ABSTRACT
The effect of carbonyl-cyanide m-chlorophenyl-hydrazone (CCCP) on intracytoplasmic membrane (ICM) assembly was examined in the purple bacterium Rhodobacter sphaeroides. CCCP blocks generation of the electrochemical proton gradient required for integral membrane protein insertion. ICM formation was induced for 8h, followed by a 4-h exposure to CCCP. Measurements of fluorescence induction/relaxation kinetics showed that CCCP caused a diminished quantum yield, a cessation in expansion of the functional absorption cross-section and a 4- to 10-fold slowing in the electron transfer turnover rate. ICM vesicles (chromatophores) and an upper-pigmented band (UPB) containing ICM growth initiation sites, were isolated and subjected to clear-native electrophoresis. Proteomic analysis of the chromatophore gel bands indicated that CCCP produced a 2.7-fold reduction in spectral counts in the preferentially assembled light-harvesting 2 (LH2) antenna, while the RC-LH1 complex, F1FO-ATPase and pyridine nucleotide transhydrogenase decreased by 1.7-1.9-fold. For 35 soluble enzymes, the ratio of 0.99 for treated/control proteins demonstrated that protein synthesis was unaffected by CCCP, suggesting that the membrane complex decline arose from the turnover of unassembled apoproteins. In the UPB fraction, an ~2-fold accumulation was observed for the preprotein translocase SecY, the SecA translocation ATPase, SecD and SecF insertion components, and chaperonins DnaJ and DnaK, consistent with the possibility that these factors, which act early in the assembly process, have accumulated in association with nascent polypeptides as stabilized assembly intermediates.
ABSTRACT
The results of a detailed structural and functional proteomic analysis of intracytoplasmic membrane (ICM) assembly in the model purple phototrophic bacterium Rhodobacter sphaeroides are reviewed in this report. Proteomics approaches have focused upon identification of membrane proteins temporally expressed during ICM development and spatially localized within the internal cell membranes, together with their structural and functional correlates. For the examination of temporal protein expression, procedures were established for the induction of ICM formation at low oxygen tension and for ICM remodeling in cells adapting to low intensity illumination, which permitted isolation by rate-zone sedimentation of ICM growth initiation sites (CM invaginations) in an upper-pigmented band (UPB), together with more mature ICM vesicles (chromatophores) as the main band. Nondenaturing clear native gel electrophoresis of the chromatophore fraction gave rise to four pigmented bands: the top and bottom bands contained the reaction center-light-harvesting 1 (RC-LH1) core complex and the LH2 peripheral antenna, respectively, while two bands of intermediate migration exhibited distinct associations of LH2 and core complexes. Proteomic analysis of the gel bands revealed developmental changes including increasing levels of LH2 polypeptides relative to those of core complexes as ICM development proceeded, as well as a large array of other associated proteins including high spectral counts for the F1FO-ATP synthase subunits, and the cytochrome bc1 complex. High counts were also observed for RSP6124, a protein of unknown function, that were correlated with increasing LH2 levels. RC-LH1-containing clear native electrophoresis gel bands from the UPB were enriched in cytoplasmic membrane (CM) markers, including electron transfer and transport proteins, as well as general membrane assembly factors (viz., preprotein translocases YidC, YajC and SecY, bacterial type 1 signal peptidase and twin arg translocation subunit TatA), thereby confirming the origin of the UPB from both peripheral respiratory membrane and sites of active CM invagination in which preferential assembly of the RC-LH1 complex occurs. Functional aspects of the photosynthetic unit assembly process were monitored by fluorescence induction/relaxation measurements of the variable fluorescence arising from LH-bacteriochlorophyll a. Slowing of the rate of RC electron transfer turnover (τQA), as assessed from the relaxation phase, was correlated with the growth of the functional absorption cross section (σ) and LH2/LH1 molar ratios. This is thought to arise from the imposition of constraints upon free diffusion of ubiquinone (UQ) redox species between the RC and cytochrome bc1 complex as the ICM bilayer becomes densely packed with LH2 rings. Such LH2 packing was confirmed in a comparison by high-resolution atomic force microscopy of ICM patches from cells grown at high and low light intensity [Adams and Hunter: Biochim Biophys Acta 2012;1817:1616-1627], in which the increasing LH2 levels form densely packed LH2-only domains, representing the light-responsive antenna complement arising under low illumination. In contrast, LH2 is initially dispersed in rows and small cluster-separating linear arrays of largely dimeric RC-LH1 core complexes, which become filled with LH2 during acclimation to reduced light intensity. In phototrophically grown cells that were transferred to oxic conditions in the dark, fluorescence induction/relaxation measurements showed that despite a growth burst independent of photosynthetic pathways, functional photosynthetic units were maintained for up to 24 h after the transition. The τQA was accelerated from â¼1 to 0.5 ms by 8 h, reflecting the decrease in LH2 levels, facilitating more rapid UQ redox species diffusion in the membrane bilayer as crowding by LH2 is overcome. Under these circumstances, UPB levels were elevated with significant increases in LH1/LH2 molar ratio. These changes indicate that vesiculation of CM growth initiation sites to form vesicular ICM was arrested under oxic conditions.
