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

The results of a detailed structural and functional proteomic analysis of intracytoplasmic membrane (ICM) assembly in the model purple phototrophic bacterium <i>Rhodobacter sphaeroides</i> 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 F₁F_O-ATP synthase subunits, and the cytochrome <i>bc</i>₁ 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 <i>a</i>. Slowing of the rate of RC electron transfer turnover (<i>τ</i>_QA), 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 <i>bc</i>₁ 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 <i>τ</i>_QA was accelerated from ∼1 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.本报告综述了模式紫色光合细菌球形红杆菌（*Rhodobacter sphaeroides*）胞内膜（intracytoplasmic membrane, ICM）组装过程的详细结构与功能蛋白质组学分析结果。蛋白质组学研究聚焦于鉴定ICM发育过程中时序表达、定位于细胞内膜内部的膜蛋白，及其结构与功能关联物。为分析时序蛋白表达情况，研究建立了在低氧张力下诱导ICM形成、以及使细胞适应低光照强度时发生ICM重塑的实验流程，借此可通过速率区带沉降法分离得到位于上层色素带（upper-pigmented band, UPB）中的ICM生长起始位点——细胞质膜（cytoplasmic membrane, CM）内陷，以及作为主带的更成熟ICM囊泡（chromatophores，载色体）。对载色体组分进行非变性透明天然凝胶电泳后，得到四条色素带：顶部与底部条带分别包含反应中心-捕光复合体1（reaction center-light-harvesting 1, RC-LH1）核心复合物与捕光复合体2（LH2）外围天线，而两条迁移速率居中的条带则呈现LH2与核心复合物的独特组合形式。对凝胶条带的蛋白质组学分析揭示了发育过程中的变化：随着ICM发育推进，LH2多肽的相对丰度逐渐高于核心复合物，同时还检测到大量其他关联蛋白，其中F₁F₀-ATP合酶（F₁F₀-ATP synthase）亚基与细胞色素bc₁复合物（cytochrome bc₁ complex）的光谱计数较高。功能未知蛋白RSP6124的计数同样较高，且其丰度与LH2水平的升高呈正相关。来自UPB的、包含RC-LH1的透明天然凝胶电泳条带富集了CM标志物，包括电子传递与转运蛋白，以及通用膜组装因子（即前蛋白转运酶YidC、YajC与SecY、细菌1型信号肽酶以及Twin精氨酸转运亚基TatA），从而证实UPB源自外周呼吸膜与活跃的CM内陷位点——后者是RC-LH1复合物优先组装的场所。光合单元组装过程的功能特性可通过LH-细菌叶绿素a（LH-bacteriochlorophyll a）产生的可变荧光的荧光诱导/弛豫测量进行监测。从弛豫阶段评估得到的RC电子传递周转速率（τ_QA）的减慢，与功能吸收截面（σ）以及LH2/LH1摩尔比的增大呈正相关。这一现象被认为源于：当ICM双层膜被LH2环密集填充时，泛醌（ubiquinone, UQ）氧化还原物种在RC与细胞色素bc₁复合物之间的自由扩散受到限制。通过对高、低光照强度下培养细胞的ICM斑块进行高分辨率原子力显微镜（atomic force microscopy, AFM）对比分析，证实了此类LH2的密集排列[Adams与Hunter：《Biochim Biophys Acta》，2012，1817：1616-1627]：在低光照条件下，LH2水平升高会形成密集的纯LH2结构域，对应光响应性天线组分。与之相反，LH2最初以线性阵列形式分散，其中大部分为二聚体RC-LH1核心复合物，且簇间存在间隔；在适应低光照强度的过程中，这些位点会被LH2填充。将光合生长的细胞转移至黑暗有氧条件后，荧光诱导/弛豫测量结果显示：尽管细胞出现不依赖光合途径的生长爆发，但功能性光合单元在转变后仍可维持长达24小时。到8小时时，τ_QA从约1毫秒加快至0.5毫秒，这反映了LH2水平的下降，随着LH2的拥挤效应被克服，泛醌氧化还原物种在膜双层中的扩散速率得以提升。在此情况下，UPB的丰度升高，且LH1/LH2摩尔比显著增大。这些变化表明：在有氧条件下，CM生长起始位点形成囊状ICM的囊泡化过程被阻断。