Deletion of Synechocystis sp. PCC 6803 Leader Peptidase LepB1 Affects Photosynthetic Complexes and Respiration

Lifang Zhang,Tiago Toscano Selão,Tatiana Pisareva,Jingru Qian,Siu Kwan Sze,Inger Carlberg,Birgitta Norling

doi:10.1074/mcp.m112.022145

Lifang Zhang, Tiago Toscano Selão + Show 5 more

Open Access

https://doi.org/10.1074/mcp.m112.022145

Copy DOI

Abstract

The cyanobacterium Synechocystis sp. PCC 6803 possesses two leader peptidases, LepB1 (Sll0716) and LepB2 (Slr1377), responsible for the processing of signal peptide-containing proteins. Deletion of the gene for LepB1 results in an inability to grow photoautotrophically and an extreme light sensitivity. Here we show, using a combination of Blue Native/SDS-PAGE, Western blotting and iTRAQ analysis, that lack of LepB1 strongly affects the cell's ability to accumulate wild-type levels of both photosystem I (PSI) and cytochrome (Cyt) b6f complexes. The impaired assembly of PSI and Cyt b6f is considered to be caused by the no or slow processing of the integral subunits PsaF and Cyt f respectively. In particular, PsaF, one of the PSI subunits, was found incorporated into PSI in its unprocessed form, which could influence the assembly and/or stability of PSI. In contrast to these results, we found the amount of assembled photosystem II (PSII) unchanged, despite a slower processing of PsbO. Thus, imbalance in the ratios of PSI and Cyt b6f to photosystem II leads to an imbalanced photosynthetic electron flow up- and down-stream of the plastoquinone pool, resulting in the observed light sensitivity of the mutant. We conclude that LepB1 is the natural leader peptidase for PsaF, PsbO, and Cyt f. The maturation of PsbO and Cyt f can be partially performed by LepB2, whereas PsaF processing is completely dependent on LepB1. iTRAQ analysis also revealed a number of indirect effects accompanying the mutation, primarily a strong induction of the CydAB oxidase as well as a significant decrease in phycobiliproteins and chlorophyll/heme biosynthesis enzymes.

Highlights

Cyanobacteria comprise a diverse group of photoautotrophic prokaryotes with an oxygen evolving photosynthetic apparatus very similar to that of higher plants
cytochrome b6f (Cyt b6f) Complexes but not photosystem II (PSII)—To gain insight into the consequences of the LepB1 deletion for the organization of the photosynthetic apparatus, total membranes from WT and ⌬LepB1 strains were analyzed by BN-PAGE (Fig. 1), which has been shown to be a powerful tool in the analysis of membrane protein complexes in cyanobacteria [36]
The LepB1 mutant is highly prone to photoinhibition unless DCMU, an inhibitor of PSII is present indicating that the electron flow from PSII is the cause for the observed photodamage

Summary

EXPERIMENTAL PROCEDURES

User defined parameters were as follows: [1] Sample Type, iTRAQ 4-plex (Peptide Labeled); [2] Cysteine alkylation, MMTS; [3] Digestion, Trypsin; [4] Instrument, QSTAR ESI; [5] Special factors, None; [6] Species, None; [7] Specify Processing, Quantitate; [8] ID Focus, biological modifications, amino acid substitutions; [9] Database, the Synechocystis PCC 6803 protein sequence database (from 2004) was downloaded from Cyanobase (http://genome.kazusa.or.jp/cyanobase/Synechocystis/genes.faa), which includes 3672 sequences and 1143081 residues; [10] Search effort, thorough; [11] MS and MS/MS mass tolerance, 0.2 Da. Protein confidence was set at 95% (equivalent to Unused ProtScore of 1.3). All individual experiments were repeated three times under the same conditions

RESULTS

Uroporphyrinogen decarboxylase

TABLE II

Rate of respiration

DISCUSSION