Reconstitution, characterisation and mass analysis of the pentacylindrical allophycocyanin core complex from the cyanobacterium Anabaena sp. PCC 7120.

The phycobilisome (PBS) of Anabaena sp. PCC 7120 was allowed to dissociate into its constituents and the resulting allophycocyanin (AP) fraction was purified. Its reconstitution yielded a complex which according to negative stain electron microscopy and spectral analysis was identical to the native pentacylindrical PBS core domain. Each cylinder of the central tricylindric unit was comprised of four AP (alphabeta)3 disks. Mass analysis using the scanning transmission electron microscope (STEM) showed the presence of 16 AP trimers in the intact reconstitute, which had a total mass of 1966(+/-66) kDa. Composition analysis indicated an AP trimer distribution of (AP-II):(AP-LCM):(AP-B):(AP-I)=6:2:2:6, i.e. an addition of two AP-I and two AP-II complexes compared to a tricylindrical PBS core domain. Therefore, we suggest that each supplementary half-core cylinder found in pentacylindrical AP core domains is comprised of one AP-I and one AP-II trimer, in agreement with the current model. The structural significance of the 127 kDa core membrane linker polypeptide was further investigated by subjecting the AP core reconstitute to mild chymotryptic degradation. After isolation, the digested complex exhibited a tricylindrical appearance while STEM mass analysis confirmed the presence of only 12 AP complexes. Polypeptide analysis by SDS-PAGE and Edman degradation related the half-cylinder loss to cleavage of the Rep4 domain of the core membrane linker polypeptide. On the basis of these data, a general model for the assembly of the three hemidiscoidal PBS types known to date is discussed.

Three C-phycoerythrin-associated linker polypeptides in the phycobilisome of green-light-grown Calothrix sp. PCC 7601 (cyanobacteria).

Microanalyses by SDS-PAGE and microsequencing demonstrate that, under green-light conditions, 3 C-phycoerythrin associated rod-linker polypeptides with different N-terminal amino acid sequences are present in phycobilisomes (PBS) from Calothrix sp. 7601 cells. Two of these polypeptides, corresponding to SDS-PAGE bands at 36 and 37 kDa, could be assigned, respectively, to the cpeC and cpcD genes found on a separate cpeCD-operon in Calothrix sp. 7601 (Federspiel, N.A. and Grossman, A.R. (1990) J. Bacteriol, 172, 4072-4081). The third C-PE rod-linker polypeptide, LR,2PE,33, requires, therefore, a third gene with the suggested locus designation 'cpeE'. A C-PE (alpha beta)6-LR,2PE,33 complex containing this third rod-linker polypeptide could be isolated from phycobilisomes and characterized. PBS from both green- and red-light cells of Calothrix contain a single, unique LRC28 rod-core linker polypeptide which is not altered during chromatic adaptation.

Reconstitution of the core complex (alpha beta)3APCLC8.9 of the phycobilisome from Mastigocladus laminosus using the Lc8.9 linker polypeptide overexpressed in Escherichia coli.

The apcC gene from Mastigocladus laminosus encodes the linker polypeptide LC8.9 located in the phycobilisome core. A T7 RNA polymerase expression system was used to express the linker polypeptide LC8.9 from M. laminosus in Escherichia coli. The apcC gene product was expressed as an inclusion body which was solubilized in a buffer containing 8M urea. Final purification was achieved by ion exchange chromatography on Fractogel TSK CM 650 (S). In addition, a method for preparative isolation of the LC8.9 linker polypeptide from M. laminosus by reverse phase chromatography is presented. Both LC8.9 isolated from M. laminosus and overexpressed in E. coli were capable of reconstituting the complex (alpha beta)3APCLC8.9. The reconstituted complex was identical to preparations isolated from M. laminosus in terms of polypeptide composition, absorption and fluorescence emission spectroscopy.