5/27/2024 - Open Access

Strong interaction of CpcL with photosystem I cores induced inheterocysts of Anabaena sp. PCC 7120
Takehiro Suzuki1, Haruya Ogawa2, Naoshi Dohmae1, Jian-Ren Shen2, Shigeki Ehira3, Ryo Nagao4§
1Biomolecular Characterization Unit, RIKEN Center for Sustainable Resource Science, Saitama 351-0198, Japan
2Research Institute for Interdisciplinary Science and Graduate School of Natural Science and Technology, Okayama
University, Okayama 700-8530, Japan
3Department of Biological Sciences, Graduate School of Science, Tokyo Metropolitan University, Tokyo 192-0397, Japan
4Faculty of Agriculture, Shizuoka University, Shizuoka 422-8529, Japan§To whom correspondence should be addressed: nagry@shizuoka.ac.jp

Abstract
Phycobilisomes (PBSs) are photosynthetic light-harvesting antennae and appear to be loosely bound to photosystem I (PSI).
We previously found unique protein bands in each PSI fraction in heterocysts of Anabaena sp. PCC 7120 by two-dimensional
blue native/SDS-PAGE; however, the protein bands have not been identified. Here we analyzed the protein bands by mass
spectrometry, which were identified as CpcL, one of the components in PBSs. As different composition and organization ofAnabaena PSI-PBS supercomplexes were observed, the expression and binding properties of PBSs including CpcL to PSIs in
this cyanobacterium may be diversified in response to its living environments.

5/27/2024 - Open AccessFigure 1. Identification of CpcL in the PSI tetramer and dimer fractions from heterocysts of Anabaena sp. PCC 7120:(A) Two-dimensional BN/SDS-PAGE of the heterocyst thylakoids. T, PSI tetramer; D, PSI dimer; M, PSI monomer. Protein
bands labeled as red arrows were analyzed by mass spectrometry. Bands labeled as purple and green arrows can be assigned toATP synthase and PBS proteins, respectively, according to Chang et al. (2015) and Srivastava et al. (2021). This figure wasmodified from Nagao et al. (2022), Copyright Elsevier. (B) Amino acid sequence of CpcL. Red and blue underlines indicate Nand C-terminal polypeptides, respectively, of CpcL detected by MS/MS analyses (C–F). (C, D) MS/MS spectra ofALPLLEYKPTTQNQR (m/z = 591.3284; z = 3+; MASCOT score = 56; Expect value = 6.2e−6) (C) andMSNWNNSANQNYDGVAILGVLLAISAGMTFLFVLNWLGISSSF (m/z = 1163.5813; z = 4+; MASCOT score = 70;
Expect value = 2.6e−7) (D) within the band labeled as a red arrow in the PSI tetramer fraction in panel A. (E, F) MS/MS
spectra of ALPLLEYKPTTQNQR (m/z = 591.3283; z = 3+; MASCOT score = 64; Expect value = 1.0e−6) (E) andMSNWNNSANQNYDGVAILGVLLAISAGMTFLFVLNWLGISSSF (m/z = 1163.5829; z = 4+; MASCOT score = 76;
Expect value = 7.8e−8) (F) within the band labeled as a red arrow in the PSI dimer fraction in panel A.

Description

5/27/2024 - Open Access
Light-harvesting antennae have been diversified in photosynthetic organisms (Blankenship 2021) and can be grouped into
membrane proteins and water-soluble proteins. Phycobilisomes (PBSs) are water-soluble pigment-protein complexes andconsist of linker proteins and pigment-binding phycobiliproteins (MacColl 1998; Adir 2005; Watanabe and Ikeuchi 2013; Rastet al. 2019; Adir et al. 2020). PBSs are primarily bound to photosystem II of cyanobacteria and red algae as revealed by cryoelectron microscopy (Rast et al. 2019; You et al. 2023). However, it is unclear how PBSs interact with photosystem I (PSI) tocatalyze light-induced electron transfer from plastocyanin or cytochrome c6 to ferredoxin (Golbeck 1992; Brettel and Leibl
2001; Fromme et al. 2001; Nelson and Junge 2015).
Watanabe et al. purified PSI-tetramer-PBS supercomplexes from the cyanobacterium Anabaena sp. PCC 7120 (hereafterreferred to as Anabaena) grown under normal conditions (Watanabe et al. 2014), whereas we have purified PSI-dimer-PBS
and PSI-monomer-PBS supercomplexes from Anabaena grown under iron-deficient conditions (Shimizu et al. 2023). The twostudies detected CpcL in the supercomplexes, which is one of the linker proteins in PBSs and was denoted by Watanabe et al.
(2014). CpcL, previously known as CpcG3 in Anabaena, is one of the rod-core linker proteins (Bryant et al. 1991). It contains
an N-terminal linker domain and a distinctive hydrophobic C-terminal domain (Kondo et al. 2007; Watanabe et al. 2014;
Hirose et al. 2017; Guo et al. 2024). In contrast, CpcL was also called CpcG2 in the cyanobacterium Synechocystis sp. PCC
6803 and its characteristic protein structure has been determined by cryo-electron microscopy (Zheng et al. 2023).
It is known that CpcL of Anabaena was highly expressed in heterocysts (Watanabe et al. 2014), which are formed under
nitrogen-starvation conditions (Wolk et al. 1994; Magnuson and Cardona 2016). We recently showed that by two-dimensional
blue-native (BN)/SDS-PAGE, an apparent protein band in a molecular-weight range of 20.1–29.0 kDa was observed in the PSI
fractions obtained from the heterocyst thylakoids of Anabaena, but this band was not observed in any PSI fractions from thevegetative thylakoids of Anabaena (Nagao et al. 2022). The protein band is presumed to be CpcL based on its apparent
molecular weight; however, it has not yet been definitively identified.
The bands presumed to be CpcL in the two-dimensional BN/SDS-PAGE of the PSI tetramer and dimer fractions from theheterocyst thylakoids (labeled as red arrows in Figure 1A) were analyzed by mass spectrometry. The results showed that theN- and C-terminal polypeptides of CpcL can be detected clearly in both PSI tetramer and dimer fractions (Figure 1B–F).
Because CpcL was hardly observed in any PSI fractions by two-dimensional BN/SDS-PAGE of vegetative thylakoids from
Anabaena (Nagao et al. 2021; Nagao et al. 2022), strong interactions of CpcL with PSI cores appear to be induced inheterocysts.
This study demonstrated that CpcL is tightly bound to at least the PSI tetramer and dimer in the Anabaena heterocysts, and it
was hardly observed in the PSI monomer fraction (Figure 1A). Under iron-deficient growth conditions of Anabaena, CpcL
was detected from the PSI dimer and monomer fractions but not the tetramer (Shimizu et al. 2023). Thus, the association ofCpcL with PSI oligomers may be changed under different conditions; e.g., iron-deficiency and nitrogen-starvation haveinduced a different association pattern of CpcL with PSI oligomers. It is interesting to note that any PBS components including
CpcL were hardly detected in the PSI fractions from the vegetative thylakoids of Anabaena by two-dimensional BN/SDSPAGE (Watanabe et al. 2011; Nagao et al. 2021; Nagao et al. 2022), although the PSI tetramer cores together with CpcL werefractionated from the vegetative thylakoids by a milder method of sucrose density gradient centrifugation (Watanabe et al.
2014). These observations suggest that the tight association of CpcL with PSI cores occurs in Anabaena grown under stressconditions, such as iron deficiency and nitrogen starvation. Thus, the expression and binding properties of PBSs including
CpcL to PSIs in Anabaena may be diversified in response to its living environments.

Methods
Heterocysts and their thylakoids were prepared as described previously (Nagao et al. 2022). Two-dimensional BN/SDS-PAGE
was carried out as described previously (Nagao et al. 2021; Nagao et al. 2022). Mass spectrometry was performed according toNagao et al. (2019) with some modifications. Briefly, the gel slices were digested with trypsin (TPCK-treated; Worthington
Biochemical), and the resultant peptides were analyzed using LC-MS/MS consisting of Easy nLC 1000 and Q Exactive(Thermo Fisher Scientific). The acquired data was searched against the database of Anabaena sp. PCC 7120 with Mascot
Version 2.8 software (Matrix Science).

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Funding:This work was supported by JSPS KAKENHI grant Nos. JP22H04916 (J.-R.S.), JP22K19138 (S.E.), and JP23H02423 (R.N.).
Author Contributions: Takehiro Suzuki: data curation. Haruya Ogawa: investigation. Naoshi Dohmae: data curation. JianRen Shen: funding acquisition, resources, writing - original draft. Shigeki Ehira: investigation, funding acquisition. RyoNagao: funding acquisition, writing - original draft, writing - review editing, conceptualization, investigation.
Reviewed By: AnonymousHistory: Received March 15, 2024 Revision Received May 16, 2024 Accepted May 23, 2024 Published Online May 27,2024 Indexed June 10, 2024
Copyright: © 2024 by the authors. This is an open-access article distributed under the terms of the Creative Commons
Attribution 4.0 International (CC BY 4.0) License, which permits unrestricted use, distribution, and reproduction in any
medium, provided the original author and source are credited.
Citation: Suzuki, T; Ogawa, H; Dohmae, N; Shen, JR; Ehira, S; Nagao, R (2024). Strong interaction of CpcL withphotosystem I cores induced in heterocysts of Anabaena sp. PCC 7120. microPublication Biology.
10.17912/micropub.biology.001183