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

Cyanobacteria comprise a diverse group of photoautotrophic prokaryotes with an oxygen evolving photosynthetic apparatus very similar to that of higher plants. Similarly to plant chloroplasts, they contain three different types of membranes, an outer membrane, a plasma membrane (PM), and a thylakoid membrane (TM). The thylakoid membrane is the site not only for photosynthesis but also the main site for respiration. The ultrastructure and organization of the membranes is however still under debate and mainly two different opinions prevail concerning the organization of plasma and thylakoid membranes. One is that the two membranes are continuous, making the periplasm and lumen a common compartment, the second that the membranes are completely separated (1–9). The complex membrane organization within the cyanobacterium implies the existence of a sophisticated system for the sorting and transport of extracytoplasmic proteins to the different membranes and compartments. Targeting of these proteins in eubacteria and archaea occurs through the well-characterized Sec and Tat translocon pathways (10). Protein substrates for the Sec and Tat systems were shown to be distinguished by specific N-terminal signal sequences (11). However, most thylakoid and plasma membrane integral proteins do not have N-terminal signal peptides. For insertion into the membrane all TM and PM integral proteins require, in addition to the Sec translocon, a protein insertase of the Alb3/Oxa1 family, YidC (12–14), as shown for the core photosystem II (PSII) protein D1 (PsbA) (15, 16). The Synechocystis sp. PCC 6803 (hereafter referred to as Synechocystis) genome, as well as all other completely sequenced cyanobacterial genomes, contains only a single set of genes encoding the subunits of the Sec and Tat translocons (17–19). This suggests that the same translocation machineries are working in plasma and thylakoid membranes and this in turn indicates that information additional to the signal sequence is needed for correct targeting (20, 21). The question of where in the intricate Synechocystis membrane system translocation and protein sorting takes place is still subject for intense research. Sec and Tat signal peptides are both cleaved by Type I signal peptidases (also called leader peptidase, Lep) (11). In contrast to other Gram-negative bacteria, almost all the cyanobacterial genomes analyzed so far contain two lep genes. Although the overall sequence identity of leader peptidases from various species is relatively low, five conserved regions were identified in the catalytic domain, called boxes B, C, D, and E, as well as the membrane anchor domain A (22, 23). Both LepB1 and LepB2 in Synechocystis have the conserved A-E boxes (21, 23), including the invariant amino acid residues demonstrated to be essential for catalytic activity. No other proteins with these conserved regions are found in the Synechocystis genome. In subproteomic analyses LepB2 was found in the plasma membrane, whereas LepB1 was found in the thylakoid membrane (7, 24, 25). Both LepB1 and LepB2 are integral membrane proteins with one transmembrane helix, present in very low amounts in the membrane. The above observations may therefore not implicate completely unique membrane localizations for the two leader peptidases. Knockout mutations of the two genes, however, give significantly different results: whereas LepB2 appears to be totally essential for cell viability, cells with a deletion in LepB1 are still able to grow under heterotrophic conditions in dim light (26). The light sensitivity disappeared when 3-(3,4-dichlorophenyl)-1,1-dimetylurea (DCMU)1 was present. DCMU inhibits the reduction of the plastoquinone (PQ) pool by photosystem II (PSII) and this indicates that the primary cause for the photosensitivity observed is the impairment in electron flow downstream of PSII. The reported decrease in the amount of cytochrome f (Cyt f), a component of the cytochrome b6f (Cyt b6f) complex, further strengthens this hypothesis (26). In the present study, we have analyzed the LepB1 mutant strain by using two-dimensional Blue Native/SDS-PAGE (2D BN/SDS-PAGE), Western blotting with specific antibodies and isobaric tags for relative and absolute quantitation (iTRAQ), with special emphasis on the integrity of the photosynthetic apparatus, to reach a better understanding of the role of LepB1 in protein maturation in Synechocystis. Our studies show that LepB1 has irreplaceable substrate specificity, as in the case of PsaF, an integral membrane subunit of the photosystem I (PSI). The processing of other photosynthetic proteins (PsbO and Cyt f) is also affected although LepB2 is able to partly compensate the loss of LepB1. This defect in processing severely disrupts the ability to maintain proper organization of the thylakoid membrane complexes and also affects the cellular proteome in a far-reaching manner.