The phycobilisome core‐membrane linkers from Synechocystis sp. PCC 6803 and red‐algae assemble in the same topology

Abstract

SUMMARY The phycobilisomes (PBSs) of cyanobacteria and red‐algae are unique megadaltons light‐harvesting protein‐pigment complexes that utilize bilin derivatives for light absorption and energy transfer. Recently, the high‐resolution molecular structures of red‐algal PBSs revealed how the multi‐domain core‐membrane linker (LCM) specifically organizes the allophycocyanin subunits in the PBS’s core. But, the topology of LCM in these structures was different than that suggested for cyanobacterial PBSs based on lower‐resolution structures. Particularly, the model for cyanobacteria assumed that the Arm2 domain of LCM connects the two basal allophycocyanin cylinders, whereas the red‐algal PBS structures revealed that Arm2 is partly buried in the core of one basal cylinder and connects it to the top cylinder. Here, we show by biochemical analysis of mutations in the apcE gene that encodes LCM, that the cyanobacterial and red‐algal LCM topologies are actually the same. We found that removing the top cylinder linker domain in LCM splits the PBS core longitudinally into two separate basal cylinders. Deleting either all or part of the helix‐loop‐helix domain at the N‐terminal end of Arm2, disassembled the basal cylinders and resulted in degradation of the part containing the terminal emitter, ApcD. Deleting the following 30 amino‐acids loop severely affected the assembly of the basal cylinders, but further deletion of the amino‐acids at the C‐terminal half of Arm2 had only minor effects on this assembly. Altogether, the biochemical data are consistent with the red‐algal LCM topology, suggesting that the PBS cores in cyanobacteria and red‐algae assemble in the same way.

Dichromic Allophycocyanin Trimer Covering a Broad Spectral Range (550–660 nm)

“…Purification and isolation of APC: APC aggregates were isolated using previously described PBS extraction methods. [6] Briefly, cells were resuspended in 0.8 M potassium phosphate buffer (pH 7.2) and disrupted using French press. After treatment with 2 %(v/v) detergent Triton X-100, the samples were loaded on sucrose gradients (2.0 M, 1.0 M, 0.75 M, 0.5 M, and 0.25 M) for ultracentrifugation at 230 000 g for 16 h at 18 °C (P40ST rotor, Hitachi CP80-WX ultracentrifuge).…”

“…The 77 K fluorescence spectra were recorded using a Fluorolog‐3 spectrofluorimeter (HORIBA Scientific). As in previous methods, [6] Synechocystis cells were suspended in 25 mM HEPES‐NaOH (pH 7.5) dissolved in BG‐11 medium before 77 K fluorescence measurement. CD spectra were recorded using a MOS‐500 CD spectrometer (Bio‐Logic, Claix) in the visible region [17,40] .…”

“…[23] Synechocystis can maintain photosynthetic autotrophy only when its PBS core sediments at � 0.5 M, while units at 0.25 M are trimers or even smaller. [6] This lack of larger rod-like structures would explain the low growth rate of PebS-ΔRod cells. The aggregates sedimenting at 0.25 M also showed a unique spectrum (Figure 2C).…”

“…In the core structure, two linker proteins, ApcC (L C ) and the repeated domains (REP domain) at C-terminal of ApcE (L CM ) locate in the central hole of trimers and interact with each other. [5,6] The light-harvesting ability of PBS results from absorption by, and efficient energy transfer among, diverse phycobilin chromophores. Based on variation of the length of the conjugated double-bond system, phycobilins absorb light over a broad spectral range.…”

“…To form the PBS super‐complex, various linker proteins are responsible for assembling the trimers together. In the core structure, two linker proteins, ApcC (L C ) and the repeated domains (REP domain) at C‐terminal of ApcE (L CM ) locate in the central hole of trimers and interact with each other [5,6] …”