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Colin Robinson - One of the best experts on this subject based on the ideXlab platform.
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binding of chloroplast signal recognition particle to a Thylakoid Membrane protein substrate in aqueous solution and delineation of the cpsrp43 substrate interaction domain
Biochemical Journal, 2011Co-Authors: Peter Cain, Iris Holdermann, Irmgard Sinning, Arthur E Johnson, Colin RobinsonAbstract:A cpSRP [chloroplast SRP (signal recognition particle)] comprising cpSRP54 and cpSRP43 subunits mediates the insertion of light-harvesting proteins into the Thylakoid Membrane. We dissected its interaction with a full-length Membrane protein substrate in aqueous solution by insertion of site-specific photoactivatable cross-linkers into in vitro-synthesized Lhcb1 (major light-harvesting chlorophyll-binding protein of photosystem II). We show that Lhcb1 residues 166-176 cross-link specifically to the cpSRP43 subunit. Some cross-link positions within Lhcb1 are in the 'L18' peptide required for targeting of cpSRP substrates, whereas other cross-linking positions define a new targeting signal in the third transMembrane span. Lhcb1 was not found to cross-link to cpSRP54 at any position, and cross-linking to cpSRP43 is unaffected by the absence of cpSRP54. cpSRP43 thus effectively binds substrates autonomously, and its ability to independently bind an extended 20+-residue substrate region highlights a major difference with other SRP types where the SRP54 subunit binds to hydrophobic target sequences. The results also show that cpSRP43 can bind to a hydrophobic, three-Membrane span, substrate in aqueous solution, presumably reflecting a role for cpSRP in the chloroplast stroma. This mode of action, and the specificity of the cpSRP43 substrate interaction, may be associated with cpSRP's unique post-translational mode of action.
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protein transport in organelles protein transport into and across the Thylakoid Membrane
FEBS Journal, 2009Co-Authors: Cassie Aldridge, Peter Cain, Colin RobinsonAbstract:The chloroplast Thylakoid is the most abundant Membrane system in nature, and is responsible for the critical processes of light capture, electron transport and photophosphorylation. Most of the resident proteins are imported from the cytosol and then transported into or across the Thylakoid Membrane. This minireview describes the multitude of pathways used for these proteins. We discuss the huge differences in the mechanisms involved in the secretory and twin-arginine translocase pathways used for the transport of proteins into the lumen, with an emphasis on the differing substrate conformations and energy requirements. We also discuss the rationale for the use of two different systems for Membrane protein insertion: the signal recognition particle pathway and the so-called spontaneous pathway. The recent crystallization of a key chloroplast signal recognition particle component provides new insights into this rather unique form of signal recognition particle.
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protein translocation into and across the bacterial plasma Membrane and the plant Thylakoid Membrane
Trends in Biochemical Sciences, 1999Co-Authors: Ross E Dalbey, Colin RobinsonAbstract:Abstract Over the past decade, some familiar themes have emerged on how proteins are inserted into or translocated across the plant chloroplast Thylakoid Membrane and bacterial inner Membranes. In the SecA and signal recognition particle (SRP) pathways, nucleotides and soluble factors are used to translocate proteins across the Membrane bilayer in the unfolded state. However, the ΔpH-dependent pathway in Thylakoids uses a radically different mechanism: transport of proteins across the Membrane is driven by the transMembrane pH gradient, and neither stromal factors nor nucleotide triphosphates are needed. In addition, this pathway, which requires the Membrane-bound protein Hcf106, appears to translocate proteins in a tightly folded form. Recently, a similar pathway has been shown to operate in eubacteria, and several of its components have been identified.
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protein translocation into and across the bacterial plasma Membrane and the plant Thylakoid Membrane
Trends in Biochemical Sciences, 1999Co-Authors: Ross E Dalbey, Colin RobinsonAbstract:Over the past decade, some familiar themes have emerged on how proteins are inserted into or translocated across the plant chloroplast Thylakoid Membrane and bacterial inner Membranes. In the SecA and signal recognition particle (SRP) pathways, nucleotides and soluble factors are used to translocate proteins across the Membrane bilayer in the unfolded state. However, the delta pH-dependent pathway in Thylakoids uses a radically different mechanism: transport of proteins across the Membrane is driven by the transMembrane pH gradient, and neither stromal factors nor nucleotide triphosphates are needed. In addition, this pathway, which requires the Membrane-bound protein Hcf106, appears to translocate proteins in a tightly folded form. Recently, a similar pathway has been shown to operate in eubacteria, and several of its components have been identified.
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the sec independent twin arginine translocation system can transport both tightly folded and malfolded proteins across the Thylakoid Membrane
Journal of Biological Chemistry, 1998Co-Authors: Peter J Hynds, David Robinson, Colin RobinsonAbstract:Abstract A subset of lumen proteins is transported across the Thylakoid Membrane by a Sec-independent translocase that recognizes a twin-arginine motif in the targeting signal. A related system operates in bacteria, apparently for the export of redox cofactor-containing proteins. In this report we describe a key feature of this system, the ability to transport folded proteins. The Thylakoidal system is able to transport dihydrofolate reductase (DHFR) when an appropriate signal is attached, and the transport efficiency is almost undiminished by the binding of folate analogs such as methotrexate that cause the protein to fold very tightly. The system is moreover able to transport DHFR into the lumen with methotrexate bound in the active site, demonstrating that the ΔpH-driven transport of large, native structures is possible by this pathway. However, correct folding is not a prerequisite for transport. Truncated, malfolded DHFR can be translocated by this system, as can physiological substrates that are severely malfolded by the incorporation of amino acid analogs.
Danja Schunemann - One of the best experts on this subject based on the ideXlab platform.
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a Thylakoid Membrane bound and redox active rubredoxin rbd1 functions in de novo assembly and repair of photosystem ii
Proceedings of the National Academy of Sciences of the United States of America, 2019Co-Authors: Jose G Garciacerdan, Ariel L. Furst, Kent L. Mcdonald, Danja Schunemann, Matthew B. Francis, Krishna K. NiyogiAbstract:Author(s): Garcia-Cerdan, Jose G; Furst, Ariel L; McDonald, Kent L; Schunemann, Danja; Francis, Matthew B; Niyogi, Krishna K | Abstract: Photosystem II (PSII) undergoes frequent photooxidative damage that, if not repaired, impairs photosynthetic activity and growth. How photosynthetic organisms protect vulnerable PSII intermediate complexes during de novo assembly and repair remains poorly understood. Here, we report the genetic and biochemical characterization of chloroplast-located rubredoxin 1 (RBD1), a PSII assembly factor containing a redox-active rubredoxin domain and a single C-terminal transMembrane α-helix (TMH) domain. RBD1 is an integral Thylakoid Membrane protein that is enriched in stroma lamellae fractions with the rubredoxin domain exposed on the stromal side. RBD1 also interacts with PSII intermediate complexes containing cytochrome b 559 Complementation of the Chlamydomonas reinhardtii (hereafter Chlamydomonas) RBD1-deficient 2pac mutant with constructs encoding RBD1 protein truncations and site-directed mutations demonstrated that the TMH domain is essential for de novo PSII assembly, whereas the rubredoxin domain is involved in PSII repair. The rubredoxin domain exhibits a redox midpoint potential of +114 mV and is proficient in 1-electron transfers to a surrogate cytochrome c in vitro. Reduction of oxidized RBD1 is NADPH dependent and can be mediated by ferredoxin-NADP+ reductase (FNR) in vitro. We propose that RBD1 participates, together with the cytochrome b 559, in the protection of PSII intermediate complexes from photooxidative damage during de novo assembly and repair. This role of RBD1 is consistent with its evolutionary conservation among photosynthetic organisms and the fact that it is essential in photosynthetic eukaryotes.
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a Thylakoid Membrane bound and redox active rubredoxin rbd1 functions in de novo assembly and repair of photosystem ii
Proceedings of the National Academy of Sciences of the United States of America, 2019Co-Authors: Jose G Garciacerdan, Ariel L. Furst, Kent L. Mcdonald, Danja Schunemann, Krishna K. Niyogi, Matthew B. FrancisAbstract:Photosystem II (PSII) undergoes frequent photooxidative damage that, if not repaired, impairs photosynthetic activity and growth. How photosynthetic organisms protect vulnerable PSII intermediate complexes during de novo assembly and repair remains poorly understood. Here, we report the genetic and biochemical characterization of chloroplast-located rubredoxin 1 (RBD1), a PSII assembly factor containing a redox-active rubredoxin domain and a single C-terminal transMembrane α-helix (TMH) domain. RBD1 is an integral Thylakoid Membrane protein that is enriched in stroma lamellae fractions with the rubredoxin domain exposed on the stromal side. RBD1 also interacts with PSII intermediate complexes containing cytochrome b 559 Complementation of the Chlamydomonas reinhardtii (hereafter Chlamydomonas) RBD1-deficient 2pac mutant with constructs encoding RBD1 protein truncations and site-directed mutations demonstrated that the TMH domain is essential for de novo PSII assembly, whereas the rubredoxin domain is involved in PSII repair. The rubredoxin domain exhibits a redox midpoint potential of +114 mV and is proficient in 1-electron transfers to a surrogate cytochrome c in vitro. Reduction of oxidized RBD1 is NADPH dependent and can be mediated by ferredoxin-NADP+ reductase (FNR) in vitro. We propose that RBD1 participates, together with the cytochrome b 559, in the protection of PSII intermediate complexes from photooxidative damage during de novo assembly and repair. This role of RBD1 is consistent with its evolutionary conservation among photosynthetic organisms and the fact that it is essential in photosynthetic eukaryotes.
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Mechanisms of protein import into Thylakoids of chloroplasts.
Biological Chemistry, 2007Co-Authors: Danja SchunemannAbstract:The Thylakoid Membrane of chloroplasts contains the major photosynthetic complexes, which consist of several either nuclear or chloroplast encoded subunits. The biogenesis of these Thylakoid Membrane complexes requires coordinated transport and subsequent assembly of the subunits into functional complexes. Nuclear-encoded Thylakoid proteins are first imported into the chloroplast and then directed to the Thylakoid using different sorting mechanisms. The cpSec pathway and the cpTat pathway are mainly involved in the transport of lumenal proteins, whereas the spontaneous pathway and the cpSRP pathway are used for the insertion of integral Membrane proteins into the Thylakoid Membrane. While cpSec-, cpTat- and cpSRP-mediated targeting can be classified as 'assisted' mechanisms involving numerous components, 'unassisted' spontaneous insertion does not require additional targeting factors. However, even the assisted pathways differ fundamentally with respect to stromal targeting factors, the composition of the translocase and energy requirements.
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a second Thylakoid Membrane localized alb3 oxai yidc homologue is involved in proper chloroplast biogenesis in arabidopsis thaliana
Journal of Biological Chemistry, 2006Co-Authors: Lars Gerdes, Jurgen Soll, Thomas Bals, Eva Klostermann, Monique Karl, Katrin Philippar, Mark Hunken, Danja SchunemannAbstract:Abstract The integral Membrane proteins Alb3, OxaI, and YidC belong to an evolutionary conserved protein family mediating protein insertion into the Thylakoid Membrane of chloroplasts, the inner Membrane of mitochondria, and bacteria, respectively. Whereas OxaI and YidC are involved in the insertion of a wide range of Membrane proteins, the function of Alb3 seems to be limited to the insertion of a subset of the light-harvesting chlorophyll-binding proteins. In this study, we identified a second chloroplast homologue of the Alb3/OxaI/YidC family, named Alb4. Alb4 is almost identical to the Alb3/OxaI/YidC domain of the previously described 110-kDa inner envelope protein Artemis. We show that Alb4 is expressed as a separate 55-kDa protein and that Artemis was identified mistakenly. Alb4 is located in the Thylakoid Membrane of Arabidopsis thaliana chloroplasts. Analysis of an Arabidopsis mutant (Salk_136199) and RNA interference lines with a reduced level of Alb4 revealed chloroplasts with an altered ultrastructure. Mutant plastids are larger and more spherical in appearance, and the grana stacks within the mutant lines are less appressed than in the wild-type chloroplasts. These data indicate that Alb4 is required for proper chloroplast biogenesis.
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the yeast split ubiquitin system to study chloroplast Membrane protein interactions
Applied Microbiology and Biotechnology, 2005Co-Authors: Jan Christoph Pasch, Jörg Nickelsen, Danja SchunemannAbstract:Each photosynthetic complex within the Thylakoid Membrane consists of several different subunits. During formation of these complexes, numerous regulatory factors are required for the coordinated transport and assembly of the subunits. Interactions between transport/assembly factors and their specific polypeptides occur in a Membraneous environment and are usually transient and short-lived. Thus, a detailed analysis of the underlying molecular mechanisms by biochemical techniques is often difficult to perform. Here, we report on the suitability of a genetic system, i.e. the yeast split-ubiquitin system, to investigate protein–protein interactions of Thylakoid Membrane proteins. The data confirm the previously established binding of the cpSec-translocase subunits, cpSecY and cpSecE, and the interaction of the cpSec-translocase from Arabidopsis thaliana with Alb3, a factor required for the insertion of the light-harvesting chlorophyll-binding proteins into the Thylakoid Membrane. In addition, the proposed interaction between D1, the reaction center protein of photosystem II and the soluble periplasmic PratA factor from Synechocystis sp. PCC 6803 was verified. A more comprehensive analysis of Alb3-interacting proteins revealed that Alb3 is able to form dimers or oligomers. Interestingly, Alb3 was also shown to bind to the PSII proteins D1, D2 and CP43, to the PSI reaction center protein PSI-A and the ATP synthase subunit CF0III, suggesting an important role of Alb3 in the assembly of photosynthetic Thylakoid Membrane complexes.
Krishna K. Niyogi - One of the best experts on this subject based on the ideXlab platform.
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a Thylakoid Membrane bound and redox active rubredoxin rbd1 functions in de novo assembly and repair of photosystem ii
Proceedings of the National Academy of Sciences of the United States of America, 2019Co-Authors: Jose G Garciacerdan, Ariel L. Furst, Kent L. Mcdonald, Danja Schunemann, Matthew B. Francis, Krishna K. NiyogiAbstract:Author(s): Garcia-Cerdan, Jose G; Furst, Ariel L; McDonald, Kent L; Schunemann, Danja; Francis, Matthew B; Niyogi, Krishna K | Abstract: Photosystem II (PSII) undergoes frequent photooxidative damage that, if not repaired, impairs photosynthetic activity and growth. How photosynthetic organisms protect vulnerable PSII intermediate complexes during de novo assembly and repair remains poorly understood. Here, we report the genetic and biochemical characterization of chloroplast-located rubredoxin 1 (RBD1), a PSII assembly factor containing a redox-active rubredoxin domain and a single C-terminal transMembrane α-helix (TMH) domain. RBD1 is an integral Thylakoid Membrane protein that is enriched in stroma lamellae fractions with the rubredoxin domain exposed on the stromal side. RBD1 also interacts with PSII intermediate complexes containing cytochrome b 559 Complementation of the Chlamydomonas reinhardtii (hereafter Chlamydomonas) RBD1-deficient 2pac mutant with constructs encoding RBD1 protein truncations and site-directed mutations demonstrated that the TMH domain is essential for de novo PSII assembly, whereas the rubredoxin domain is involved in PSII repair. The rubredoxin domain exhibits a redox midpoint potential of +114 mV and is proficient in 1-electron transfers to a surrogate cytochrome c in vitro. Reduction of oxidized RBD1 is NADPH dependent and can be mediated by ferredoxin-NADP+ reductase (FNR) in vitro. We propose that RBD1 participates, together with the cytochrome b 559, in the protection of PSII intermediate complexes from photooxidative damage during de novo assembly and repair. This role of RBD1 is consistent with its evolutionary conservation among photosynthetic organisms and the fact that it is essential in photosynthetic eukaryotes.
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a Thylakoid Membrane bound and redox active rubredoxin rbd1 functions in de novo assembly and repair of photosystem ii
Proceedings of the National Academy of Sciences of the United States of America, 2019Co-Authors: Jose G Garciacerdan, Ariel L. Furst, Kent L. Mcdonald, Danja Schunemann, Krishna K. Niyogi, Matthew B. FrancisAbstract:Photosystem II (PSII) undergoes frequent photooxidative damage that, if not repaired, impairs photosynthetic activity and growth. How photosynthetic organisms protect vulnerable PSII intermediate complexes during de novo assembly and repair remains poorly understood. Here, we report the genetic and biochemical characterization of chloroplast-located rubredoxin 1 (RBD1), a PSII assembly factor containing a redox-active rubredoxin domain and a single C-terminal transMembrane α-helix (TMH) domain. RBD1 is an integral Thylakoid Membrane protein that is enriched in stroma lamellae fractions with the rubredoxin domain exposed on the stromal side. RBD1 also interacts with PSII intermediate complexes containing cytochrome b 559 Complementation of the Chlamydomonas reinhardtii (hereafter Chlamydomonas) RBD1-deficient 2pac mutant with constructs encoding RBD1 protein truncations and site-directed mutations demonstrated that the TMH domain is essential for de novo PSII assembly, whereas the rubredoxin domain is involved in PSII repair. The rubredoxin domain exhibits a redox midpoint potential of +114 mV and is proficient in 1-electron transfers to a surrogate cytochrome c in vitro. Reduction of oxidized RBD1 is NADPH dependent and can be mediated by ferredoxin-NADP+ reductase (FNR) in vitro. We propose that RBD1 participates, together with the cytochrome b 559, in the protection of PSII intermediate complexes from photooxidative damage during de novo assembly and repair. This role of RBD1 is consistent with its evolutionary conservation among photosynthetic organisms and the fact that it is essential in photosynthetic eukaryotes.
Himadri B. Pakrasi - One of the best experts on this subject based on the ideXlab platform.
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a novel redoxin in the Thylakoid Membrane regulates the titer of photosystem i
Journal of Biological Chemistry, 2016Co-Authors: Yuehui Zhu, Michelle Liberton, Himadri B. PakrasiAbstract:In photosynthetic organisms like cyanobacteria and plants, the main engines of oxygenic photosynthesis are the pigment-protein complexes photosystem I (PSI) and photosystem II (PSII) located in the Thylakoid Membrane. In the cyanobacterium Synechocystis sp. PCC 6803, the slr1796 gene encodes a single cysteine thioredoxin-like protein, orthologs of which are found in multiple cyanobacterial strains as well as chloroplasts of higher plants. Targeted inactivation of slr1796 in Synechocystis 6803 resulted in compromised photoautotrophic growth. The mutant displayed decreased chlorophyll a content. These changes correlated with a decrease in the PSI titer of the mutant cells, whereas the PSII content was unaffected. In the mutant, the transcript levels of genes for PSI structural and accessory proteins remained unaffected, whereas the levels of PSI structural proteins were severely diminished, indicating that Slr1796 acts at a posttranscriptional level. Biochemical analysis indicated that Slr1796 is an integral Thylakoid Membrane protein. We conclude that Slr1796 is a novel regulatory factor that modulates PSI titer.
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global proteomic analysis reveals an exclusive role of Thylakoid Membranes in bioenergetics of a model cyanobacterium
Molecular & Cellular Proteomics, 2016Co-Authors: Michelle Liberton, Rajib Saha, Jon M Jacobs, Amelia Y Nguyen, Marina A Gritsenko, Richard D Smith, David W Koppenaal, Himadri B. PakrasiAbstract:Cyanobacteria are photosynthetic microbes with highly differentiated Membrane systems. These organisms contain an outer Membrane, plasma Membrane, and an internal system of Thylakoid Membranes where the photosynthetic and respiratory machinery are found. This existence of compartmentalization and differentiation of Membrane systems poses a number of challenges for cyanobacterial cells in terms of organization and distribution of proteins to the correct Membrane system. Proteomics studies have long sought to identify the components of the different Membrane systems in cyanobacteria, and to date about 450 different proteins have been attributed to either the plasma Membrane or Thylakoid Membrane. Given the complexity of these Membranes, many more proteins remain to be identified, and a comprehensive catalogue of plasma Membrane and Thylakoid Membrane proteins is needed. Here we describe the identification of 635 differentially localized proteins in Synechocystis sp. PCC 6803 by quantitative iTRAQ isobaric labeling; of these, 459 proteins were localized to the plasma Membrane and 176 were localized to the Thylakoid Membrane. Surprisingly, we found over 2.5 times the number of unique proteins identified in the plasma Membrane compared with the Thylakoid Membrane. This suggests that the protein composition of the Thylakoid Membrane is more homogeneous than the plasma Membrane, consistent with the role of the plasma Membrane in diverse cellular processes including protein trafficking and nutrient import, compared with a more specialized role for the Thylakoid Membrane in cellular energetics. Thus, our data clearly define the two Membrane systems with distinct functions. Overall, the protein compositions of the Synechocystis 6803 plasma Membrane and Thylakoid Membrane are quite similar to that of the plasma Membrane of Escherichia coli and Thylakoid Membrane of Arabidopsis chloroplasts, respectively. Synechocystis 6803 can therefore be described as a Gram-negative bacterium with an additional internal Membrane system that fulfills the energetic requirements of the cell.
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photosynthetic pigment localization and Thylakoid Membrane morphology are altered in synechocystis 6803 phycobilisome mutants
Plant Physiology, 2012Co-Authors: Aaron M Collins, Michelle Liberton, Himadri B. Pakrasi, Howland D T Jones, Omar Fidel Garcia, Jerilyn A TimlinAbstract:Cyanobacteria are oxygenic photosynthetic prokaryotes that are the progenitors of the chloroplasts of algae and plants. These organisms harvest light using large Membrane-extrinsic phycobilisome antenna in addition to Membrane-bound chlorophyll-containing proteins. Similar to eukaryotic photosynthetic organisms, cyanobacteria possess Thylakoid Membranes that house photosystem (PS) I and PSII, which drive the oxidation of water and the reduction of NADP+, respectively. While Thylakoid morphology has been studied in some strains of cyanobacteria, the global distribution of PSI and PSII within the Thylakoid Membrane and the corresponding location of the light-harvesting phycobilisomes are not known in detail, and such information is required to understand the functioning of cyanobacterial photosynthesis on a larger scale. Here, we have addressed this question using a combination of electron microscopy and hyperspectral confocal fluorescence microscopy in wild-type Synechocystis species PCC 6803 and a series of mutants in which phycobilisomes are progressively truncated. We show that as the phycobilisome antenna is diminished, large-scale changes in Thylakoid morphology are observed, accompanied by increased physical segregation of the two photosystems. Finally, we quantified the emission intensities originating from the two photosystems in vivo on a per cell basis to show that the PSI:PSII ratio is progressively decreased in the mutants. This results from both an increase in the amount of photosystem II and a decrease in the photosystem I concentration. We propose that these changes are an adaptive strategy that allows cells to balance the light absorption capabilities of photosystems I and II under light-limiting conditions.
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photochemical competence of assembled photosystem ii core complex in cyanobacterial plasma Membrane
Journal of Biological Chemistry, 2005Co-Authors: Nir Keren, Michelle Liberton, Himadri B. PakrasiAbstract:Abstract Cyanobacterial cells have two autonomous internal Membrane systems, plasma Membrane and Thylakoid Membrane. In these oxygenic photosynthetic organisms the assembly of the large Membrane protein complex photosystem II (PSII) is an intricate process that requires the recruitment of numerous protein subunits and cofactors involved in excitation and electron transfer processes. Precise control of this assembly process is necessary because electron transfer reactions in partially assembled PSII can lead to oxidative damage and degradation of the protein complex. In this communication we demonstrate that the activation of PSII electron transfer reactions in the cyanobacterium Synechocystis sp. PCC 6803 takes place sequentially. In this organism partially assembled PSII complexes can be detected in the plasma Membrane. We have determined that such PSII complexes can undergo light-induced charge separation and contain a functional electron acceptor side but not an assembled donor side. In contrast, PSII complexes in Thylakoid Membrane are fully assembled and capable of multiple turnovers. We conclude that PSII reaction center cores assembled in the plasma Membrane are photochemically competent and can catalyze single turnovers. We propose that upon transfer of such PSII core complexes to the Thylakoid Membrane, additional proteins are incorporated followed by binding and activation of various donor side cofactors. Such a stepwise process protects cyanobacterial cells from potentially harmful consequences of performing water oxidation in a partially assembled PSII complex before it reaches its final destination in the Thylakoid Membrane.
Michelle Liberton - One of the best experts on this subject based on the ideXlab platform.
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a novel redoxin in the Thylakoid Membrane regulates the titer of photosystem i
Journal of Biological Chemistry, 2016Co-Authors: Yuehui Zhu, Michelle Liberton, Himadri B. PakrasiAbstract:In photosynthetic organisms like cyanobacteria and plants, the main engines of oxygenic photosynthesis are the pigment-protein complexes photosystem I (PSI) and photosystem II (PSII) located in the Thylakoid Membrane. In the cyanobacterium Synechocystis sp. PCC 6803, the slr1796 gene encodes a single cysteine thioredoxin-like protein, orthologs of which are found in multiple cyanobacterial strains as well as chloroplasts of higher plants. Targeted inactivation of slr1796 in Synechocystis 6803 resulted in compromised photoautotrophic growth. The mutant displayed decreased chlorophyll a content. These changes correlated with a decrease in the PSI titer of the mutant cells, whereas the PSII content was unaffected. In the mutant, the transcript levels of genes for PSI structural and accessory proteins remained unaffected, whereas the levels of PSI structural proteins were severely diminished, indicating that Slr1796 acts at a posttranscriptional level. Biochemical analysis indicated that Slr1796 is an integral Thylakoid Membrane protein. We conclude that Slr1796 is a novel regulatory factor that modulates PSI titer.
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global proteomic analysis reveals an exclusive role of Thylakoid Membranes in bioenergetics of a model cyanobacterium
Molecular & Cellular Proteomics, 2016Co-Authors: Michelle Liberton, Rajib Saha, Jon M Jacobs, Amelia Y Nguyen, Marina A Gritsenko, Richard D Smith, David W Koppenaal, Himadri B. PakrasiAbstract:Cyanobacteria are photosynthetic microbes with highly differentiated Membrane systems. These organisms contain an outer Membrane, plasma Membrane, and an internal system of Thylakoid Membranes where the photosynthetic and respiratory machinery are found. This existence of compartmentalization and differentiation of Membrane systems poses a number of challenges for cyanobacterial cells in terms of organization and distribution of proteins to the correct Membrane system. Proteomics studies have long sought to identify the components of the different Membrane systems in cyanobacteria, and to date about 450 different proteins have been attributed to either the plasma Membrane or Thylakoid Membrane. Given the complexity of these Membranes, many more proteins remain to be identified, and a comprehensive catalogue of plasma Membrane and Thylakoid Membrane proteins is needed. Here we describe the identification of 635 differentially localized proteins in Synechocystis sp. PCC 6803 by quantitative iTRAQ isobaric labeling; of these, 459 proteins were localized to the plasma Membrane and 176 were localized to the Thylakoid Membrane. Surprisingly, we found over 2.5 times the number of unique proteins identified in the plasma Membrane compared with the Thylakoid Membrane. This suggests that the protein composition of the Thylakoid Membrane is more homogeneous than the plasma Membrane, consistent with the role of the plasma Membrane in diverse cellular processes including protein trafficking and nutrient import, compared with a more specialized role for the Thylakoid Membrane in cellular energetics. Thus, our data clearly define the two Membrane systems with distinct functions. Overall, the protein compositions of the Synechocystis 6803 plasma Membrane and Thylakoid Membrane are quite similar to that of the plasma Membrane of Escherichia coli and Thylakoid Membrane of Arabidopsis chloroplasts, respectively. Synechocystis 6803 can therefore be described as a Gram-negative bacterium with an additional internal Membrane system that fulfills the energetic requirements of the cell.
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photosynthetic pigment localization and Thylakoid Membrane morphology are altered in synechocystis 6803 phycobilisome mutants
Plant Physiology, 2012Co-Authors: Aaron M Collins, Michelle Liberton, Himadri B. Pakrasi, Howland D T Jones, Omar Fidel Garcia, Jerilyn A TimlinAbstract:Cyanobacteria are oxygenic photosynthetic prokaryotes that are the progenitors of the chloroplasts of algae and plants. These organisms harvest light using large Membrane-extrinsic phycobilisome antenna in addition to Membrane-bound chlorophyll-containing proteins. Similar to eukaryotic photosynthetic organisms, cyanobacteria possess Thylakoid Membranes that house photosystem (PS) I and PSII, which drive the oxidation of water and the reduction of NADP+, respectively. While Thylakoid morphology has been studied in some strains of cyanobacteria, the global distribution of PSI and PSII within the Thylakoid Membrane and the corresponding location of the light-harvesting phycobilisomes are not known in detail, and such information is required to understand the functioning of cyanobacterial photosynthesis on a larger scale. Here, we have addressed this question using a combination of electron microscopy and hyperspectral confocal fluorescence microscopy in wild-type Synechocystis species PCC 6803 and a series of mutants in which phycobilisomes are progressively truncated. We show that as the phycobilisome antenna is diminished, large-scale changes in Thylakoid morphology are observed, accompanied by increased physical segregation of the two photosystems. Finally, we quantified the emission intensities originating from the two photosystems in vivo on a per cell basis to show that the PSI:PSII ratio is progressively decreased in the mutants. This results from both an increase in the amount of photosystem II and a decrease in the photosystem I concentration. We propose that these changes are an adaptive strategy that allows cells to balance the light absorption capabilities of photosystems I and II under light-limiting conditions.
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unique Thylakoid Membrane architecture of a unicellular n2 fixing cyanobacterium revealed by electron tomography
Plant Physiology, 2011Co-Authors: Michelle Liberton, Howard R BergAbstract:Cyanobacteria, descendants of the endosymbiont that gave rise to modern-day chloroplasts, are vital contributors to global biological energy conversion processes. A thorough understanding of the physiology of cyanobacteria requires detailed knowledge of these organisms at the level of cellular architecture and organization. In these prokaryotes, the large Membrane protein complexes of the photosynthetic and respiratory electron transport chains function in the intracellular Thylakoid Membranes. Like plants, the architecture of the Thylakoid Membranes in cyanobacteria has direct impact on cellular bioenergetics, protein transport, and molecular trafficking. However, whole-cell Thylakoid organization in cyanobacteria is not well understood. Here we present, by using electron tomography, an in-depth analysis of the architecture of the Thylakoid Membranes in a unicellular cyanobacterium, Cyanothece sp. ATCC 51142. Based on the results of three-dimensional tomographic reconstructions of near-entire cells, we determined that the Thylakoids in Cyanothece 51142 form a dense and complex network that extends throughout the entire cell. This Thylakoid Membrane network is formed from the branching and splitting of Membranes and encloses a single lumenal space. The entire Thylakoid network spirals as a peripheral ring of Membranes around the cell, an organization that has not previously been described in a cyanobacterium. Within the Thylakoid Membrane network are areas of quasi-helical arrangement with similarities to the Thylakoid Membrane system in chloroplasts. This cyanobacterial Thylakoid arrangement is an efficient means of packing a large volume of Membranes in the cell while optimizing intracellular transport and trafficking.
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photochemical competence of assembled photosystem ii core complex in cyanobacterial plasma Membrane
Journal of Biological Chemistry, 2005Co-Authors: Nir Keren, Michelle Liberton, Himadri B. PakrasiAbstract:Abstract Cyanobacterial cells have two autonomous internal Membrane systems, plasma Membrane and Thylakoid Membrane. In these oxygenic photosynthetic organisms the assembly of the large Membrane protein complex photosystem II (PSII) is an intricate process that requires the recruitment of numerous protein subunits and cofactors involved in excitation and electron transfer processes. Precise control of this assembly process is necessary because electron transfer reactions in partially assembled PSII can lead to oxidative damage and degradation of the protein complex. In this communication we demonstrate that the activation of PSII electron transfer reactions in the cyanobacterium Synechocystis sp. PCC 6803 takes place sequentially. In this organism partially assembled PSII complexes can be detected in the plasma Membrane. We have determined that such PSII complexes can undergo light-induced charge separation and contain a functional electron acceptor side but not an assembled donor side. In contrast, PSII complexes in Thylakoid Membrane are fully assembled and capable of multiple turnovers. We conclude that PSII reaction center cores assembled in the plasma Membrane are photochemically competent and can catalyze single turnovers. We propose that upon transfer of such PSII core complexes to the Thylakoid Membrane, additional proteins are incorporated followed by binding and activation of various donor side cofactors. Such a stepwise process protects cyanobacterial cells from potentially harmful consequences of performing water oxidation in a partially assembled PSII complex before it reaches its final destination in the Thylakoid Membrane.