Abstract: Twin arginine translocation (Tat) systems transport folded proteins using the proton motive force as sole energy source. The thylakoid Tat system comprises three membrane components. A complex composed of cpTatC and Hcf106 is the twin arginine signal peptide receptor. Signal peptide binding triggers assembly of Tha4 for the translocation step. Tha4 is thought to serve as the protein-conducting element, and the topology it adopts during transport produces the transmembrane passageway. We analyzed Tha4 topology and conformation in actively transporting translocases and compared that with Tha4 in non-transporting membranes. Using cysteine accessibility labeling techniques and diagnostic protease protection assays, we confirm an overall NOUT-CIN topology for Tha4 that is maintained under transport conditions. Significantly, the APH and C-tail exhibited substantial changes in accessibility when actively engaged in protein transport. Compared to resting state, cysteines within the APH became less accessible to stromally applied modifying reagent. The APH proximal C-tail, although still accessible to Cys-directed reagents, was much less accessible to protease. We attribute these changes in accessibility to indicate the conformation Tha4 adopts in the translocase primed for translocation. We propose that in the primed translocase, the APH partitions more extensively and uniformly into the membrane interface and the C-tails pack closer together in a mesh-like network. Implications for the mode by which the substrate protein crosses the bilayer are discussed.
Abstract: 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.
Abstract: The thylakoid membrane forms stacked thylakoids interconnected by 'stromal' lamellae. Little is known about the mobility of proteins within this system. We studied a stromal lamellae protein, Hcf106, by targeting an Hcf106-GFP fusion protein to the thylakoids and photobleaching. We find that even small regions fail to recover Hcf106-GFP fluorescence over periods of up to 3 min after photobleaching. The protein is thus either immobile within the thylakoid membrane, or its diffusion is tightly restricted within distinct regions. Autofluorescence from the photosystem II light-harvesting complex in the granal stacks likewise fails to recover. Integral membrane proteins within both the stromal and granal membranes are therefore highly constrained, possibly forming 'microdomains' that are sharply separated.
Abstract: Cyanobacteria possess a differentiated membrane system and transport proteins into both the periplasm and thylakoid lumen. We have used green fluorescent protein (GFP)-tagged constructs to study the Tat protein transporter and Rieske Tat substrates in Synechocystis PCC6803. The Tat system has been shown to operate in the plasma membrane; we show here that it is also relatively abundant in the thylakoid membrane network, indicating that newly synthesized Tat substrates are targeted to both membrane systems. Synechocystis contains three Rieske iron-sulphur proteins, all of which contain typical twin-arginine signal-like sequences at their N-termini. We show that two of these proteins (PetC1 and PetC2) are obligate Tat substrates when expressed in Escherichia coli. The Rieske proteins exhibit differential localization in Synechocystis 6803; PetC1 and PetC2 are located in the thylakoid membrane, while PetC3 is primarily targeted to the plasma membrane. The combined data show that Tat substrates are directed with high precision to both membrane systems in this cyanobacterium, raising the question of how, and when, intracellular sorting to the correct membrane is achieved.
Abstract: Plastids are essential plant organelles vital for life on earth, responsible not only for photosynthesis but for many fundamental intermediary metabolic reactions. Plastids are not formed de novo but arise by binary fission from pre-existing plastids, and plastid division therefore represents an important process for the maintenance of appropriate plastid populations in plant cells. Plastid division comprises an elaborate pathway of co-ordinated events which include division machinery assembly at the division site, the constriction of envelope membranes, membrane fusion and, ultimately, the separation of the two new organelles. Because of their prokaryotic origin bacterial cell division has been successfully used as a paradigm for plastid division. This has resulted in the identification of the key plastid division components FtsZ, MinD, and MinE, as well as novel proteins with similarities to prokaryotic cell division proteins. Through a combination of approaches involving molecular genetics, cell biology, and biochemistry, it is now becoming clear that these proteins act in concert during plastid division, exhibiting both similarities and differences compared with their bacterial counterparts. Recent efforts in the cloning of the disrupted loci in several of the accumulation and replication of chloroplasts mutants has further revealed that the division of plastids is controlled by a combination of prokaryote-derived and host eukaryote-derived proteins residing not only in the plastid stroma but also in the cytoplasm. Based on the available data to date, a working model is presented showing the protein components involved in plastid division, their subcellular localization, and their protein interaction properties.
Abstract: Plastids arise by division from pre-existing organelles, and with the recent characterization of several new components of plastid division our understanding of the division process in higher plants has improved dramatically. However, it is still not known how these different protein components act together during division. Here we analyse protein-protein interactions between all known stromal plastid division proteins. Using a combination of quantitative yeast two-hybrid assays, in planta co-localization studies, fluorescence resonance energy transfer and bimolecular fluorescence complementation assays we show that these proteins do not act in isolation but rather in protein complexes to govern appropriate plastid division. We have previously shown that AtMinD1 forms functional homodimers and we show here that in addition to homodimerization AtMinD1 also interacts with AtMinE1. Furthermore, AtMinE1 has the ability to homodimerize. We also demonstrate that proteins from both FtsZ families (AtFtsZ1-1 and AtFtsZ2-1) not only interact with themselves but also with each other, and we show that these interactions are not dependent on correct Z-ring formation. Further to this we demonstrate that ARC6 specifically interacts with the core domain of AtFtsZ2-1, but not with AtFtsZ1-1, providing in planta evidence for a functional difference between the two FtsZ protein families in plants. Our studies have enabled us to construct a meaningful intraplastidic protein-protein interaction map of all known stromal plastid division proteins in Arabidopsis.
Abstract: Bacteria and plastids divide symmetrically through binary fission by accurately placing the division site at midpoint, a process initiated by FtsZ polymerization, which forms a Z-ring. In Escherichia coli precise Z-ring placement at midcell depends on controlled oscillatory behavior of MinD and MinE: In the presence of ATP MinD interacts with the FtsZ inhibitor MinC and migrates to the membrane where the MinD-MinC complex recruits MinE, followed by MinD-mediated ATP hydrolysis and membrane release. Although correct Z-ring placement during Arabidopsis plastid division depends on the precise localization of the bacterial homologs AtMinD1 and AtMinE1, the underlying mechanism of this process remains unknown. Here we have shown that AtMinD1 is a Ca2+-dependent ATPase and through mutation analysis demonstrated the physiological importance of this activity where loss of ATP hydrolysis results in protein mislocalization within plastids. The observed mislocalization is not due to disrupted AtMinD1 dimerization, however; the active site AtMinD1(K72A) mutant is unable to interact with the topological specificity factor AtMinE1. We have shown that AtMinE1, but not E. coli MinE, stimulates AtMinD1-mediated ATP hydrolysis, but in contrast to prokaryotes stimulation occurs in the absence of membrane lipids. Although AtMinD1 appears highly evolutionarily conserved, we found that important biochemical and cell biological properties have diverged. We propose that correct intraplastidic AtMinD1 localization is dependent on AtMinE1-stimulated, Ca2+-dependent AtMinD1 ATP hydrolysis, ultimately ensuring precise Z-ring placement and symmetric plastid division.
