Towards Functional Proteomics of Membrane Protein Complexes in Synechocystis sp. PCC 6803

Abstract

The composition and dynamics of membrane protein complexes were studied in the cyanobacterium Synechocystis sp. PCC 6803 by two-dimensional blue native/SDS-PAGE followed by matrix-assisted laser-desorption ionization time of flight mass spectrometry. Approximately 20 distinct membrane protein complexes could be resolved from photoautotrophically grown wild-type cells. Besides the protein complexes involved in linear photosynthetic electron flow and ATP synthesis (photosystem [PS] I, PSII, cytochrome b 6 f, and ATP synthase), four distinct complexes containing type I NAD(P)H dehydrogenase (NDH-1) subunits were identified, as well as several novel, still uncharacterized protein complexes. The dynamics of the protein complexes was studied by culturing the wild type and several mutant strains under various growth modes (photoautotrophic, mixotrophic, or photoheterotrophic) or in the presence of different concentrations of CO 2 , iron, or salt. The most distinct modulation observed in PSs occurred in iron-depleted conditions, which induced an accumulation of CP43Ј protein associated with PSI trimers. The NDH-1 complexes, on the other hand, responded readily to changes in the CO 2 concentration and the growth mode of the cells and represented an extremely dynamic group of membrane protein complexes. Our results give the first direct evidence, to our knowledge, that the NdhF3, NdhD3, and CupA proteins assemble together to form a small low CO 2 -induced protein complex and further demonstrate the presence of a fourth subunit, Sll1735, in this complex. The two bigger NDH-1 complexes contained a different set of NDH-1 polypeptides and are likely to function in respiratory and cyclic electron transfer. Pulse labeling experiments demonstrated the requirement of PSII activity for de novo synthesis of the NDH-1 complexes.Photosynthetic organisms experience continuous fluctuation in their growth environment, including changes in light conditions, temperature, and nutrient availability. Cyanobacteria, the probable progenitors of chloroplasts, possess a remarkable capacity to adapt to a wide range of environmental conditions. Cyanobacteria are primarily photoautotrophic, but some strains, such as Synechocystis sp. PCC 6803, can also grow mixotrophically or photoheterotrophically in the presence of external Glc. This, together with their flexibility to genetic manipulation and simple nutrient requirements for growth, has rendered cyanobacteria into a favored model in deciphering the structurefunction relationships and acclimation strategies of the photosynthetic machinery.Energy-transducing thylakoid membranes are the targets for modification by a number of environmental variables. Light, the most important environmental factor for photosynthesis, induces a well-defined acclimation response, including changes in the amount and ratio of the two PSs (Fujita, 1997;Hihara, 1999) and in the abundance and composition of the light-harvesting phycobilisome antenna (Grossman et al., 1993(Grossman et al., , 2001. Similar responses in th...

Role of Type 2 NAD(P)H Dehydrogenase NdbC in Redox Regulation of Carbon Allocation in Synechocystis

“…In contrast, marked up-regulation was observed for NdhD2 (Table III), the specific subunit of NDH-1 2 . The latter complex is present in Synechocystis cells in much smaller amounts than NDH-1 1 (Herranen et al, 2004), but, despite a low abundance, NDH-1 2 that accumulated in the DndbC mutant contributed to the significant increase in CET (Fig. 4, C and D).…”

Operon flv4-flv2 Provides Cyanobacterial Photosystem II with Flexibility of Electron Transfer

“…Protein complexes in the membrane and soluble fractions were analyzed by BN-PAGE, which was performed as described by Herranen et al (2004) with minor modifications. The membrane samples were washed with 50 mM BisTris, pH 7.0, 330 mM sorbitol, and 1 mM pefabloc, pelleted by centrifugation at 18,000g for 10 min, and resuspended in 25 mM BisTris, pH 7.0, 20% glycerol (w/v), 1 mM pefabloc, and 10 mM MgCl 2 to a protein concentration 15 mg/mL.…”

Proteomic analyses of the response of cyanobacteria to different stress conditions

“…Synechocystis is a moderately halotolerant cyanobacterium and has actually become a model system for investigating the environmental stress responses of photosynthetic organisms. Most of these studies are mainly focused on the soluble protein fraction, but some others deal with the membrane protein fraction [2,3].…”