic adaptation group II, no evidence for the synthesis of red light-inducible phycocyanin subunits was found in either strain. Phycobilisomes isolated from MAC and R-MAC contained linker polypeptides with relative molecular masses of 95, 34.5, 34, 32, and 29 kilodaltons. When grown in red light, phycobilisomes of the mutant R-MAC appeared to contain a slightly higher amount of the 32-kilodalton linker polypeptide than did the phycobilisomes isolated from the parental strain under the same conditions. The 34.5-kilodalton linker polypeptide was totally absent from phycobilisomes isolated from cells of either MAC or R-MAC grown in green light.