The thylakoid proton antiporter KEA3 regulates photosynthesis in response to the chloroplast energy status.

Plant photosynthesis contains two functional modules, the light-driven reactions in the thylakoid membrane and the carbon-fixing reactions in the chloroplast stroma. In nature, light availability for photosynthesis often undergoes massive and rapid fluctuations. Efficient and productive use of such variable light supply requires an instant crosstalk and rapid synchronization of both functional modules. Here, we show that this communication involves the stromal exposed C-terminus of the thylakoid K+-exchange antiporter KEA3, which regulates the ΔpH across the thylakoid membrane and therefore pH-dependent photoprotection. By combining in silico, in vitro, and in vivo approaches, we demonstrate that the KEA3 C-terminus senses the energy state of the chloroplast in a pH-dependent manner and regulates transport activity in response. Together our data pinpoint a regulatory feedback loop by which the stromal energy state orchestrates light capture and photoprotection via multi-level regulation of KEA3.

Chloroplast Ribosomes Interact With the Insertase Alb3 in the Thylakoid Membrane.

Members of the Oxa1/YidC/Alb3 protein family are involved in the insertion, folding, and assembly of membrane proteins in mitochondria, bacteria, and chloroplasts. The thylakoid membrane protein Alb3 mediates the chloroplast signal recognition particle (cpSRP)-dependent posttranslational insertion of nuclear-encoded light harvesting chlorophyll a/b-binding proteins and participates in the biogenesis of plastid-encoded subunits of the photosynthetic complexes. These subunits are cotranslationally inserted into the thylakoid membrane, yet very little is known about the molecular mechanisms underlying docking of the ribosome-nascent chain complexes to the chloroplast SecY/Alb3 insertion machinery. Here, we show that nanodisc-embedded Alb3 interacts with ribosomes, while the homolog Alb4, also located in the thylakoid membrane, shows no ribosome binding. Alb3 contacts the ribosome with its C-terminal region and at least one additional binding site within its hydrophobic core region. Within the C-terminal region, two conserved motifs (motifs III and IV) are cooperatively required to enable the ribosome contact. Furthermore, our data suggest that the negatively charged C-terminus of the ribosomal subunit uL4c is involved in Alb3 binding. Phylogenetic analyses of uL4 demonstrate that this region newly evolved in the green lineage during the transition from aquatic to terrestrial life.

Ribosome-Associated Chloroplast SRP54 Enables Efficient Cotranslational Membrane Insertion of Key Photosynthetic Proteins.

Key proteins of the photosynthetic complexes are encoded in the chloroplast genome and cotranslationally inserted into the thylakoid membrane. However, the molecular details of this process are largely unknown. Here, we demonstrate by ribosome profiling that the conserved chloroplast signal recognition particle subunit (cpSRP54) is required for efficient cotranslational targeting of several central photosynthetic proteins, such as the PSII PsbA (D1) subunit, in Arabidopsis (Arabidopsis thaliana). High-resolution analysis of membrane-associated and soluble ribosome footprints revealed that the SRP-dependent membrane targeting of PsbA is already initiated at an early translation step before exposure of the nascent chain from the ribosome. In contrast to cytosolic SRP, which contacts the ribosome close to the peptide tunnel exit site, analysis of the cpSRP54/ribosome binding interface revealed a direct interaction of cpSRP54 and the ribosomal subunit uL4, which is not located at the tunnel exit site but forms a part of the internal peptide tunnel wall by a loop domain. The plastid-specific C-terminal tail region of cpSRP54 plays a crucial role in uL4 binding. Our data indicate a novel mechanism of SRP-dependent membrane protein transport with the cpSRP54/uL4 interaction as a central element in early initiation of cotranslational membrane targeting.

Molecular mechanism of SRP-dependent light-harvesting protein transport to the thylakoid membrane in plants.

The light-harvesting chlorophyll a/b binding proteins (LHCP) belong to a large family of membrane proteins. They form the antenna complexes of photosystem I and II and function in light absorption and transfer of the excitation energy to the photosystems. As nuclear-encoded proteins, the LHCPs are imported into the chloroplast and further targeted to their final destination-the thylakoid membrane. Due to their hydrophobicity, the formation of the so-called 'transit complex' in the stroma is important to prevent their aggregation in this aqueous environment. The posttranslational LHCP targeting mechanism is well regulated through the interaction of various soluble and membrane-associated protein components and includes several steps: the binding of the LHCP to the heterodimeric cpSRP43/cpSRP54 complex to form the soluble transit complex; the docking of the transit complex to the SRP receptor cpFtsY and the Alb3 translocase at the membrane followed by the release and integration of the LHCP into the thylakoid membrane in a GTP-dependent manner. This review summarizes the molecular mechanisms and dynamics behind the posttranslational LHCP targeting to the thylakoid membrane of Arabidopsis thaliana.

From bacteria to chloroplasts: evolution of the chloroplast SRP system.

Chloroplasts derive from a prokaryotic symbiont that lost most of its genes during evolution. As a result, the great majority of chloroplast proteins are encoded in the nucleus and are posttranslationally imported into the organelle. The chloroplast genome encodes only a few proteins. These include several multispan thylakoid membrane proteins which are synthesized on thylakoid-bound ribosomes and cotranslationally inserted into the membrane. During evolution, ancient prokaryotic targeting machineries were adapted and combined with novel targeting mechanisms to facilitate post- and cotranslational protein transport in chloroplasts. This review focusses on the chloroplast signal recognition particle (cpSRP) protein transport system, which has been intensively studied in higher plants. The cpSRP system derived from the prokaryotic SRP pathway, which mediates the cotranslational protein transport to the bacterial plasma membrane. Chloroplasts contain homologs of several components of the bacterial SRP system. The function of these conserved components in post- and/or cotranslational protein transport and chloroplast-specific modifications of these transport mechanisms are described. Furthermore, recent studies of cpSRP systems in algae and lower plants are summarized and their impact on understanding the evolution of the cpSRP system are discussed.

The Chloroplast SRP Systems of Chaetosphaeridium globosum and Physcomitrella patens as Intermediates in the Evolution of SRP-Dependent Protein Transport in Higher Plants.

The bacterial signal recognition particle (SRP) mediates the cotranslational targeting of membrane proteins and is a high affinity complex consisting of a SRP54 protein subunit (Ffh) and an SRP RNA. The chloroplast SRP (cpSRP) pathway has adapted throughout evolution to enable the posttranslational targeting of the light harvesting chlorophyll a/b binding proteins (LHCPs) to the thylakoid membrane. In spermatophytes (seed plants), the cpSRP lacks the SRP RNA and is instead formed by a high affinity interaction of the conserved 54-kD subunit (cpSRP54) with the chloroplast-specific cpSRP43 protein. This heterodimeric cpSRP recognizes LHCP and delivers it to the thylakoid membrane. However, in contrast to spermatophytes, plastid SRP RNAs were identified within all streptophyte lineages and in all chlorophyte branches. Furthermore, it was shown that cpSRP43 does not interact with cpSRP54 in chlorophytes (e.g., Chlamydomonas reinhardtii). In this study, we biochemically characterized the cpSRP system of the charophyte Chaetosphaeridium globosum and the bryophyte Physcomitrella patens. Interaction studies demonstrate low affinity binding of cpSRP54 to cpSRP43 (Kd ~10 µM) in Chaetosphaeridium globosum and Physcomitrella patens as well as relatively low affinity binding of cpSRP54 to cpSRP RNA (Kd ~1 µM) in Physcomitrella patens. CpSRP54/cpSRP43 complex formation in charophytes is supported by the finding that specific alterations in the second chromodomain of cpSRP43, that are conserved within charophytes and absent in land plants, do not interfere with cpSRP54 binding. Furthermore, our data show that the elongated apical loop structure of the Physcomitrella patens cpSRP RNA contributes to the low binding affinity between cpSRP54 and the cpSRP RNA.

Chloroplast SRP54 Was Recruited for Posttranslational Protein Transport via Complex Formation with Chloroplast SRP43 during Land Plant Evolution.

In bacteria, membrane proteins are targeted cotranslationally via a signal recognition particle (SRP). During the evolution of higher plant chloroplasts from cyanobacteria, the SRP pathway underwent striking adaptations that enable the posttranslational transport of the abundant light-harvesting chlorophyll-a/b-binding proteins (LHCPs). The conserved 54-kDa SRP subunit in higher plant chloroplasts (cpSRP54) is not bound to an SRP RNA, an essential SRP component in bacteria, but forms a stable heterodimer with the chloroplast-specific cpSRP43. This heterodimeric cpSRP recognizes LHCP and delivers it to the thylakoid membrane whereby cpSRP43 plays a central role. This study shows that the cpSRP system in the green alga Chlamydomonas reinhardtii differs significantly from that of higher plants as cpSRP43 is not complexed to cpSRP54 in Chlamydomonas and cpSRP54 is not involved in LHCP recognition. This divergence is attributed to altered residues within the cpSRP54 tail and the second chromodomain of cpSRP43 that are crucial for the formation of the binding interface in Arabidopsis. These changes are highly conserved among chlorophytes, whereas all land plants contain cpSRP proteins with typical interaction motifs. These data demonstrate that the coevolution of LHCPs and cpSRP43 occurred independently of complex formation with cpSRP54 and that the interaction between cpSRP54 and cpSRP43 evolved later during the transition from chlorophytes to land plants. Furthermore, our data show that in higher plants a heterodimeric form of cpSRP is required for the formation of a low molecular weight transit complex with LHCP.

The peptide microarray "ChloroPhos1.0" identifies new phosphorylation targets of plastid casein kinase II (pCKII) in Arabidopsis thaliana.

We report the development of a peptide microarray based on previously determined phosphorylation sites in chloroplast proteins. Altogether, 905 peptides were spotted as 15mers in nine replicates onto glass slides. We used the microarray for in vitro phosphorylation experiments and specifically assessed the peptide substrate spectrum of chloroplast casein kinase II (pCKII). To this end, native pCKII from Arabidopsis thaliana and Sinapis alba chloroplasts was enriched by Heparin-Sepharose chromatography and its activity on the microarray was compared to the activity of a recombinant Arabidopsis pCKII. All three kinase preparations phosphorylated a similar set of peptides that were clearly distinct from those phosphorylated by bovine heart protein kinase A (PKA) in control experiments. The majority of the pCKII phosphorylation targets are involved in plastid gene expression, supporting the earlier denomination of pCKII as plastid transcription kinase (PTK). In addition we identified Alb3 as pCKII substrate that is essential for the integration of light-harvesting complex subunits (LHC) into the thylakoid membrane. Plastid CKII phosphorylation activity was characterized in greater detail in vitro with recombinant wildtype Alb3 and phosphorylation site mutants as substrates, establishing S424 as the pCKII phosphorylation site. Our data show that the peptide microarray ChloroPhos1.0 is a suitable tool for the identification of new kinase downstream targets in vitro that can be validated subsequently by in vivo experiments.

Interaction studies between the chloroplast signal recognition particle subunit cpSRP43 and the full-length translocase Alb3 reveal a membrane-embedded binding region in Alb3 protein.

Posttranslational targeting of the light-harvesting chlorophyll a,b-binding proteins depends on the function of the chloroplast signal recognition particle, its receptor cpFtsY, and the translocase Alb3. The thylakoid membrane protein Alb3 of Arabidopsis chloroplasts belongs to the evolutionarily conserved YidC/Oxa1/Alb3 protein family; the members of this family facilitate the insertion, folding, and assembly of membrane proteins in bacteria, mitochondria, and chloroplasts. Here, we analyzed the interaction sites of full-length Alb3 with the cpSRP pathway component cpSRP43 by using in vitro and in vivo studies. Bimolecular fluorescence complementation and Alb3 proteoliposome studies showed that the interaction of cpSRP43 is dependent on a binding domain in the C terminus of Alb3 as well as an additional membrane-embedded binding site in the fifth transmembrane domain (TMD5) of Alb3. The C-terminal binding domain was mapped to residues 374-388, and the binding domain within TMD5 was mapped to residues 314-318 located close to the luminal end of TMD5. A direct binding between cpSRP43 and these binding motifs was shown by pepspot analysis. Further studies using blue-native gel electrophoresis revealed that full-length Alb3 is able to form dimers. This finding and the identification of a membrane-embedded cpSRP43 binding site in Alb3 support a model in which cpSRP43 inserts into a dimeric Alb3 translocation pore during cpSRP-dependent delivery of light-harvesting chlorophyll a,b-binding proteins.

Interplay between the cpSRP pathway components, the substrate LHCP and the translocase Alb3: an in vivo and in vitro study.

The chloroplast signal recognition particle (cpSRP) and its receptor, cpFtsY, posttranslationally target the nuclear-encoded light-harvesting chlorophyll-binding proteins (LHCPs) to the translocase Alb3 in the thylakoid membrane. In this study, we analyzed the interplay between the cpSRP pathway components, the substrate protein LHCP and the translocase Alb3 by using in vivo and in vitro techniques. We propose that cpSRP43 is crucial for the binding of LHCP-loaded cpSRP and cpFtsY to Alb3. In addition, our data suggest that a direct interaction between Alb3 and LHCP contributes to the formation of this complex.