Proteomic characterization of the Rhodobacter sphaeroides 2.4.1 photosynthetic membrane: identification of new proteins

Abstract

The Rhodobacter sphaeroides intracytoplasmic membrane (ICM) is an inducible membrane that is dedicated to the major events of bacterial photosynthesis, including harvesting light energy, separating primary charges, and transporting electrons. In this study, multichromatographic methods coupled with Fourier transform ion cyclotron resonance mass spectrometry, combined with subcellular fractionation, was used to test the hypothesis that the photosynthetic membrane of R. sphaeroides 2.4.1 contains a significant number of heretofore unidentified proteins in addition to the integral membrane pigment-protein complexes, including light-harvesting complexes 1 and 2, the photochemical reaction center, and the cytochrome bc(1) complex described previously. Purified ICM vesicles are shown to be enriched in several abundant, newly identified membrane proteins, including a protein of unknown function (AffyChip designation RSP1760) and a possible alkane hydroxylase (RSP1467). When the genes encoding these proteins are mutated, specific photosynthetic phenotypes are noted, illustrating the potential new insights into solar energy utilization to be gained by this proteomic blueprint of the ICM. In addition, proteins necessary for other cellular functions, such as ATP synthesis, respiration, solute transport, protein translocation, and other physiological processes, were also identified to be in association with the ICM. This study is the first to provide a more global view of the protein composition of a photosynthetic membrane from any source. This protein blueprint also provides insights into potential mechanisms for the assembly of the pigment-protein complexes of the photosynthetic apparatus, the formation of the lipid bilayer that houses these integral membrane proteins, and the possible functional interactions of ICM proteins with activities that reside in domains outside this specialized bioenergetic membrane.

FIG. 1.

Proteomic profiles of 70 ICM- and IM-associated proteins identified in ICM vesicles of R. sphaeroides grown anaerobically in the light at 3 W/m². (A and B) Comparison of the numbers of AMT tags for the 70 ICM- and IM-associated proteins identified in the ICM with those identified in the IM of the photosynthetic cells and with those identified in the IM of aerobic cells, respectively. (C and D) Comparison of protein coverage percentages for 70 ICM- and IM-associated proteins identified in the ICM with those identified in the IM of photosynthetic cells and with those identified in the IM of aerobic cells, respectively. (E) Relative RNA abundance (microarray data) from genes corresponding to 70 ICM- and IM-associated proteins in the photosynthetic cells (http://www.rhodobacter.org) (24). The same number is used for each gene or its protein. 1 to 10, the ICM-unique proteins; 11 to 28, the ICM-localized proteins; and 29 to 70, the CM-enriched proteins. AE, aerobic cells; 3W, photosynthetic cells.

FIG. 2.

HPLC analysis of carotenoids extracted from 100-ml cultures of cells of R. sphaeroides 2.4.1 and mutant strains ΔRSP1760 and ΔRSP1467 according to the method described in Materials and Methods. The peak indicates an elution profile monitored at 486 nm, and the elution times for spheroidenone (SO) and spheroidene (SE) are 14.5 min and 15.5 min, respectively. Semi, semiaerobic.

FIG. 3.

Western blot analysis of His-tagged alkane hydroxylase (46.0 kDa; RSP1467) and an unknown protein (25 kDa; RSP1760) in IM and ICM of the cells of mutant strains ΔRSP1760(pR1760his) and ΔRSP1467(pR1467his) grown aerobically or anaerobically in the presence of light intensity at 3 W/m². Lane 1, protein standards; lane 2, RSP1760-His in aerobic cells; lane 3, RSP1760-His in anaerobic cells; lane 4, RSP1467-His in aerobic cells; lane 5, RSP1467-His in anaerobic cells; lane 6, RSP1760-His in the IM of anaerobic cells; lane 7, RSP1760-His in ICM of anaerobic cells; lane 8, RSP1467-His in the IM of anaerobic cells; lane 9, RSP1467-His in ICM of anaerobic cells.

FIG. 4.

Growth curves for R. sphaeroides 2.4.1 and mutant strains ΔRSP1467 and ΔRSP1467(pR1467his). Cells were grown anaerobically in the presence of light at 100 W/m².

FIG. 5.

SDS-PAGE analysis of ICM vesicles and IM (100 μg protein). Membranes were solubilized and separated on a 15% acrylamide gel before being stained with Coomassie blue. The arrows on the right of the gel lane represent the protein sizes predicted from molecular weight, c-type cyt staining, or partial purification of membrane complexes, and the band positions for Possible alkane hydroxylase (46.0 kDa; RSP1467) and an unknown protein (25 kDa; RSP1760) were detected by anti-histidine antibodies. The relative intensity of each band in ICM vesicles or IM is also indicated; the numbers on the left indicate the sizes of the protein standards.