Structural and catalytic properties of the oxygen-evolving complex. Correlation of polypeptide and manganese release with the behavior of Z+ in chloroplasts and a highly resolved preparation of the PS II complex

Treatment of intact thylakoid membranes with Triton X-100 at pH 6 produces a preparation of the PS II complex capable of high rates of O₂ evolution. The preparation contains four managanese, one cytochrome b-559, one Signal II_f and one Signal II_s per 250 chlorophylls. By selective manipulation of the preparation polypeptides of approximate molecular weights of 33, 23 and 17 kDa can be removed from the complex. Release of 23 and 17 kDa polypeptides does not release functional manganese. Under these conditions Z⁺ is not readily and directly accessible to an added donor (benzidine) and it appears as if at least some of the S-state transitions occur. Evidence is presented which indicates that benzidine does have increased access to the oxygen-evolving complex in these polypeptide depleted preparations. Conditions which release the 33 kDa species along with Mn and the 23 and 17 kDa polypeptides generate an alteration in the structure of the oxidizing side of PS II, which becomes freely accessible to benzidine. These findings are examined in relationship to alterations of normal S-state behavior (induced by polypeptide release) and a model is proposed for the organization of functional manganese and polypeptides involved in the oxygen-evolving reaction.     

The detection,isolation and characterization of a light-harvesting complex which is specifically associated with Photosystem I

10% of the chlorophyll associated with a ‘native’ Photosystem (PS) I complex (110 chlorophylls/P-700) is chlorophyll (Chl) b. The Chl b is associated with a specific PS I antenna complex which we designate as LHC-I (i.e., a light-harvesting complex serving PS I). When the native PS I complex is degraded to the core complex by LHC-I extraction, there is a parallel loss of Chl b, fluorescence at 735 nm, together with 647 and 686 nm circular dichroism spectral properties, as well as a group of polypeptides of 24-19 kDa. In this paper we present a method by which the LHC-I complex can be dissociated from the native PS I. The isolated LHC-I contains significant amounts of Chl b (Chl $\text{a}\text{b ? 3.7}$). The long-wavelength fluorescence at 730 nm and circular dichroism signal at 686 nm observed in native PS I are maintained in this isolated complex. This isolated fraction also contains the low molecular weight polypeptides lost in the preparation of PS I core complex. We conclude that we have isolated the PS I antenna in an intact state and discuss its in vivo function.     

Structural studies on cyanobacterial photosystem I

The cyanobacterial photosystem, I complex from Synechococcus sp. PCC6301 contains polypeptides of apparent M_r of 70,000, 18,000, 17,700, 16,000 and 10,000. Procedures were developed for the purification of the M_r 17,700 and 10,000 polypeptides. Amino acid analyses showed the absence of cystine and cysteine from these polypeptides. Amino-terminal sequences of 98 residues for the M_r 17,700 polypeptide and of 42 residues for the M_r 10,000 polypeptide were determined. Studies of pigment distribution within the photosystem I complex indicated that the binding of chlorophyll a and -carotene is in part dependent on the presence of these polypeptides.Abbreviations PSI photosystem I - P700 reaction center of PSI - SDS sodium dodecylsulfate - TBS tris-buffered saline - TTBS TBS containing Tween-20     

The b Subunits in the Peripheral Stalk of F1F0 ATP Synthase Preferentially Adopt an Offset Relationship

The peripheral stalk of F₁F₀ ATP synthase is essential for the binding of F₁ to F_O and for proper transfer of energy between the two sectors of the enzyme. The peripheral stalk of Escherichia coli is composed of a dimer of identical b subunits. In contrast, photosynthetic organisms express two b-like genes that form a heterodimeric peripheral stalk. Previously we generated chimeric peripheral stalks in which a portion of the tether and dimerization domains of the E. coli b subunits were replaced with homologous sequences from the b and b′ subunits of Thermosynechococcus elongatus (Claggett, S. B., Grabar, T. B., Dunn, S. D., and Cain, B. D. (2007) J. Bacteriol. 189, 5463–5471). The spatial arrangement of the chimeric b and b′ subunits, abbreviated Tb and Tb′, has been investigated by Cu²⁺-mediated disulfide cross-link formation. Disulfide formation was studied both in soluble model polypeptides and between full-length subunits within intact functional F₁F₀ ATP synthase complexes. In both cases, disulfides were preferentially formed between Tb_A83C and Tb′_A90C, indicating the existence of a staggered relationship between helices of the two chimeric subunits. Even under stringent conditions rapid formation of disulfides between these positions occurred. Importantly, formation of this cross-link had no detectable effect on ATP-driven proton pumping, indicating that the staggered conformation is compatible with normal enzymatic activity. Under less stringent reaction conditions, it was also possible to detect b subunits cross-linked through identical positions, suggesting that an in-register, nonstaggered parallel conformation may also exist.F₁F₀ ATP synthases are found in the inner mitochondrial membrane, the thylakoid membrane of chloroplasts, and the cytoplasmic membrane of bacteria (1–4). These enzymes are responsible for harnessing an electrochemical gradient of protons across the membranes for the synthesis of ATP. In Escherichia coli F₁F₀ ATP synthase, the membrane-embedded F₀ sector is composed of subunits ab₂c₁₀ and a soluble F₁ portion composed of subunits α₃β₃γδϵ. The F₀ sector houses a proton channel located principally in the a subunit, and the flow of protons through F₀ generates torque used to rotate the c₁₀ subunit ring relative to the ab₂ subunits. The F₁ γ and ϵ subunits are bound to the c₁₀ ring and form the central or rotor stalk. Catalytic sites are located at the interfaces of each αβ pair in F₁. The γ subunit extends into the center of the α₃β₃ hexamer, creating an asymmetry in the conformations of the αβ pairs (5). It is the rotation of the γ subunit and the resulting sequential conformational changes in each αβ pair that provides the driving force for the synthesis of ATP at the catalytic sites. The α₃β₃ hexamer is held stationary relative to the rotary stalk by the peripheral stalk consisting of the b₂δ subunits.The peripheral stalk is essential for binding F₁ to F₀ and for coupling proton translocation to catalytic activity (6–8). In the E. coli enzyme, the peripheral stalk is a dimer of identical b subunits. The stalk has been conceptually divided into functional domains called the membrane domain (b_M1-I33), the tether domain (b_E34-A61), the dimerization domain (b_T62-K122), and the F₁-binding domain (b_Q123-L156) (9). Although there is ample evidence of direct protein-protein interactions between b subunits within the membrane, dimerization, and F₁-binding domains, there is remarkably little evidence of tight packing between the b subunits in the tether domain. In fact, electron spin resonance studies suggested that the tether domains of the two b subunits may be separated by more than 20 Å in the F₁F₀ complex (10, 11). Much of what is known about the structure of the stalk has been inferred from analysis of the properties of polypeptides modeling segments of the b subunit. The structure of a peptide modeling the membrane domain, b_M1-E34, has been determined by NMR (12), and a peptide based on the dimerization domain, b_T62-K122, has been determined by x-ray diffraction (13). Both polypeptides assumed α-helical conformations, but neither structure directly revealed b subunit dimerization interactions. Recently, Priya et al. (14) reported a low resolution structure of a b_M22-L156 dimer, but the extended conformation appears to be slightly too long to accurately reflect the dimensions of the peripheral stalk within the F₁F₀ complex.Molecular modeling efforts supported by a variety of biochemical and biophysical experiments have yielded competing right-handed coiled coil (15, 16) and left-handed coiled coil (17, 18) models for the peripheral stalk. The parallel two-stranded left-handed coiled coli is a well known structure characterized by knobs-into-holes packing of the side chains of the two helices that are aligned in-register. An in-register conformation implies that any specific amino acid on the b subunit-subunit interface would occupy a position immediately adjacent to its counterpart in the other b subunit. In contrast, del Rizzo et al. (16) proposed a novel parallel right-handed coiled coil with the helices of the two b subunits offset by approximately one and a half turns of an α helix. This staggered model positions the two identical residues contributed by each of the b subunits in a homodimer into differing environments and at considerable distance from one another. Sequence analyses have been offered in support of both models (16, 18). In terms of experimental support, cross-linking studies of polypeptides have provided evidence that dimer packing could be in-register at many sites starting from residue Ala⁵⁹ and continuing to the carboxyl termini in model b_Y24–L156 dimers and in b_D53–K122 dimers (9, 16). Cross-linking at a few of the carboxyl-proximal positions has been confirmed within intact F₁F₀ ATP synthase complexes (19, 20). Recent electron spin resonance distance measurements on b_24–156 have also been interpreted as support for the in-register arrangement (17, 18). Conversely, work with polypeptides modeling the b subunit has generated evidence favoring a staggered conformation in this section of the dimer (15, 16, 21). In mixtures of dimerization domain polypeptides with cysteines incorporated at different sites, disulfides preferentially formed between positions that were 4–7 residues apart. For example, b_D53–L156 dimers were covalently locked into the offset conformation by the formation of disulfide bridges between cysteines introduced at positions b_A79 and b_R83 as well as b_R83 and b_A90 (15). These staggered dimers were more stable, melted with higher cooperativity, and bound soluble F₁ with higher affinity than b_D53–L156 dimers fixed in the in-register arrangement. Moreover, active and coupled F₁F₀ complexes were assembled with heterodimeric peripheral stalks using b subunits with tether domains varying in length by as many as 14 amino acids (22). These F₁F₀ complexes had peripheral stalks that were by definition out of register, at least within the tether domain.In contrast to the homodimer of identical b subunits observed in the peripheral stalk of E. coli, photosynthetic organisms express two b-like subunits, b and b′, that are thought to form heterodimeric peripheral stalks in F₁F₀ ATP synthase. Previously, we generated heterodimeric peripheral stalks within the E. coli F₁F₀ by constructing chimeric b subunits (23). Segments of the tether and dimerization domains of the E. coli b subunit were replaced with the homologous regions of the Thermosynechococcus elongatus b and b′ subunits. The chimeric subunits formed heterodimeric peripheral stalks that were incorporated into intact, functional F₁F₀ ATP synthase complexes. The most active chimeric enzymes had T. elongatus primary sequences replacing residues b_E39–I86 of the E. coli b subunit. For simplicity, these chimeric subunits will be referred to here as Tb and Tb′.The ability to generate F₁F₀ ATP synthases with Tb/Tb′ heterodimeric peripheral stalks provided a means to investigate the positions of the two subunits in the peripheral stalk. In the present work, we show that the Tb and Tb′ subunits assumed preferred positions relative to one another within the F₁F₀ complex. The staggered conformation appears to be a favored and functional conformation for the peripheral stalk. However, within a population of F₁F₀ complexes, some complexes with peripheral stalks in the in-register conformation are likely to exist.     

Partial purification,subunit structure and thermal stability of the photochemical reaction center of the thermophilic green bacterium Chloroflexus aurantiacus

Spectrally pure reaction center preparations from Chloroflexus aurantiacus have been obtained in a stable form; however, the product contained several contaminating polypeptides. The reaction center pigment molecules (probably three bacteriochlorophyll a and three bacteriopheophytin a molecules) are associated with two polypeptides (M_r = 30000 and 28000) in a reaction center complex of M_r = 52000. No carotenoid is present in the complex. These data together with previous spectral data suggest that the Chloroflexus reaction center represents a more primitive evolutionary form of the purple bacterial reaction center, and that it has little if any relationship to the green bacterial component. A reaction center preparation from Rhodopseudomonas sphaeroides R26 was fully denatured at 50°C while the Chloroflexus reaction center required higher temperatures (70–75°C) for complete denaturation. Thus, an intrinsic membrane protein of a photosynthetic thermophile has been demonstrated to have greater thermal stability than the equivalent component of a mesophile.     

Ectopic F0F1 ATP synthase contains both nuclear and mitochondrially-encoded subunits

Over the past few years, several reports have described the presence of F₀F₁ ATP synthase subunits at the surface of hepatocytes, where the hydrolytic activity of F₁ sector faces outside and triggers HDL endocytosis. An intriguing question is whether the ectopic enzyme has same subunit composition and molecular mass as that of the mitochondrial ATP synthase. Also due to the polar nature of hepatocytes, the enzyme may be localized to a particular cell boundary. Using different methods to prepare rat liver plasma membranes, which have been subjected to digitonin extraction, hr CN PAGE, immunoblotting, and mass spectrometry analysis, we demonstrate the presence of ecto-F₀F₁ complexes which have a similar molecular weight to the monomeric form of the mitochondrial complexes, containing both nuclear and mitochondrially-encoded subunits. This finding makes it unlikely that the enzyme assembles on the plasma membranes, but suggest it to be transported whole after being assembled in mitochondria by still unknown pathways. Moreover, the plasma membrane preparation enriched in basolateral proteins contains much higher amounts of complete and active F₀F₁ complexes, consistent with their specific function to modulate the HDL uptake on hepatocyte surface.     

Isolation and characterization of precursors in T4 baseplate assembly the complex of gene 10 and gene 11 products

The first multi-protein precursor in the assembly of the radial arms of the T4 baseplate has been purified to homogeneity. The complex was isolated from cells infected with a mutant blocked in the subsequent step in baseplate arm assembly. The assay for this precursor exploited the fact that the complex contains the target antigen of the neutralizing antibodies found in antibaseplate serum (Berget & King, 1978).The complex is composed of gene 10 protein (M_r, 88,000) and gene 11 protein (M_r, 24,000). Analytical ultracentrifugation experiments revealed a molecular weight of 258,000 and a sedimentation coefficient of 9.3 S for the complex. The overall and single polypeptide chain molecular weights are consistent with the complex containing two gene 10 polypeptides and four gene 11 polypeptides. Visualization of the complex in the electron microscope revealed an asymmetric angular structure. The shape, together with the previous identification of gene 11 product as the tail-spike protein (Crowther et al., 1977), indicates that the complex forms the body of the spikes and vertices of the hexagonal baseplate.Using an in vitro baseplate assembly assay, it was possible to demonstrate that the complex contains both the assembly-active gene 10 and gene 11 products. Gene 11 product (from 10^? extracts) can convert 11^? particles to viable phage. However, the complex lacked this activity, indicating that it does not readily dissociate. The precursor complex could be dissociated with denaturing solvents. Upon returning to physiological conditions, both the antigenic and biological activities of the gene 11 product could be recovered. The biological activity of the gene 10 product was not regained.     

F0F1 ATP synthase of Streptomycetes: Modulation of activity and oligomycin resistance by protein Ser/Thr kinases

Inverted membrane vesicles of Gram-positive actinobacteria Streptomyces fradiae, S. lividans, and S. avermitilis have been prepared and membrane-bound F₀F₁ ATP synthase has been biochemically characterized. It has been shown that the ATPase activity of membrane-bound F₀F₁ complex is Mg²⁺-dependent and moderately stimulated by high concentrations of Ca²⁺ ions (10–20 mM). The ATPase activity is inhibited by N,N′-dicyclohexylcarbodiimide and oligomycin A, typical F₀F₁ ATPase inhibitors that react with the membrane-bound F₀ complex. The assay of biochemical properties of the F₀F₁ ATPases of Streptomycetes in all cases showed the presence of ATPase populations highly susceptible and insensitive to oligomycin A. The in vitro labeling and inhibitory assay showed that the inverted phospholipid vesicles of S. fradiae contained active membrane-bound Ser/Thr protein kinase(s) phosphorylating the proteins of the F₀F₁ complex. Inhibition of phosphorylation leads to decrease of the ATPase activity and increase of its susceptibility to oligomycin. The in vivo assay confirmed the enhancement of actinobacteria cell sensitivity to oligomycin after inhibition of endogenous phosphorylation. The sequencing of the S. fradiae genes encoding oligomycin-binding A and C subunits of F₀F₁ ATP synthase revealed their close phylogenetic relation to the genes of S. lividans and S. avermitilis.     

Structural organization of ribonucleoproteins containing small nuclear RNAs from HeLa cells. Proteins interact closely with a similar structural domain of U1, U2, U4 and U5 small nuclear RNAs

U₁ snRNP² isolated from HeLa cells and purified by centrifugation in cesium chloride contains a set of proteins that may be resolved into four/five polypeptides by gel electrophoresis. When this particle was submitted to extensive digestion with micrococcal nuclease, RNA fragments of about 25 nucleotides in length were obtained. Sequence analyses showed that these highly protected fragments were derived from the same region of the U₁ molecule, spanning positions 119 to 143. At low concentrations of nuclease, a longer fragment, from nucleotide 119 to the 3′ OH end, was also detected. U₁ core-resistant snRNP, isolated by high performance liquid chromatography, still contains all the protein components of the intact particle.When a less drastically purified U₁ snRNP containing, beside the four/five polypeptides remaining after centrifugation in cesium chloride, a set of at least three polypeptides of larger size, was digested with the nuclease, no other protected RNA fragment was detected.When a mixture of U₁, U₂, U₄, U₅ and U₆ snRNPs, which contains the same four/five polypeptides as U₁ snRNP, was treated with micrococcal nuclease, protected fragments of snRNAs U₂, U₄ and U₅ were found in addition to those derived from U₁. No fragment derived from U₆ was found.In all cases, the region of snRNA shielded from nuclease attack corresponds to a distinctive feature of the molecule. It is a single-stranded region, comprising the sequence A(U)_nG with n ≥ 3, bordered by two double-stranded stems. One of these stems includes the 3′ terminus of the RNA, except in the case of U₂, where there are two stems instead of one on the 3′ side of the single-stranded stretch. Although a comparable structural domain exists also in U₆ snRNA, it does not contain the sequence A(U)_nG which correlates well with the fact that no U₆ snRNA fragment seems to resist micrococcal nuclease digestion.     

Photoaffinity Labeling of Mature and Precursor Forms of the Small Subunit of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase after Expression in Escherichia coli

The small subunit (SSU) of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) possesses a binding site that can be photoaffinity labeled with [³²P]8-azidoadenosine 5′ triphosphate (N₃ATP). In the present study, photoaffinity labeling was used to compare the nucleotide analog binding properties of SSU in the Rubisco holoenzyme complex (holoE SSU) with the properties of isolated SSU and the precursor form (pSSU) that contains a transit peptide. To facilitate these studies, the complete coding regions of tobacco (Nicotiana tabacum L.) SSU and pSSU were cloned into pET expression vectors and the polypeptides were synthesized in Escherichia coli. Protein import studies showed that cloned pSSU polypeptides were imported into intact chloroplasts, where they were processed to the mature form and assembled into the Rubisco holoenzyme. Cloned SSU and pSSU isolated from E. coli were photoaffinity labeled with N₃ATP. The apparent K_d value for SSU and pSSU, 18 micromolar N₃ATP, was identical to the value determined for holoE SSU. However, differences in photolabeling between cloned SSU or pSSU and holoE SSU were apparent in the level of protection afforded by ATP and UTP, in the response of photolabeling to free Mg²⁺, and in the higher photolabeling efficiency that characterized the cloned SSU. Treatment of the Rubisco holoenzyme with a concentration of urea sufficient to disassociate the subunits markedly increased photoincorporation into SSU, indicating that intersubunit associations within the holoenzyme complex may be the major factor influencing photolabeling efficiency of SSU. Thus, differences in SSU conformation between the isolated and assembled states affect photolabeling efficiency and other nucleotide analog binding properties of the SSU, but not the apparent affinity for N₃ATP.     

Biosynthetic approach to the structure of F1-ATPase (BF1 factor) from Micrococcus lysodeikticus membranes: in vivo labeling of the polypeptides and study of their relationship

The F₁-ATPase or BF₁ factor was purified from Micrococcus lysodeikticus substrain B grown in a synthetic medium in the presence of tritiated amino acids. When analyzed in sodium dodecyl sulfate-7% polyacrylamide gels, the fresh purified preparation contained α, β, γ subunits (referred as the intrinsic subunits) and two other polypeptides (designated as X and component of relative mobility 1.0) whose status as subunits remains to be established. This overall polypeptide composition was similar to that of the F₁-ATPase isolated from the same strain grown in complex medium (J. Carreira, J. M. Andreu, M. Nieto, and E. Muñoz., 1976 Mol. Cell. Biochem.10, 67–76). The distribution of ³H-labeled amino acids into purified F₁-ATPase and its constituent polypeptides under different stages of growth was used to investigate the biosynthetic relationship between the different polypeptides. The incorporation of amino acids into purified BF₁ factor was slower than that of cytoplasmic and other membrane proteins. In isotope-dilution and chase experiments, F₁-ATPase showed one of the slowest rates of decay of the incorporated label. These results point out that F₁-ATPase of M. lysodeikticus undergoes slower turnover than the overall cytoplasmic and membrane proteins. Pulse and chase experiments allowed us to conclude that the α, β, γ subunits and the components of relative mobility 1.0 are independent with differences in their turnover and therefore do not bear any apparent relation as precursors-products. The two major subunits represent seemingly the “core” of ATPase, the β subunit behaving like the most stable component. On the other hand, the γ subunit appears to be synthesized independently from this α + β complex.     

Photocontrol of chloroplast and mitochondrial polypeptide levels in euglena

Two dimensional polyacrylamide gel electrophoresis resolved protein from intact chloroplasts of wild type Euglena gracilis Klebs var. bacillaris Cori into 185 polypeptides of which 55 were localized on the whole cell polypeptide map. Of these chloroplast polypeptides, the relative amounts of 49 increased, the relative amounts of two decreased, and the relative amounts of four polypeptides were unaltered by exposure of dark grown resting cells to light for 72 hours. Proteins from intact purified mitochondria obtained from a bleached mutant (W₁₀BSmL) lacking plastids were resolved into 193 polypeptides of which 44 were localized on the whole cell polypeptide map from wild type cells. Of these mitochondrial polypeptides, the relative amount of one increased, the relative amounts of 12 were unaltered, and the relative amounts of 31 decreased after exposure of the dark grown resting cells to light. Since it is known that the development of the chloroplast in Euglena occurs without a net increase in total cellular protein and without a change in the size of the cellular amino acid pools, the degradation of mitochondrial polypeptides represents a major source of amino acids for the synthesis of chloroplast polypeptides.     

The occurrence of artemocyanin in Branchiopoda (Crustacea)

Artemocyanin, the extracellular hemolymph biliprotein of Artemia, is demonstrated in the fairy shrimp Streptocephalus, the clam shrimp Leptestheria and the water flea Daphnia. Artemocyanins can be purified from hemolymph as intact polypeptides (M_r 170–190,000), but are degraded upon homogenization of the whole animal by partial proteolysis to polypeptides with M_r 102,000 and 85,000. The aminoterminal sequence of the intact artemocyanin polypeptide was determined, but no clear-cut relationships with arthropod biliproteins or other protein families could be demonstrated.     

Biogenesis of the chloroplast phosphate translocator

Calcium-dependent proteolysis of several polypeptides from rat brain and synaptosomal cytosol was observed including proteolysis of polypeptides of M_r 340 000 and 300 000. These latter polypeptides comigrated with high-M_r microtubule-associated proteins of microtubule preparations from brain or synaptosomal cytosol. Calcium influx into intact synaptosomes due to depolarisation with high potassium or veratridine or treatment with the ionophore A23187 did not result in Ca²⁺-dependent proteolysis of any polypeptides. This may be due to the low calcium sensitivity of the protease since no proteolysis of the M_r 340 000 and 300 000 polypeptides was seen in synaptosomal cytosal at < 10 μM free Ca²⁺.     

Isolation and Characterization of an Fe(III)-Chelating Compound Produced by Pseudomonas syringae

The phytopathogenic bacterium Pseudomonas syringae produces a fluorescent pigment when it is grown in iron-deficient media. This pigment forms a very stable Fe(III) complex that was purified in this form by using a novel procedure based on ultrafiltration and column chromatography. The Fe(III) complex has a molecular weight of 1,100 and contains 1 mol of Fe(III). The pigment is composed of an amino acid moiety with three threonines, three serines, one lysine, δ-N-hydroxyornithine, and a quinoline-type fluorescent chromophore. These features and its stability constant (in the range of 10³²) suggest that the fluorescent pigment of P. syringae is related to the siderophores produced by another Pseudomonas species.     

Pigment organization in the photosynthetic apparatus of Roseiflexus castenholzii

The light-harvesting-reaction center (LHRC) complex from the chlorosome-lacking filamentous anoxygenic phototroph (FAP), Roseiflexus castenholzii (R. castenholzii) was purified and characterized for overall pigment organization. The LHRC is a single complex that is comprised of light harvesting (LH) and reaction center (RC) polypeptides as well as an attached c-type cytochrome. The dominant carotenoid found in the LHRC is keto-γ-carotene, which transfers excitation to the long wavelength antenna band with 35% efficiency. Linear dichroism and fluorescence polarization measurements indicate that the long wavelength antenna pigments absorbing around 880 nm are perpendicular to the membrane plane, with the corresponding Q_y transition dipoles in the plane of the membrane. The antenna pigments absorbing around 800 nm, as well as the bound carotenoid, are oriented at a large angle with respect to the membrane. The antenna pigments spectroscopically resemble the well-studied LH2 complex from purple bacteria, however the close association with the RC makes the light harvesting component of this complex functionally more like LH1.     

Structure of Dimeric F1F0-ATP Synthase

The structure of the dimeric ATP synthase from yeast mitochondria was analyzed by transmission electron microscopy and single particle image analysis. In addition to the previously reported side views of the dimer, top view and intermediate projections served to resolve the arrangement of the rotary c₁₀ ring and the other stator subunits at the F₀-F₀ dimeric interface. A three-dimensional reconstruction of the complex was calculated from a data set of 9960 molecular images at a resolution of 27 Å. The structural model of the dimeric ATP synthase shows the two monomers arranged at an angle of ∼45°, consistent with our earlier analysis of the ATP synthase from bovine heart mitochondria (Minauro-Sanmiguel, F., Wilkens, S., and Garcia, J. J. (2005) Proc. Natl. Acad. Sci. U.S.A. 102, 12356–12358). In the ATP synthase dimer, the two peripheral stalks are located near the F₁-F₁ interface but are turned away from each other so that they are not in contact. Based on the three-dimensional reconstruction, a model of how dimeric ATP synthase assembles to form the higher order oligomeric structures that are required for mitochondrial cristae biogenesis is discussed.     

Coenzyme Q supplementation or over-expression of the yeast Coq8 putative kinase stabilizes multi-subunit Coq polypeptide complexes in yeast coq null mutants

Coenzyme Q biosynthesis in yeast requires a multi-subunit Coq polypeptide complex. Deletion of any one of the COQ genes leads to respiratory deficiency and decreased levels of the Coq4, Coq6, Coq7, and Coq9 polypeptides, suggesting that their association in a high molecular mass complex is required for stability. Over-expression of the putative Coq8 kinase in certain coq null mutants restores steady-state levels of the sensitive Coq polypeptides and promotes the synthesis of late-stage Q-intermediates. Here we show that over-expression of Coq8 in yeast coq null mutants profoundly affects the association of several of the Coq polypeptides in high molecular mass complexes, as assayed by separation of digitonin extracts of mitochondria by two-dimensional blue-native/SDS PAGE. The Coq4 polypeptide persists at high molecular mass with over-expression of Coq8 in coq3, coq5, coq6, coq7, coq9, and coq10 mutants, indicating that Coq4 is a central organizer of the Coq complex. Supplementation with exogenous Q₆ increased the steady-state levels of Coq4, Coq7, and Coq9, and several other mitochondrial polypeptides in select coq null mutants, and also promoted the formation of late-stage Q-intermediates. Q supplementation may stabilize this complex by interacting with one or more of the Coq polypeptides. The stabilizing effects of exogenously added Q₆ or over-expression of Coq8 depend on Coq1 and Coq2 production of a polyisoprenyl intermediate. Based on the observed interdependence of the Coq polypeptides, the effect of exogenous Q₆, and the requirement for an endogenously produced polyisoprenyl intermediate, we propose a new model for the Q-biosynthetic complex, termed the CoQ-synthome.     

Photosynthetic functions and thylakoid membrane polypeptide composition in light-sensitive mutants of Chlamydomonas reinhardii

In this paper we compared the pigment composition, photochemical activity, chloroplast ultrastructure, thylakoid membrane polypeptide composition and ribosomal content of wild-type and seven light-sensitive mutants of Chlamydomonas reinhardii.All the mutants had low chlorophyll and carotenoid content compared to wild-type. Mutants lts-30 and lts-135 were also characterized by a complete absence of visible carotenoids, while mutant lts-19 was fully deficient in chlorophylls.In most mutants, the chloroplast fragment could not carry out any DCIP photoreduction and O₂ evolution was also blocked. The PSI/P₇₀₀/activity was decreased in most cases.The mutant strains contained mostly single lamellae in their plastids, that is the stacking capacity of the thylakoid membranes was very decreased or fully absent. In most cases the number of lamellae was also very low.The relative amounts of 70 S ribosomes were decreased in all of the mutants. The thylakoid membranes showed anomalies in the region of 24 000–30 000 dalton polypeptides. The common characteristic for them was the relatively higher amount of the 30 000 dalton polypeptide and considerably decreased level of the 27 000 and 24 000 dalton polypeptides relative to the wild-type. These polypeptides were probably constituents of the chlorophyll-protein complex II which has been suggested to be the light harvesting pigment complex for PSII. The polypeptide of 30 000 daltons is the precursor for the LHCP apoprotein (24 000 dalton protein). It may be that the lighstimulated conversion of this precursor into LHCP apoprotein was blocked in our pigment-deficient mutants.Abbreviations CPI Chlorophyll-protein complex I - PSI Photosystem I - PSII Photosystem II - LHCP Light-harvesting pigment complex - DCIP 2,6-dichlorophenolindophenol - RuDPC-ase Ribulose-1,5-biphosphate-carboxylase - SDS Sodium dodecyl sulfate - LIDS Lithium dodecyl sulfate - PAG Polyacrylamide gel - TKM buffer 25 mM Tris-HCl, pH 7.S; 25 mM KCl; 25 mM Mg acetate     

Detection of Sendai virus fusion with human erythrocytes by fluorescence photobleaching recovery

The subunit stoichiometry of the ATP synthetase (CF₁-CF₀) immunoprecipitated from Triton X-100 extracts of chloroplast thylakoid membranes was determined to be α₃, β₃, γ, δ, ? (CF₁) and I_0.3, II_0.6–0.9, III₄₍₆₎ (CF₀). Antibodies against the polypeptides α, β, γ, δ, I, II and ? combined specifically with the isolated subunits as analysed by the protein blotting method. Applying this technique, antibodies against the CF₁ subunits were found to form complexes with the corresponding polypeptides of thylakoids, whereas those against I (M_r 20 000) and II (M_r 17 000) combined with M_r 26 000 and M_r 24 500 membrane polypeptides, respectively. The M_r 26 000 polypeptide was identified as the major subunits of the light-harvesting chlorophyll a/b-protein (LHCP) complex and the M_r 24 500 component seems to be functionally connected with this complex. From the results it is concluded that the chloroplast ATP synthetase consists of the subunit of the α, β, γ, δ, ? and III (proteolipid only and that proteolytically altered LHCP polypeptides bind artifically to the protein complex during isolation.