N-Proximal Sequence Motif in Light-Harvesting Chlorophyll a/b-Binding Protein Is Essential for the Trimerization of Light-Harvesting Chlorophyll a/b Complex

Abstract

The major light-harvesting complex (LHCII) of photosystem I1 can be reconstituted in its native, trimeric form starting from its apoprotein light-harvesting chlorophyll alb-binding protein (LHCP), pigments, and thylakoid lipids. In this paper we identify segments in the LHCP polypeptide that are essential for the formation of stable LHCII trimers by analyzing N-and C-terminal deletion mutants of LHCP and mutants carrying point-specific amino acid exchanges. C-Terminal deletions that do not abolish pigment binding to LHCP do not affect trimerization either. By contrast, on the N-terminus of LHCP, where as many as 61 amino acids can be deleted without significant effects on pigment binding, only 15 amino acids are dispensible for LHCII trimer formation . This indicates that structural elements between amino acids 16 and 61 are involved in the stabilization of LHCII trimers but not monomers. Closer inspection of this protein domain in a more detailed mutation analysis revealed that amino acids W16 and/or Y17 as well as R21 are essential for the formation of LHCII trimers. These amino acids are conserved in virtually all known sequences of LHCII apoproteins but only in some of the minor chlorophyll alb complexes. Possible functions of the crucial residues are discussed.In the photosynthetic apparatus in higher plants, both photosystem I and photosystem I1 (PSI and PSII, respectively)' contain a number of peripheral, chlorophyll aib-

A comparison of the three isoforms of the light-harvesting complex II using transient absorption and time-resolved fluorescence measurements

“…However, Caffarri et al did not report significant differences in the Chl b content of the three isoforms. In addition, there is also some discrepancy in the reported Chl a/b ratio for the three reconstituted isoforms, which is closer to the value of 1.33 observed in the new crystal structures of LHC II (Liu et al 2004;Standfuss et al 2005) in the samples prepared by Caffarri et al The protein residues which are needed for trimerization of LHC II complexes (Hobe et al 1995) are conserved in all the isoforms, but in contrast to Lhcb1 and Lhcb2, Lhcb3 alone does not form stable homotrimers in vitro (Standfuss and Ku¨hlbrandt 2004;Caffarri et al 2004). Furthermore, the N terminus of Lhcb3 is 10 residues shorter and lacks the phosphorylation site.…”

Plants lacking the main light-harvesting complex retain photosystem II macro-organization

“…After pelleting and washing in 0.3 M sorbitol, 20 mM Tricine NaOH, pH 7.6, 5 mM MgCl 2 , chloroplasts were osmotically shocked for 30 s with 5 mM MgCl 2 , pH 7.6. Pelleted thylakoids were incubated in stacking medium (5 mM MgCl 2 ,15mMN aCland2mM MES, pH 6.3) for 1 h, after which we treated them with 2.5% Triton X-100 at a chlorophyll concentration of 3 mg ml 21 . The PSII membranes were sedimented at 30,000g for 30 min, resuspended in buffer containing 20 mM Bis-Tris (pH 6.5) and 5 mM MgCl 2 ,and solubilized with 0.4% n-dodecyl-a-D-maltoside at a chlorophyll concentration of 1.4 mg ml 21 .…”

“…17), greater than 40%, as compared with roughly 30% or less for the other Lhc genes. Lhcb5 is the only gene that clusters with the Lhcb1-Lhcb3 genes 17 .I n addition, a WYXXXR motif near the amino terminus of Lhcb1, which is necessary for LHCII trimerization 21 , is conserved in Lhcb5, the apoprotein of CP26, but not in the apoproteins of CP29 and CP24. But another LHCII trimerization domain near the carboxy terminus (Trp 222) 22 is not found in Lhcb5, which could explain the reduced efficiency of trimerization for CP26.…”

<em>In Vitro</em> Reconstitution of Light-harvesting Complexes of Plants and Green Algae

“…The importance of different protein domains on the stability and folding of the complexes, or their involvement in the protein-protein interactions, have been determined by truncating the apoprotein or performing random mutagenesis 8,[41][42][43][44] . Single amino acid residues important for the coordination of different pigments can be altered through site-directed mutagenesis in order to analyze the properties of individual pigments or assess their contribution to the function and stability of the complex 10,28,29,[45][46][47][48][49][50][51][52] .…”