Role of the Colorless Polypeptides in Phycobilisome Assembly in

We have identified the function of the 'extra' polypeptides involved in phycobilisome assembly in Nostoc sp. These phycobilisomes, as those of other cyanobacteria, are composed of an allophycocyanin core, phycoerythrinand phycocyanin-containing rods, and five additional polypeptides of 95, 34.5, 34, 32, and 29 kilodaltons. The 95 kilodalton polypeptide anchors the phycobilisome to the thylakoid membrane (Rusckowski, Zilinskas 1982 Plant Physiol 70. 1055-1059); the 29 kilodalton polypeptide attaches the phycoerythrinand phycocyanin-containing rods to the allophycocyanin core (Glick, Zilinskas 1982 Plant Physiol 69: 991-997). Two populations of rods can exist simultaneously or separately in phycobilisomes, depending upon illumination conditions. In white light, only one type of rod with phycoerythrin and phycocyanin in a 2:1 molar ratio is synthesized. Associated with this rod are the 29, 32, and 34 kilodalton colorless polypeptides; the 32 kilodalton polypeptide links the two phycoerythrin hexamers, and the 34 kilodalton polypeptide attaches a phycoerythrin hexamer to a phycocyanin hexamer. The second rod, containing predominantly phycocyanin, and the 34.5 and 29 kilodalton polypeptides, is synthesized by redUght-adapted celis; the 34.5 kilodalton polypeptide links two phycocyanin hexamers. These assignments are based on isolation of rods, dissociation of these rods into their component biliproteins, and analysis of colorless polypeptide composition, followed by investigation of complexes formed or not formed upon their recombination. Blue-green algae (Cyanobacteria) have a remarkable ability to adapt to available light through complementary chromatic adaptation, where genes for specific light-harvesting phycobiliproteins appear to be transcribed upon exposure of the cells to red or green light. PE2, which absorbs green light very effectively, is preferentially synthesized in green light, while PC, which absorbs orangered light, is synthesized upon exposure to red light (1). Tandeau de Marsac and Cohen-Bazire (19) first showed that in addition to the differential synthesis of the a and ,B chromophore-containing subunits of PE or PC during chromatic adaptation, there was also a change observed in the quantity and quality of several additional colorless polypeptides isolated with the variously adapted phyco' Supported in part by the Science and Education Administration of the United States Department of Agriculture under Grant 5901-0410-8-01850 from the Competitive Research Grants Office, by a National Science Foundation Grant PCM-80-18740, by a Busch Research Grant, and by a Rutgers University Research Council Grant. New Jersey Agricultural Experiment Station, Publication No. D-01 104-3-82, supported by State funds and by the United States Hatch Act. 2 Abbreviations: PE, phycoerythrin; PC, phycocyanin; PBsomes, phycobilisomes; APC, allophycocyanin; PMSF, phenylmethylsulfonylfluoride. bilisomes (PBsomes). Their paper was the first to suggest a role of these polypeptides in linking the phycobiliproteins to each other in the multiprotein PBsome aggregate as well as a role in attachment of the PBsome to the thylakoid membrane. Additional evidence has since accumulated to support this original suggestion and was reviewed recently (6). The approaches taken to the understanding of the function of these polypeptides have been varied. The first approach involved studying the parallel change in colorless polypeptide composition and biliprotein content during chromatic adaptation (2, 3, 19). The color of light during growth can dramatically influence not only the colored biliprotein composition but also the type and quantity of colorless biliproteins synthesized. Others have looked at the role of these 'extra' polypeptides through isolation of the PBsome components, e.g. the PEand PC-containing rods (3, 13, 25, 26) and the APC core (18, 26, 27), and then identified by gel electrophoresis the associated linker polypeptides. A third approach employs mutants (8, 21, 23, 24) or environmental conditions (22, 23), e.g. N03 or CO2 deprivation, which result in the synthesis of incomplete PBsomes; correlations are then drawn between the absence of certain colorless polypeptides and the resultant PBsome structure. The last strategy involves analysis of colorless polypeptides in reassembling PBsomes or PBsome components (e.g. rods) from separated parts. This includes reassembly of PBsomes from separated rods and cores (5, 1 1), reassociation of rods or biliprotein complexes from separated biliproteins (15, 26), and in the most elaborate case, reassembly of rods of PC upon addition of certain colorless polypeptides to purified PC (16). In all of these experiments, polypeptides in the 36 to 27 kD range, called group II polypeptides, are involved in association of rod components and attachment of the rod to tbe APC core. Polypeptides which vary in size from 100 to 75 kD, depending on the organism, called group I polypeptides, are involved in attaching the APC to the thylakoid membrane (see 6, 11, 18). In this paper, PBsome rods containing PE and PC were isolated from chromatically adapted cells of Nostoc sp. The function of each of the accompanying colorless polypeptides was determined by dissociation of these rods into their component biliproteins, followed by analysis of their colorless polypeptide composition and then by an investigation of rods formed or not formed upon their recombination. This work was presented earlier in preliminary form (28). MATERIALS AND METHODS Nostoc sp. (strain Mac) was grown at 37°C in medium CG 10 (12) with 5% CO2 in air in a 14-L New Brunswick Scientific fermentor with General Electric Gro and Sho (Fl5T8/PL) pink fluorescent lamps (light intensity, 10 w/m2, measured with Yellow Springs Instrument model 65A Radiometer). The organism was also grown in 1.5-L low-form culture flasks aerated with 5% CO2 in air on a New Brunswick Scientific shaker in cool-white fluo-