Biogenesis of Phycobiliproteins: I. cpcS-I AND cpcU MUTANTS OF THE CYANOBACTERIUM SYNECHOCOCCUS sp. PCC 7002 DEFINE A HETERODIMERIC PHYOCOCYANOBILIN LYASE SPECIFIC FOR β-PHYCOCYANIN AND ALLOPHYCOCYANIN SUBUNITS

Gaozhong Shen,Wendy M Schluchter,Donald A Bryant

doi:10.1074/jbc.m708164200

Abstract

Phycobilin lyases covalently attach phycobilin chromophores to apo-phycobiliproteins (PBPs). Genome analyses of the unicellular, marine cyanobacterium Synechococcus sp. PCC 7002 identified three genes, denoted cpcS-I, cpcU, and cpcV, that were possible candidates to encode phycocyanobilin (PCB) lyases. Single and double mutant strains for cpcS-I and cpcU exhibited slower growth rates, reduced PBP levels, and impaired assembly of phycobilisomes, but a cpcV mutant had no discernable phenotype. A cpcS-I cpcU cpcT triple mutant was nearly devoid of PBP. SDS-PAGE and mass spectrometry demonstrated that the cpcS-I and cpcU mutants produced an altered form of the phycocyanin (PC) beta subunit, which had a mass approximately 588 Da smaller than the wild-type protein. Some free PCB (mass = 588 Da) was tentatively detected in the phycobilisome fraction purified from the mutants. The modified PC from the cpcS-I, cpcU, and cpcS-I cpcU mutant strains was purified, and biochemical analyses showed that Cys-153 of CpcB carried a PCB chromophore but Cys-82 did not. These results show that both CpcS-I and CpcU are required for covalent attachment of PCB to Cys-82 of the PC beta subunit in this cyanobacterium. Suggesting that CpcS-I and CpcU are also required for attachment of PCB to allophycocyanin subunits in vivo, allophycocyanin levels were significantly reduced in all but the CpcV-less strain. These conclusions have been validated by in vitro experiments described in the accompanying report (Saunée, N. A., Williams, S. R., Bryant, D. A., and Schluchter, W. M. (2008) J. Biol. Chem. 283, 7513-7522). We conclude that the maturation of PBP in vivo depends on three PCB lyases: CpcE-CpcF, CpcS-I-CpcU, and CpcT.

Highlights

IntroductionMARCH 21, 2008 VOLUME 283 NUMBER 12 energy to photosynthetic reaction centers (predominantly photosystem II) in cyanobacteria and red algae [1,2,3]
MARCH 21, 2008 VOLUME 283 NUMBER 12 energy to photosynthetic reaction centers in cyanobacteria and red algae [1,2,3]
Comparative Bioinformatics Analysis—Paralogs of the CpeS and CpeT proteins of F. diplosiphon are found among the core set of proteins common to all cyanobacteria that produce PBSs, and most interestingly, paralogs occur in strains that do not synthesize PE (Fig. 1 and supplemental Fig. S3)

Summary

Introduction

MARCH 21, 2008 VOLUME 283 NUMBER 12 energy to photosynthetic reaction centers (predominantly photosystem II) in cyanobacteria and red algae [1,2,3]. CpcS-I and CpcU: a Cys-81/82 Phycobiliprotein Lyase strains that do not synthesize PE revealed paralogs, whose products displayed very high sequence similarity to CpeS and CpeT [23]. Paralogs of cpeS and cpeT were present in all PBPcontaining cyanobacteria, were absent in organisms that do not synthesize PBP, and were encoded within operons with other PBP-related genes. PCC 7002 with sequence similarity to CpeS were named CpcS-I, CpcU, and CpcV, and the single protein with similarity to CpeT was named CpcT [23, 24]. In this report we describe the characterization of single, double, and triple mutants of the cpcS-I, cpcU, cpcV, and cpcT genes of Synechococcus sp. The companion report [27] describes in vitro biochemical studies of the CpcS-I and CpcU proteins that validate these conclusions

Results

Discussion

Conclusion