Proc. Natl. Acad. Sci. USA Vol. 76, No. 12, pp. 6162-6166, December 1979 Biochemistry Characterization of cyanobacterial phycobilisomes in zwitterionic detergents (Synechococcus/photosynthetic accessory pigments/sedimentation/electron microscopy/aggregation) ALEXANDER N. GLAZER*, ROBLEY C. WILLIAMSt, GREGORY YAMANAKA*, AND H. K. SCHACHMANt *Department of Microbiology and Immunology, and tDepartment of Molecular Biology, University of California, Berkeley, California 94720 Contributed by Robley C. Williams, September 10, 1979 ABSTRACT Properties of cyanobacterial phycobilisomes preparations of nearly uniform-sized phycobilisomes were (from Synechococcus spp. 6301 and 6312 and Synechocystis sp. obtained. Ultrastructural studies of the particles prepared in 6701) prepared in the presence of two different zwitterionic detergents were compared to those of phycobilisomes detached zwitterionic detergents were facilitated by the marked decrease from membranes with the nonionic detergent Triton X-100 and in aggregation. Such studies show that phycobilisomes from then freed from Triton by sedimentation through high-salt su- different organisms have certain characteristics in common, crose density gradients. The absorption spectra, polypeptide as concluded by others (1, 7, 8), but also exhibit distinctive composition, and ultrastructure of phycobilisomes were inde- structural features. pendent of the detergent used during the preparation. Phyco- bilisomes from certain cyanobacteria aggregated in the absence MATERIALS AND METHODS of detergent. Such aggregation was not seen in preparations containing zwitterionic detergents. Aggregation of phycobili- Strains and Culture Conditions. Synechococcus spp. 6301 somes led to a partial quenching of their fluorescence. Electron (ATCC 27144) and 6312 (ATCC 27167) and Synechocystis sp. microscopy showed the three types of phycobilisomes to have 6701 (ATCC 27170) (9) were grown as 1-liter cultures in me- a hemidisc appearance, although their detailed structures were dium BG-ll (10) with continuous stirring under warm white quite different. fluorescent illumination. Cells were grown at 30°C in the Cells of cyanobacteria and chloroplasts of red algae contain high presence of air or at 40°C gassed with 1% C02/99% N2. concentrations of photosynthetic accessory proteins-the Phycobilisome Isolation. Phycobilisomes were isolated as phycobiliproteins. These proteins are assembled into particles described (3). In some experiments, Triton was replaced by the (phycobilisomes) that are attached in regular arrays to the outer zwitterionic detergent Deriphat 160 (gift of General Mills surface of the thylakoids (1). The polypeptide composition of Chemical Division, Kankakee, IL) or Miranol S2M-SF (gift of phycobilisomes is complex. In addition to multiple copies of the Miranol Chemical Corp., Irvington, NJ). In these experiments, subunits of the phycobiliproteins, these particles contain detergent was used at a final concentration of 1-1.5% (wt/vol) polypeptides believed to function in the assembly of the chro- to solubilize phycobilisomes from membrane particles in broken moproteins and in the attachment of the particles to the cell suspensions; it was also present in the sucrose gradients at membrane (2, 3). Preparation of phycobilisomes with unim- 0.1%. paired energy transfer properties has invariably involved Sedimentation Studies. Sedimentation coefficients were breakage of the cells in solutions of 0.65-0.75 M NaH2PO4 ti- determined with a Spinco model E analytical ultracentrifuge trated to pH 7.0-8.0 with K2HPO4 (NaK-PO4), incubation with equipped with absorption optics. Experiments were performed 1-2% Triton X-100, and isolation by centrifugation in density at protein concentrations between 0.13 and 1.5 mg/ml in 0.75 gradients of sucrose in the same buffer (4, 5). Triton X-100 is M NaK-PO4, using 12-mm double-sector cells in an AN-D rotor insoluble in this buffer and is totally removed from the phy- at speeds of 26,000-30,000 rpm. Corrections of sobs to s20,w were cobilisomes during sedimentation (4, 6). made based on the determination of relative density and vis- It is generally recognized that membrane proteins, isolated cosity values of 1.0988 and 1.3556, respectively, for the refer- with detergents, form aggregates upon removal of the deter- ence buffer, 0.75 M NaK-PO4. No correction was made for gents. Such aggregates are an artifact of the isolation procedure possible preferential hydration of the phycobilisomes; however, and do not represent structures present in the cell. The proce- such behavior is not unlikely in 0.75 M NaK-PO4 (see ref. 11). dure for phycobilisome preparation (here called the "Triton A partial specific volume of 0.73 ml/g was estimated for phy- procedure") may be anticipated to lead to the formation of cobilisomes from strain 6301, based on the known amino acid aggregates. Indeed, although phycobilisome preparations from compositions of phycocyanin (12), and allophycocyanin (13), certain cyanobacteria sediment as a single component in and the Mr 30,000 and-33,000 polypeptides (unpublished data). high-salt sucrose density gradients, those from other organisms The above components account for about 94% of the protein give either two bands or a continuous smear covering a mo- by weight in these phycobilisomes (3). lecular weight range of several million (ref. 7; unpublished Electron Microscopy. Copper grids (400 mesh) coated with observations). What, then, is the relationship between the a film of carbon over Formvar were used as specimen supports. aggregation state of phycobilisomes isolated by the Triton just before sample application they were exposed for about 5 procedure and that of particles isolated in the continuous sec to a 500 V/cm glow discharge in a partial vacuum (100 presence of detergent? To answer this question, we have pre- millitorrs). The phycobilisome specimens were diluted from pared phycobilisomes in two zwitterionic detergents that are their initial concentration of about 2 mg/ml to 20 ,g/ml im- readily soluble in the high-salt buffers. In such detergents, mediately prior to application to the support films in 5-iA drops. Samples isolated with the aid of Triton X-100 were diluted in The publication costs of this article were defrayed in part by page 0.75 M NaK-PO4 buffer. The diluent for those isolated in charge payment. This article must therefore be hereby marked "ad- vertisement" in accordance with 18 U. S. C. §1734 solely to indicate Abbreviation: NaK-PO4, NaH2PO4 titrated to pH 8.0 with this fact. K2HPO4. 6162 Downloaded by guest on September 29, 2021 Biochemistry: Glazer et al. Proc. Natl. Acad. Sci. USA 76 (1979) 6163 zwitterionic detergents contained, additionally, the appropriate emission and decrease in energy transfer to allophycocyanin detergent at 0.1%. After about 30-sec residence time, each 5-,ul and allophycocyanin B (3) when phycobilisome solutions are sample drop was almost entirely withdrawn by suction friom incubated at low protein concentrations (data not shown). a fine-tip pipet (14), and the residual liquid film was overlaid Attempts to isolate phycobilisomes from strain 6312 by the with a 5-,ul drop of glutaraldehyde that had been freshly diluted Triton procedure used with strain 6301 (3) resulted in the dis- to 0.3% in the buffer used for sample dilution. After 5 min, the tribution of phycobilisome material on sucrose density gradients grid was serially rinsed in 100 mM and 10 mM ammonium illustrated in tube A of Fig. 1 Inset. Two deep blue bands of acetate, care being taken to wet only the sample side of the grid. protein were seen against a continuum of blue material, ex- After removal of the 10 mM rinse solution, a 5-,Ml drop of 1-2% tending from a free phycobiliprotein fraction at the top to aqueous uranyl formate was applied, left on for several seconds, nearly the bottom of the centrifuge tube. Samples from bands and thoroughly removed by suction applied to one edge of the 1 and 2 from such a gradient had virtually identical absorption grid. The uranyl formate stain was unbuffered (pH nz2.0) and spectra (Fig. 1, solid line). In the analytical ultracentrifuge, band had been passed through a 25-nm Millipore filter within a few 1 represented a single component of about 55 S. Band 2, how- hours of use. Each specimen was observed in a JEOL 100 B ever, gave rise to two distinct species: a small component of electron microscope immediately after preparation. Primary about 55 S, probably identical to that present in band 1, and a magnifications were X20,000-X45,000. All micrographs were large, more polydisperse component (as judged from the shape obtained by use of the technique of minimal beam exposure (15) of the boundary) with an average sedimentation coefficient of as adapted to the JEOL instrument. 120-130 S. In both of the experiments described in Table 1, the Other Methods. Density measurements were performed in smaller component in band 2 represented only about 10% of a Precision density meter (DMA 02C) and viscosity measure- the total material in the sample. ments were made in an Ostwald capillary viscometer. Gel Phycobilisome Aggregation During Isolation. In investi- electrophoresis and all other measurements were performed gating the question of phycobilisome aggregation during su- as described (3). crose gradient centrifugation, we tested two zwitterionic de- tergents, Miranol and Deriphat, in place of the nonionic de- RESULTS tergent Triton X-100. It has been shown recently that these The phycobilisomes of Synechococcus spp. 6301 and 6312 were detergents efficiently solubilize the protein-chlorophyll com- chosen for comparative study because they represent extremes plexes of photosynthetic membranes (17). Unlike Triton X-100, in the relative ratios of the major phycobiliproteins, C-phyco- which formed a floating detergent layer, the zwitterionic de- cyanin and allophycocyanin. The phycocyanin/allophyco- tergents were soluble in 0.75 M NaK-PO4 and could thus be cyanin ratios are -6:1 (3) and ;1.5:1 (16) in Synechococcus spp. added to sucrose gradients in high-salt buffer.