Proteomic Analysis of the Developing Intracytoplasmic Membrane in Rhodobacter sphaeroides During Adaptation to Low Light Intensity

Abstract

Although the primary photochemical events in the facultative photoheterotrophic purple bacterium Rhodobacter sphaeroides are now well understood, comparatively little is known about how their photosynthetic apparatus is assembled. Here we present a proteomic analysis of the intracytoplasmic membrane (ICM) assembly process during adaptation to lowered light intensity, in which the size of the photosynthetic units is greatly expanded by addition of the light-harvesting 2 (LH2) peripheral antenna complex. When the isolated ICM-derived chromatophore vesicles were subjected to clear native gel electrophoresis (CNE), four pigmented bands appeared; the top and bottom bands contained the reaction center - light-harvesting 1 (RC-LH1) core complex and LH2 peripheral antenna, respectively, while the two bands of intermediate migration contained associations of the LH2 and core complexes. Proteomic analysis revealed a large array of other proteins associated with the CNE gel bands - in particular, several F(1)F(O)-ATP synthase subunits gave unexpectedly high spectral counts, given the inability to detect this coupling factor, as well as the more abundant cytochrome bc (1) complex, by atomic force microscopy. Significant levels of general membrane assembly factors were also found, as well as numerous proteins of unknown function including high counts for RSP6124 that were correlated with LH2 levels. When combined with further AFM and spectroscopic studies, these proteomic approaches are expected to provide a much-improved understanding of the overall assembly process.

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

“…2 f), which in addition demonstrate an essentially complete separation of LH2 from RC-LH1 cores in the respective bottom and top CNE band lanes. The intermediate complexes are thought to represent detergent-protein micelles arising from ICM regions seen in AFM where the LH2 and LH1 complexes are associated [Woronowicz and Niederman, 2010]. From their apparent aggregation states as estimated from electrophoretic migration behavior in CNE, the lower intermediate band was suggested to consist of associations of monomeric LH2 and RC-LH1 core complexes, with the upper one representing a core monomer associated with an LH2 dimer [Woronowicz et al, 2012].…”

“…2 f) were identified tentatively by Woronowicz and Niederman [2010]. These included the α-, β-and γ-polypeptides of the F 1 -ATPase (55.2, 50.4 and 31.2 kDa, respectively), well resolved in LH2-enriched CNE bands, cytochrome b (50.1 kDa), migrating to the same position as in Western blots [Sturgis et al, 2009], and the hypothetical protein RSP6124 (11.4 kDa), that was prominent in 3-and 24-hour bottom bands and correlated with the increasing LH2 levels.…”

“…This means that proteins that are not highly abundant with a large number of sites would give higher counts than more abundant proteins with far fewer sites; in extreme cases where no sites are available, no counts are found in the resulting pivot table and it is necessary to use other proteases such as chymotrypsin. Nevertheless, valid comparisons can be made between the same components in different gel bands and the designated clusters of orthologous groups into which they are classified [Woronowicz and Niederman, 2010]. By loading equal amounts of protein from membrane preparations taken at different time points it is possible to determine the changing levels of individual protein components and to make comparisons between the UPB and chromatophore bands.…”

“…Owing to the unique combination of numerous metabolic capabilities and accessible molecular genetics with an intracytoplasmic membrane (ICM) that is amenable to an unparalleled variety of biochemical, spectroscopic and ultrastructural probes, the purple facultatively photoheterotrophic bacterium Rhodobacter sphaeroides has provided an ideal experimental system for examining the biogenesis and assembly of energy-transducing membranes [Drews and Niederman, 2002;Hunter et al, 2005;Koblízek et al, 2005;Woronowicz et al, 2012;Woronowicz and Niederman, 2010]. When grown under anoxic conditions in the light, an ICM is formed which houses photosynthetic units that are seen in atomic force microscopy (AFM) to largely consist of arrays of dimeric reaction center-light-harvesting 1 (RC-LH1) core structures, interspersed with narrow lanes of the peripheral LH2 antenna complex [Bahatyrova et al, 2004].…”

“…In an effort to obtain a more complete understanding of the ICM assembly process we have initiated a detailed analysis of the relevant R. sphaeroides proteome [Woronowicz et al, 2011a[Woronowicz et al, , 2012Woronowicz and Niederman, 2010]. R. sphaeroides provides a unique system for such a structural and functional proteomic analyses insofar as the assembled proteins can be temporally expressed and spatially localized within the internal structure of the cell that can be assessed structurally at submolecular resolution by AFM and functionally characterized by informative fluorescence techniques.…”