Quantitative proteomic analysis of intracytoplasmic membrane development in Rhodobacter sphaeroides

Abstract

SummaryThe purple phototrophic bacteria elaborate a specialized intracytoplasmic membrane (ICM) system for the conversion of solar energy to ATP. Previous radiolabelling and ultrastructural experiments have shown that ICM assembly in Rhodobacter sphaeroides is initiated at indentations of the cytoplasmic membrane, termed UPB. Here, we report proteomic analyses of precursor (UPB) and mature (ICM) fractions. Qualitative data identified 387 proteins, only 43 of which were found in the ICM, reflecting its specialized role within the cell, the conversion of light into chemical energy; 236 proteins were found in the significantly more complex UPB proteome. Metabolic labelling was used to quantify the relative distribution of 173 proteins between the UPB and ICM fractions. Quantification reveals new information on assembly of the RC-LH1-PufX, ATP synthase and NAD(P)H transhydrogenase complexes, as well as showing that the UPB is enriched in enzymes for lipid, carbohydrate and amino acid metabolism, tetrapyrrole biosynthesis and proteins representing a wide range of other metabolic and biosynthetic functions. Proteins involved in light harvesting, photochemistry, electron transport and ATP synthesis are all enriched in ICM, consistent with the spatial proximity of energy capturing and transducing functions. These data provide further support to the developmental precursor-product relationship between UPB and ICM.

Effects of the protonophore carbonyl-cyanide m-chlorophenylhydrazone on intracytoplasmic membrane assembly in Rhodobacter sphaeroides

“…sphaeroides membranes has recently been reported [37] in which 43 unique proteins were identified in the highly specialized chromatophore membrane as compared to 236 making up the more complex membrane components isolated in the UPB fraction. A meatabolically stable isotope labeling procedure permitted determination of the relative distribution of proteins between the chromatophores and the UPB, revealing that the UPB is enriched in enzymes of lipid and tetrapyrrole biosynthesis.…”

Overall energy conversion efficiency of a photosynthetic vesicle

“…Early chromatophore models prior to (Cartron et al, 2014) account only for LH proteins, whereas in proteomics studies, hundreds of different types of non-LH peptides are actually identified, including ATP synthase, cytbc 1 , membrane assembly factors, as well as proteins of unknown function (Jackson et al, 2012;Woronowicz and Niederman, 2010). Most of these components are notably unresolved in AFM images.…”

“…sphaeroides the basic photosynthetic unit is the chromatophore (Cogdell et al, 2006;Strümpfer et al, 2011;Cartron et al, 2014), a 50-70 nm diameter vesicle, as shown in Figure 1, formed through invagination of the intracytoplasmic membrane (Tucker et al, 2010;Gubellini et al, 2007) and comprising over a hundred protein complexes (Jackson et al, 2012;Woronowicz and Niederman, 2010;Woronowicz et al, 2013). The proteins that constitute the chromatophore are primarily the light harvesting (LH) complexes, photosynthetic reaction centers (RCs), cytbc 1 complexes, and ATP synthases, which cooperate to harvest light energy for photophosphorylation.…”

“…The proteins that constitute the chromatophore are primarily the light harvesting (LH) complexes, photosynthetic reaction centers (RCs), cytbc 1 complexes, and ATP synthases, which cooperate to harvest light energy for photophosphorylation. The architecture of the chromatophore, reported in (Ş ener et al, 2007, 2010Cartron et al, 2014), has been determined by combining atomic force microscopy (AFM) (Bahatyrova et al, 2004;Olsen et al, 2008), cryo-electron microscopy (cryo-EM) (Qian et al, 2005;Cartron et al, 2014), crystallography (Koepke et al, 1996;McDermott et al, 1995;Papiz et al, 2003;Jamieson et al, 2002), optical spectroscopy Sener et al, 2010), mass spectroscopy (Cartron et al, 2014), and proteomics (Jackson et al, 2012;Woronowicz and Niederman, 2010;Woronowicz et al, 2013) data. The composition of the chromatophore depends on growth conditions such as light intensity (Adams and Hunter, 2012;Woronowicz et al, 2011bWoronowicz et al, , 2011a) and can also be influenced by mutations Hsin et al, 2010b).…”

Structural and Functional Proteomics of Intracytoplasmic Membrane Assembly in <b><i>Rhodobacter sphaeroides</i></b>

“…This may reflect different degradation rates for the LH2 and RC-LH1 complexes, arising from a preferential protection of the cores from proteolysis. Alternatively, LH1 may be formed from accumulated polypeptide pools identified in the quantitative proteomics studies of Jackson et al [2012], as well as chromophores that may be directed toward LH1 rather than LH2 assembly. Surprisingly, fluorescence induction/relaxation measurements revealed that considerable photosynthetic activities were retained after transfer to oxic conditions (online suppl.…”