skip to main content
OSTI.GOV title logo U.S. Department of Energy
Office of Scientific and Technical Information

Title: Structural and Functional Proteomic Analysis of a Developing Energy Transducing Membrane

Abstract

While much is known about the light reactions of photosynthesis in purple bacteria, comparatively little information is available on how the requisite integral membrane proteins are assembled, their patterns of cellular localization are established or their apoproteins cooperate with numerous assembly factors in their insertion into the growing intracytoplasmic membrane (ICM). This problem was approached through a detailed structural and functional proteomic analysis of ICM assembly process in the well-characterized purple bacterium Rhodobacter sphaeroides. Proteomic approaches have focused upon identification of membrane proteins temporally expressed during ICM development and spatially localized in both membrane growth initiation sites and in mature ICM vesicles. Protocols were established for ICM induction under reduced aeration and ICM remodeling in cells adapting to low intensity illumination, which permitted isolation, in sucrose density gradients, of ICM growth initiation sites as an upper pigmented band (UPB) and mature ICM vesicles as the main (chromatophore) band. Non-denaturing clear native gel electrophoresis (CNE) of these isolated membrane fractions gave rise to pigmented bands containing the peripheral light-harvesting 2 (LH2) antenna and the reaction center-light-harvesting 1 (RC-LH1) core complex, together with a full array of other ICM proteins, which were subjected to proteomic analysis. Proteomic analysis of the gel bandsmore » from chromatophores revealed developmental changes including increasing levels of the LH2 complex as ICM development proceeded, as well as a large array of other associated proteins including high spectral counts for the F1FO ATP synthase subunits, given the inability to detect this coupling factor, as well as the more abundant cytochrome bc1 complex by atomic force microscopy (AFM). Significant levels of general membrane assembly factors were encountered, as well as high counts for RSP6124, a protein of unknown function, correlated with increasing LH2 levels. RC-LH1-containing CNE 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 relative to chromatophore bands. This confirms the origin of the UPB from both peripheral respiratory membrane and sites of CM invagination. Significant levels of preprotein translocases YidC, YajC and SecY, bacterial type 1 signal peptidase and twin arg translocation subunit TatA were found. Such general membrane assembly factors were significantly enriched in the UPB RC-LH1 gel bands, confirming the active role of membrane invagination sites in pigment-protein complex assembly. Functional correlates of proteomics approaches were provided by near-IR fluorescence induction/relaxation transients arising from LH-BChl components. A linear relation was found between increasing functional absorption cross-section and slowing of RC electron transfer turnover rate, thought to arise from the imposition of constraints upon free UQ diffusion between the RC and cytochrome bc1 complex as the membrane became saturated with new LH2 rings. In cells undergoing ICM induction in which generation of the electrochemical proton gradient was uncoupled with CCCP, blockage in membrane insertion of the LH and RC polypetides was demonstrated. This was reflected in a diminution of quantum yield of the primary charge separation, a cessation in expansion of functional absorption cross-section and a >4-fold slowing in RC electron transfer turnover. The ICM insertion of ATP synthase and transhydrogenase was also significantly diminished. Importantly, for the UPB fraction, CCCP treatment resulted in accumulation of ~2-fold greater levels of the preprotein translocase SecY, the SecA translocation ATPase, Sec D and SecF insertion components, and chaperonins DnaJ and DnaK, suggesting that these general membrane assembly factors had accumulated in association with nascent LH and RC assembly intermediates. In addition to spectrally homogeneous bands containing LH2 and RC-LH1 complexes, CNE also gave rise to two bands of intermediate migration, an upper LH2 and a lower LH1-enriched band, which has facilitated in vivo assessment of LH2:LH1 associations existing in the membrane. For chromatophores from cells adapting to low light intensity, the LH2 CNE gel band was enriched in LH2 polypeptides encoded by the puc1 light-responsive operon, suggesting that these LH2 complexes arise from LH2-only domains formed when LH2-LH1 associations are complete [7]. In contrast, high levels of LH2 polypeptides encoded by the puc2 operon and the absence of alterations in Puc polypeptide distribution in the upper intermediate LH2-LH1 gel band indicate that it arises from ICM regions laid down early in the ICM remodeling process, in which LH2 is sandwiched between rows of aligned dimeric LH1-RC core complexes.« less

Authors:
Publication Date:
Research Org.:
Rutgers University/New Brunswick
Sponsoring Org.:
USDOE
OSTI Identifier:
1041400
Report Number(s):
DE-FG02-08ER15957
DOE Contract Number:  
FG02-08ER15957
Resource Type:
Technical Report
Country of Publication:
United States
Language:
English
Subject:
14 SOLAR ENERGY; Membrane assembly, Photosynthesis, Light harvesiting, Proteomics, Atomic force microscopy, Linear dichroism, Fluorescence

Citation Formats

Niederman, Robert A. Structural and Functional Proteomic Analysis of a Developing Energy Transducing Membrane. United States: N. p., 2012. Web. doi:10.2172/1041400.
Niederman, Robert A. Structural and Functional Proteomic Analysis of a Developing Energy Transducing Membrane. United States. https://doi.org/10.2172/1041400
Niederman, Robert A. Mon . "Structural and Functional Proteomic Analysis of a Developing Energy Transducing Membrane". United States. https://doi.org/10.2172/1041400. https://www.osti.gov/servlets/purl/1041400.
@article{osti_1041400,
title = {Structural and Functional Proteomic Analysis of a Developing Energy Transducing Membrane},
author = {Niederman, Robert A},
abstractNote = {While much is known about the light reactions of photosynthesis in purple bacteria, comparatively little information is available on how the requisite integral membrane proteins are assembled, their patterns of cellular localization are established or their apoproteins cooperate with numerous assembly factors in their insertion into the growing intracytoplasmic membrane (ICM). This problem was approached through a detailed structural and functional proteomic analysis of ICM assembly process in the well-characterized purple bacterium Rhodobacter sphaeroides. Proteomic approaches have focused upon identification of membrane proteins temporally expressed during ICM development and spatially localized in both membrane growth initiation sites and in mature ICM vesicles. Protocols were established for ICM induction under reduced aeration and ICM remodeling in cells adapting to low intensity illumination, which permitted isolation, in sucrose density gradients, of ICM growth initiation sites as an upper pigmented band (UPB) and mature ICM vesicles as the main (chromatophore) band. Non-denaturing clear native gel electrophoresis (CNE) of these isolated membrane fractions gave rise to pigmented bands containing the peripheral light-harvesting 2 (LH2) antenna and the reaction center-light-harvesting 1 (RC-LH1) core complex, together with a full array of other ICM proteins, which were subjected to proteomic analysis. Proteomic analysis of the gel bands from chromatophores revealed developmental changes including increasing levels of the LH2 complex as ICM development proceeded, as well as a large array of other associated proteins including high spectral counts for the F1FO ATP synthase subunits, given the inability to detect this coupling factor, as well as the more abundant cytochrome bc1 complex by atomic force microscopy (AFM). Significant levels of general membrane assembly factors were encountered, as well as high counts for RSP6124, a protein of unknown function, correlated with increasing LH2 levels. RC-LH1-containing CNE 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 relative to chromatophore bands. This confirms the origin of the UPB from both peripheral respiratory membrane and sites of CM invagination. Significant levels of preprotein translocases YidC, YajC and SecY, bacterial type 1 signal peptidase and twin arg translocation subunit TatA were found. Such general membrane assembly factors were significantly enriched in the UPB RC-LH1 gel bands, confirming the active role of membrane invagination sites in pigment-protein complex assembly. Functional correlates of proteomics approaches were provided by near-IR fluorescence induction/relaxation transients arising from LH-BChl components. A linear relation was found between increasing functional absorption cross-section and slowing of RC electron transfer turnover rate, thought to arise from the imposition of constraints upon free UQ diffusion between the RC and cytochrome bc1 complex as the membrane became saturated with new LH2 rings. In cells undergoing ICM induction in which generation of the electrochemical proton gradient was uncoupled with CCCP, blockage in membrane insertion of the LH and RC polypetides was demonstrated. This was reflected in a diminution of quantum yield of the primary charge separation, a cessation in expansion of functional absorption cross-section and a >4-fold slowing in RC electron transfer turnover. The ICM insertion of ATP synthase and transhydrogenase was also significantly diminished. Importantly, for the UPB fraction, CCCP treatment resulted in accumulation of ~2-fold greater levels of the preprotein translocase SecY, the SecA translocation ATPase, Sec D and SecF insertion components, and chaperonins DnaJ and DnaK, suggesting that these general membrane assembly factors had accumulated in association with nascent LH and RC assembly intermediates. In addition to spectrally homogeneous bands containing LH2 and RC-LH1 complexes, CNE also gave rise to two bands of intermediate migration, an upper LH2 and a lower LH1-enriched band, which has facilitated in vivo assessment of LH2:LH1 associations existing in the membrane. For chromatophores from cells adapting to low light intensity, the LH2 CNE gel band was enriched in LH2 polypeptides encoded by the puc1 light-responsive operon, suggesting that these LH2 complexes arise from LH2-only domains formed when LH2-LH1 associations are complete [7]. In contrast, high levels of LH2 polypeptides encoded by the puc2 operon and the absence of alterations in Puc polypeptide distribution in the upper intermediate LH2-LH1 gel band indicate that it arises from ICM regions laid down early in the ICM remodeling process, in which LH2 is sandwiched between rows of aligned dimeric LH1-RC core complexes.},
doi = {10.2172/1041400},
url = {https://www.osti.gov/biblio/1041400}, journal = {},
number = ,
volume = ,
place = {United States},
year = {2012},
month = {6}
}