Membranes of Rhodopseudomonas sphaeroides. VI. Isolation of a fraction enriched in newly synthesized bacteriochlorophyll a-protein complexes

Radioactivity eventually destined for the chromatophore membrane of Rhodopseudomonas sphaeroides was shown in pulse-chase studies to appear first in a distinct pigmented fraction. This material formed an upper pigmented band which sedimented more slowly than chromatophores when cell-free extracts were subjected directly to rate-zone sedimentation on sucrose density gradients. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that the purified fraction contained polypeptide bands of the same mobility as light-harvesting bacteriochlorophyll a and reaction center-associated protein components of chromatophores; these were superimposed upon cytoplasmic membrane polypeptides. The pulse-chase relation was confined mainly to the polypeptide components of these pigment-protein complexes. It is suggested that the isolated fraction may be derived from sites at which new membrane invagination is initiated.     

Role of apparent membrane growth initiation sites during photosynthetic membrane development in synchronously dividing Rhodopseudomonas sphaeroides.

Sites of intracytoplasmic membrane growth and temporal relations in the assembly of photosynthetic units were examined in synchronously dividing Rhodopseudomonas sphaeroides cells. After rate-zone sedimentation of cell-free extracts, apparent sites of initiation of intracytoplasmic membrane growth formed an upper pigmented band that sedimented more slowly than the intracytoplasmic membrane-derived chromatophore fraction. Throughout the cell cycle, the levels of the peripheral B800-850 light-harvesting pigment-protein complex relative to those of the core B875 complex in the upper pigmented fraction were only about half those of chromatophores. Pulse-labeling studies with L-[35S]methionine indicated that the rates of assembly of proteins in the upper pigmented fraction were much higher than those of chromatophores throughout the cell cycle; rates for the reaction center polypeptides were estimated to be approximately 3.5-fold higher than in chromatophores when the two membrane fractions were equalized on a protein basis. In pulse-chase studies, radioactivity of the reaction center and B875 polypeptides increased significantly in chromatophores and decreased in the upper pigmented band during cell division. These data suggest that the B875 reaction center cores of the photosynthetic units are inserted preferentially into sites of membrane growth initiation isolated in the upper pigmented band and that the incomplete photosynthetic units are transferred from their sites of assembly into the intracytoplasmic membrane during cell division. These results suggested further that B800-850 is added directly to the intracytoplasmic membrane throughout the cell cycle.     

Membranes of Rhodopseudomonas sphaeroides. VII. Photochemical properties of a fraction enriched in newly synthesized bacteriochlorophyll a-protein complexes.

Previous pulse-chase studies have shown that bacteriochlorophyll a-protein complexes destined eventually for the photosynthetic (chromatophore) membrane of Rhodopseudomonas sphaeroides appear first in a distinct pigmented fraction. This rapidly labeled material forms an upper band when extracts of phototrophically grown cells are subjected directly to rate-zone sedimentation. In the present investigation, flash-induced absorbance changes at 605 nm have demonstrated that the upper fraction is enriched two-fold in photochemical reaction center activity when compared to chromotophores; a similar enrichment in the reaction center-associated B-875 antenna bacteriochlorophyll complex was also observed. Although b- and c-type cytochromes were present in the upper pigmented band, no photoreduction of the b-type components could be demonstrated. The endogenous c-type cytochrome (Em = +345 mV) was photooxidized slowly upon flash illumination. The extent of the reaction was increased markedly with excess exogenous ferrocytochrome c but only slightly in chromatophores. Only a small light-induced carotenoid band shift was observed. These results indicate that the rapidly labeled fraction contains photochemically competent reaction centers associated loosely with c-type and unconnected to b-type cytochrome. It is suggested that this fraction arises from new sites of cytoplasmic membrane invagination which fragment to form leaky vesicles upon cell disruption.     

Development and growth of photosynthetic membranes of Rhodospirillum rubrum

In cell-free extracts from low-aeration suspensions of Rhodospirillum rubrum strain G-9, bacteriochlorophyll a was distributed in two bands after rate-zone sedimentation in sucrose density gradients. From the physicochemical properties of these fractions, it was concluded that the upper band consisted of small membrane fragments, whereas the major band was composed of fragmented vesicular intracytoplasmic membrane (chromatophores). After a pulse with L-[35S]methionine, apparent polypeptide subunits of the reaction center and light-harvesting complexes within the upper pigmented fraction were labeled more rapidly than those of chromatophores; after a chase with excess unlabeled L-methionine, radioactivity from these components within the upper band appeared to be chased into the corresponding polypeptides of chromatophores. These labeling patterns are interpreted to reflect growth initiation and maturation of the photosynthetic apparatus and may, in part, represent a general mechanism for the development of vesicular intracytoplasmic membranes.     

Membranes of Rhodopseudomonas sphaeroides VII. Photochemical properties of a fraction enriched in newly synthesized bacteriochlorophyll a-protein complexes

Previous pulse-chase studies have shown that bacteriochlorophyll a-protein complexes destined eventually for the photosynthetic (chromatophore) membrane of Rhodopseudomonas sphaeroides appear first in a distinct pigmented fraction. This rapidly labeled material forms an upper band when extracts of phototrophically grown cells are subjected directly to rate-zone sedimentation. In the present investigation, flash-induced absorbance changes at 605 nm have demonstrated that the upper fraction is enriched two-fold in photochemical reaction center activity when compared to chromatophores; a similar enrichment in the reaction center-associated B-875 antenna bacteriochlorophyll complex was also observed. Although b- and c-type cytochromes were present in the upper pigmented band, no photoreduction of the b-type components could be demonstrated. The endogenous c-type cytochrome (E_m = +345 mV) was photooxidized slowly upon flash illumination. The extent of the reaction was increased markedly with excess exogenous ferrocytochrome c but only slightly in chromatophores. Only a small light-induced carotenoid band shift was observed. These results indicate that the rapidly labeled fraction contains photochemically competent reaction centers associated loosely with c-type and unconnected to b-type cytochrome. It is suggested that this fraction arises from new sites of cytoplasmic membrane invagination which fragment to form leaky vesicles upon cell disruption.     

Photosynthetic membrane development in Rhodopseudomonas sphaeroides. Spectral and kinetic characterization of redox components of light-driven electron flow in apparent photosynthetic membrane growth initiation sites

The kinetics of light-driven electron flow and the nature of redox centers at apparent photosynthetic membrane growth initiation sites in Rhodopseudomans sphaeroides were compared to those of intracytoplasmic photosynthetic membranes. In sucrose gradients, these membrane growth sites sediment more slowly than intracytoplasmic membrane-derived chromatophores and form an upper pigmented band. Cytochromes c1, c2, b561, and b566 were demonstrated in the upper fraction by redox potentiometry; c-type cytochromes were also detected electrophoretically. Signals characteristic of light-induced reaction center bacteriochlorophyll triplet and photooxidized reaction center bacteriochlorophyll dimer states were observed by EPR spectroscopy but the Rieske iron-sulfur signal of the ubiquinol-cytochrome c2 oxidoreductase was present at a 3-fold reduced level on a reaction center basis in comparison to chromatophores. Flash-induced absorbance measurements of the upper pigmented fraction demonstrated reaction center primary and secondary semiquinone anion acceptor signals, but cytochrome b561 photoreduction and cytochrome c1/c2 reactions occurred at slow rates. This fraction was enriched approximately 2- and 4-fold in total b- and c-type cytochromes, respectively, per reaction center over chromatophores, but photoreducible b-type cytochrome was lower. Measurements of respiratory activity indicated a 1.6-fold higher level of succinate-cytochrome c oxidoreductase/reaction center than in chromatophores, but the apparent turnover rates in both preparations were low. Overall, the results suggest that complete cycles of rapid, light-driven electron flow do not occur merely by introduction of newly synthesized reaction centers into respiratory membrane, but that subsequent synthesis and assembly of appropriate components of the ubiquinol-cytochrome c2 oxidoreductase is required.     

Membranes of Rhodopseudomonas sphaeroides. V. Identification of bacteriochlorophyll alpha-depleted cytoplasmic membrane in phototrophically grown cells.

The separation of membrane fragments was investigated in extracts of phototropically grown Rhodopseudomonas sphaeroides to determine if the plasma membrane contains discrete regions. A highly purified fraction of bacteriochlorophyll alpha-deficient membrane fragments was isolated by differential centrifugation, chromatography on Sepharose 2B, reaggregation, and isopycnic sedimentation on sucrose gradients. Significant levels of b- and c-type cytochromes and succinate dehydrogenase were demonstrated in the isolated membrane fragments and their appearance in electron micrographs, their polypeptide profile in dodecyl sulfate-polyacrylamide gel electrophoresis, and overall chemical composition were essentially identical to a similar fraction isolated from aerobically grown cells. Their polypeptide profiles were distinct from those of the intracytoplasmic chromatophore and outer membranes, and on the basis of bacteriochlorophyll content the phototrophic fraction was contaminated with chromatophores by less than 9%. The membrane fragments contained no diaminopimelic acid or glucosamine. It is condluded that the membrane fragments isolated from phototrophically growing Rp. sphaeroides have arisen from photosynthetic pigment-depleted regions of the plasma membrane structurally and functionally differentiated from the intracytoplasmic chromatophore membrane. These regions represent conserved chemotrophic cytoplasmic membrane whose synthesis continues under photoheterotrophic conditions.     

The location and function of cytochrome c2 in Rhodopseudomonas capsulate membranes.

Two fractions of membrane preparations, a heavy and a light one were isolated from mildly broken Rhodopseudomonas capsulata cells. The light fraction which contained vesicles similar to the regular chromatophores obtained by sonication and a heavy fraction which appeared in electron micrographs to consist of cell fragments which were designated as heavy chromatophores and were composed of broken cell envelopes containing closely packed vesicles enclosed within the cytoplasmic membrane. Both types of chromatophores catalyzed photophosphorylation. However, cytochrome c2 could be washed out only from the heavy chromatophores. Photophosphorylation activity which was lost by the removal of the cytochrome could be restored by addition of either cytochrome c2 or phenazine methosulphate. Light induced proton efflux in heavy chromatophores in contrast to proton influx in regular chromatophores. The washed heavy chromatophores did not lose the light induced proton movement. Light induced quenching of 9-aminoacridine and atebrin fluorescence in chromatophores, while the fluorescence was enhanced in the heavy chromatophores. The washing did not affect the fluorescence changes of the heavy chromatophores but caused a reduction of the steady state of the carotenoid absorbance shift. It is suggested that the membrane in the heavy chromatophores is oriented inside out with respect to the membrane in regular chromatophores. Cytochrome c2 which is attached to that side of the membrane facing the outside medium could be removed from the heavy chromatophors and reconstituted to them. The role of cytochrome c2 in photophosphorylation is discussed.     

Membranes of Rhodopseudomonas sphaeroides. V. Identification of bacteriochlorophyll a-depleted cytoplasmic membrane in phototrophically grown cells

The separation of membrane fragments was investigated in extracts of phototropically grown Rhodopseudomonas sphaeroides to determine if the plasma membrane contains discrete regions. A highly purified fraction of bacteriochlorophyll a-deficient membrane fragments was isolated by differential centrifugation, chromatography on Sepharose 2B, reaggregation, and isopycnic sedimentation on sucrose gradients. Significant levels of b- and c-type cytochromes and succinate dehydrogenase were demonstrated in the isolated membrane fragments and their appearance in electron micrographs, their polypeptide profile in dodecyl sulfate-polyacrylamide gel electrophoresis, and overall chemical composition were essentially identical to a similar fraction isolated from aerobically grown cells. Their polypeptide profiles were distinct from those of the intracytoplasmic chromatophore and outer membranes, and on the basis of bacteriochlorophyll content the phototrophic fraction was contaminated with chromatophores by <9%. The membrane fragments contained no diaminopimelic acid or glucosamine. It is concluded that the membrane fragments isolated from phototrophically growing Rp. sphaeroides have arisen from photosynthetic pigment-depleted regions of the plasma membrane structurally and functionally differentiated from the intracytoplasmic chromatophore membrane. These regions represent conserved chemotrophic cytoplasmic membrane whose synthesis continues under photoheterotrophic conditions.     

Structure of a bacterial photosynthetic membrane. Isolation, polypeptide composition, and selective proteolysis

A procedure for the isolation of highly purified bacterial photosynthetic membranes from Rhodopseudomonas viridis is described. The purity of the final membrane fraction has been confirmed by electron microscopy. Seven major polypeptide bands are associated with the photosynthetic membranes, and all seven are resistant to solubilization in Triton X-100 detergent. Two pigmented bands with apparent molecular weights of 44K and 41K are thought to be cytochromes. The three polypeptides with apparent molecular weights of 38K, 32K, and 28K have been reported in reaction center preparations of other laboratories. Two low-molecular-weight (16K and 11K) bands bind bacteriochlorophyll b and may represent light-harvesting bacteriochlorophyll-protein complexes. The structures that were isolated seem to represent complete photosynthetic membranes, consisting of reaction center, electron transport, and light-harvesting components, all arranged in the regular lattice characteristic of viridis. Selective proteolysis of these membranes indicates that all membrane components are accessible to digestion by trypsin and pronase, except for the light-harvesting complexes.     

Membranes of Rhodospirillum rubrum: physicochemical properties of chromatophore fractions isolated from osmotically and mechanically disrupted cells.

Isolation of highly purified membrane fractions from phototrophically grown Rhodospirillum rubrum was achieved by velocity and isopyknic sedimentation under carefully controlled ionic conditions. Bacteriochlorophyll-rich and succinic dehydrogenase-rich chromatophores that were essentially devoid of contamination by non-chromatophore protein were separated from a denser fraction in extracts disrupted in a French pressure cell. Highly purified chromatophores and a nearly photopigment-free envelope fraction were also obtained from cells lysed by treatment with ethylenediaminetetraacetate-lysozyme-Brij 58. After lysis with lysozyme and ethylenediaminetetraacetate alone, about 50% of the total photosynthetic pigment was released in chromatophores similar to those isolated by the above procedures. Chromatophores prepared by each method were found to have very similar near-infrared absorption spectra, overall chemical composition, equilibrium buoyant densities in CsCl, and protein patterns in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The protein profiles of the dense, outer membrane-rich fractions were different from those of the chromatophores. The release of much of the photosynthetic apparatus as discrete chromatophores is osmotically lysed extracts necessitates a reevaluation of the concept that isolated chromatophores arise only from mechanical comminution of a larger membrane structure.     

Light-harvesting pigment-protein complexes of Rhodopseudomonas sphaeroides forma sp. denitrificans

The composition of the light-harvesting system of Rhodopseudomonas sphaeroides forma sp. denitrificans was investigated. When chromatophores were solubilized by sodium dodecyl sulfate (SDS) at 0 degrees C and subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE), at least two B800-B850 pigment-protein complexes, three B870 pigment-protein complexes, a reaction center (RC) complex and two pigmented bands which contained B800, B850, and B870 were resolved. In the re-electrophoresis, the B870 pigment-protein complexes gave rise to a series of multiple pigmented bands. All of these multiple pigment-protein complexes showed almost the same polypeptide composition and absorption spectrum characteristic of the B870 complex. The apparent molecular weights of these B870 complexes showed a regular interval of about 7,000 indicating that these complexes were oligomers of a subunit. It was also found that a predominant B800-B850 pigment-protein complex could be degraded into a small complex via some intermediates. These results indicate that essentially two kinds of pigment-protein complexes construct the light-harvesting system of this bacteria and, upon treatment with SDS, these complexes are degraded into many classes of subunit aggregates showing a complicated profile of pigmented bands on the gel. Pigmented bands which contained both of B800-B850 and B870 complexes were considered to arise from occasional co-migration of distinct B800-B850 and B870 pigment-protein complexes.     

Transverse membrane topography of the B875 light-harvesting polypeptides of wild-type Rhodobacter sphaeroides.

Purified B875 light-harvesting complex, chromatophores, and spheroplast-derived vesicles from wild-type Rhodobacter sphaeroides were treated with proteinase K or trypsin, and the alpha and beta polypeptides were analyzed by electrophoretic, immunochemical, and protein-sequencing methods. With the purified complex, proteinase K digested both polypeptides and completely eliminated the A875 peak. Trypsin digested the alpha polypeptide and reduced the A875 by 50%. Proteinase K cleaved the beta polypeptide of chromatophores and the alpha polypeptide of spheroplast-derived vesicles. Sequence analyses of polypeptides extracted from proteinase K-treated chromatophores revealed that the beta polypeptide was cleaved between amino acids 4 and 5 from the N terminus. The N terminus of the alpha polypeptide was intact. We concluded that the N terminus of the beta polypeptide is exposed on the cytoplasmic membrane surface, and the difference in the digestion patterns between the spheroplast-derived vesicles and chromatophores suggested that the C terminus of the alpha polypeptide is exposed on the periplasmic surface.     

Isolation of the carotenoid-containing cell wall of three unicellular cyanobacteria.

A carotenoid-containing membrane fraction devoid of chlorophyll and phycobiliproteins was isolated from three unicellular cyanobacteria, Synechococcus sp., Synechococcus leopoliensis UTEX 625, and Anacystis nidulans R-2, by aqueous-phase separation, hydrophobic chromatography, and differential centrifugation. The presence of 2-keto-3-deoxyoctonate, muramic acid, and diaminopimelic acid suggests that the preparation is highly enriched in cell wall. Electron micrographs of thin sections of this material showed C-shaped membrane profiles similar to those seen in other gram-negative cell wall preparations. The inactivation of cyanophage AS-1 by this fraction confirmed its identity as cell wall. The cell wall contained approximately equal weights of total carbohydrate and protein. Absorption maxima at 434, 452, and 488 nm indicated the presence of carotenoids. These were in the outer membrane and were not due to contaminating cytoplasmic or thylakoid membranes. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the preparations showed a broad band of approximately 50,000 molecular weight which contained 35% of the total outer membrane protein. This band was resolved into at least two components running at approximately 50,000 and 52,000 molecular weight. The smaller of these polypeptides was a glycoprotein. The polypeptide components were unaffected by protease or detergent treatment in either whole cells or isolated cell wall preparations, indicating that the polypeptide components were not exposed to the surface or easily removed from the hydrophobic environment.     

The Separation of Chromatophores from the Cell Envelope in Rhodopseudomonas Spheroides

When subcellular particles from. Rhodopseudophas spheroices wore laced on sucrose density gradients, the separation of chromatophores from the cell envelope was markedly affected by the presence of ionic species. In gradients that contained Tris buffer plus 0.01’ magnesium, chromatophores were distributed nearly equally between an upper and a lower pigmented band. About half of the chronatopnores were release from the lower band when magnesium was excluded from the gradients. exclusion of both Tris and magnesium resulted in a quantitative separation of chromatophores (upper band) from the cell envelope (lower ‘and). Thus, the photosynthotic apparatus in Rps. spheroides resides on a membrane system separable from the cell wall-cell membrane complex.     

Isolation and fractionation of the photosynthetic membranous organelles from Rhodopseudomonas spheroides

Molecular sieve chromatography and sucrose gradient centrifugation were used to prepare large quantities of purified chromatophores from Rhodopseudomonas spheroides. Electron micrographs of these chromatophores revealed that the final preparations were very homogeneous and free of non-chromatophore particulate material. As an additional check on purity, (14)C-l-phenylalanine-labeled aerobic cells, devoid of chromatophores, were mixed with unlabeled photosynthetic cells. The resulting preparation contained less than 1% of the radioactivity, originally located in non-chromatophore protein. The purified chromatophores were solubilized in 2-chloroethanol and separated into two fractions. Fraction P(1) contained 3 to 5% of the total chromatophore protein and could be resolved into 10 electrophoretic components. The second fraction, P(II), contained five electrophoretic components. One of these components had associated with it all of the pigment and phospholipid present in P(II). Preliminary immunochemical studies on these fractions are also reported.     

Studies on mitochondrial proteins I. Separation and characterization by polyacrylamide gel electrophoresis

Rat liver mitochondria were fractionated into inner and outer membranes and soluble intermembrane space and matrix. The protein components of these fractions were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Mitochondria contained at least 20 components ranging in molecular weights from 10 000 to 140 000. Inner membranes differed markedly from outer membranes both in number of components and size distribution. The intermembrane space contained a few polypeptide species. These were of low molecular weight. The matrix was characterized by a high molecular weight component (130 000) which comprised 30% of this fraction. A major carbohydrate-containing polypeptide with an approximate molecular weight of 93 000 was detected in outer membrane preparations.     

Intracellular localization of photosynthetic membrane growth initiation sites in Rhodopseudomonas sphaeroides

Putative membrane invagination sites at which intracytoplasmic photosynthetic membrane growth is initiated in Rhodopseudomonas sphaeroides can be isolated in an upper pigmented fraction by rate-zone sedimentation. The intracellular localization of membranes present in the isolated fraction was investigated with the impermeant surface-labeling reagent pyridoxal 5'-phosphate, which has been shown to diffuse into the periplasmic space and to label proteins of both the peripheral cytoplasmic membrane and the mature intracytoplasmic membrane. A comparison of the extent of labeling at 25 and 0 degrees C was consistent with the possibility that membranes present in the upper pigmented fraction arise from sites near the cell periphery. Pronase digestion of the surface-labeled membranes suggested further that the purified upper fraction consisted largely of open membrane fragments and that the majority of the intracytoplasmic membrane is labeled by this procedure. The pigmented membrane growth initiation sites were separated partially from undifferentiated respiratory cytoplasmic membrane also present in the upper fraction.     

Probing the topology of proteins in the chromatophore membrane of Rhodospirillum rubrum G-9 with proteinase K

All the major membrane proteins of isolated chromatophore vesicles are eventually degraded upon incubation with the unspecific proteinase K. These proteins must therefore be exposed at least partially or temporarily on the cytosolic surface of the membrane which is exclusively accessible to the proteinase in intact chromatophore vesicles. That the vesicles are intact during the incubation with proteinase is demonstrated by the finding that cytochrome c₂, which is located in the interior of the vesicles, is protected from proteolytic attack. The degree of degradation of the various chromatophore proteins and the time taken for degradation differ characteristically. From the changes in intensity of the gel bands during the course of digestion it appears that reaction center subunit H is digested first, much faster than are subunits M and L. The near-infrared absorption spectrum of the chromatophores changes only after proteolytic degradation of these two pigment-carrying subunits. Fading of the band of the light-harvesting polypeptide is evident only after prolonged incubation. It seems that this is the most stable component of the chromatophore membrane. The light-harvesting polypeptide appears to be somewhat shortened eventually, leaving the protein conformation necessary for holding the pigments unchanged, as shown by the absorption spectrum. The possible topology of these major membrane components is discussed in the light of these findings.     

Localization of reaction center and B800-850 antenna pigment proteins in membranes of Rhodobacter sphaeroides.

The localization of the N- and C-terminal regions of pigment-binding polypeptides of the bacterial photosynthetic apparatus of Rhodobacter sphaeroides was investigated by proteinase K treatment of chromatophore and spheroplast-derived vesicles and amino acid sequence determination. Under conditions of proteinase K treatment of chromatophores, which left the in vivo absorption spectrum and the membrane intact, 15 and 46 amino acyl residues from the N-terminal regions of the L and M subunits, respectively, of the reaction center polypeptides were removed. The N termini are therefore exposed on the cytoplasmic surface of the membrane. The C-terminal domain of the light-harvesting B800-850 alpha and B870 alpha polypeptides was found to be exposed on the periplasmic surface of the membrane. A total of 9 and 13 amino acyl residues were cleaved from the B800-850 alpha and B870 alpha polypeptides, respectively, when spheroplasts were treated with proteinase K. The N-terminal regions of the alpha polypeptides were not digested in either membrane preparation and were apparently protected from proteolytic attack. Seven N-terminal amino acyl residues of the B800-850 beta polypeptide were removed after the digestion of chromatophores. C-terminal residues were not removed after the digestion of chromatophores or spheroplasts. The C termini seem to be protected from protease attack by interaction with the membrane. Therefore, the N-terminal regions of the beta polypeptides are exposed on the cytoplasmic membrane surface. The C termini of the beta polypeptides are believed to point to the periplasmic space.