Mónica Balsera

Characterization of Tic110, a Channel-forming Protein at the Inner Envelope Membrane of Chloroplasts, Unveils a Response to Ca2+ and a Stromal Regulatory Disulfide Bridge

Tic110 has been proposed to be a channel-forming protein at the inner envelope of chloroplasts whose function is essential for the import of proteins synthesized in the cytosol. Sequence features and topology determination experiments presently summarized suggest that Tic110 consists of six transmembrane helices. Its topology has been mapped by limited proteolysis experiments in combination with mass spectrometric determinations and cysteine modification analysis. Two hydrophobic transmembrane helices located in the N terminus serve as a signal for the localization of the protein to the membrane as shown previously. The other amphipathic transmembrane helices are located in the region composed of residues 92–959 in the pea sequence. This results in two regions in the intermembrane space localized to form supercomplexes with the TOC machinery and to receive the transit peptide of preproteins. A large region also resides in the stroma for interaction with proteins such as molecular chaperones. In addition to characterizing the topology of Tic110, we show that Ca2+ has a dramatic effect on channel activity in vitro and that the protein has a redox-active disulfide with the potential to interact with stromal thioredoxin.

TIC62 Redox-regulated Translocon Composition and Dynamics

The preprotein translocon at the inner envelope of chloroplasts (Tic complex) facilitates the import of nuclear-encoded preproteins into the organelle. Seven distinct subunits have been identified so far. For each of those, specific functions have been proposed based on structural prediction or experimental evidence. Three of those subunits possess modules that could act as redox-active regulatory components in the import process. To date, however, the mode of redox regulation of the import process remains enigmatic. To investigate how the chloroplast redox state influences translocon behavior and composition, we studied the Tic component and the putative redox sensor Tic62 in more detail. The experimental results provide evidence that Tic62 can act as a bona fide dehydrogenase in vitro, and that it changes its localization in the chloroplast dependent on the NADP+/NADPH ratio in the stroma. Moreover, the redox state influences the interactions of Tic62 with the translocon and the flavoenzyme ferredoxin-NADP+ oxidoreductase. Additionally, we give initial experimental insights into the Tic62 structure using circular dichroism measurements and demonstrate that the protein consists of two structurally different domains. Our results indicate that Tic62 possesses redox-dependent properties that would allow it to fulfill a role as redox sensor protein in the chloroplast.

Three-dimensional electron cryo-microscopy study of the extrinsic domains of the oxygen-evolving complex of spinach: Assignment of the PsbO protein

Three independent three-dimensional reconstructions of the spinach photosystem II-light-harvesting complex supercomplex were derived from single particle analyses of non-stained, vitrified samples imaged by electron microscopy. Each reconstruction was found to differ significantly in the composition of the lumenal oxygen-evolving complex extrinsic proteins. From difference mapping, aided by electron microscopy of negatively stained selectively washed samples, regions of density were assigned to the PsbO and PsbP/PsbQ proteins. Interpretation of the density assigned to the PsbO protein was explored using computer-aided structural predictions. PsbO is calculated to be mainly a β-protein (38% β) composed of two domains within an overall elongated shape (Pazos, F., Heredia, P., Valencia, A., and De Las Rivas, J. (2001) Proteins Struct. Funct. Genet. 45, 372–381). The positioning and fitting of the proposed structural model for the PsbO protein within the three-dimensional map indicated that there is a single copy per reaction center. Moreover, the structural model derived for PsbO, together with difference mapping, indicates that this protein stretches across the surface of the reaction center with its N- and C-terminal domains located toward the CP47 and CP43 side, respectively. This structural assignment is discussed in terms of the recent x-ray-derived cyanobacterial model of PSII (Zouni, A., Witt, H.-T., Kern, J., Fromme, P., Krauss, N., Saenger, W., and Orth, P. (2001) Nature 409, 739–743).

Tic62: a protein family from metabolism to protein translocation.

BackgroundThe function and structure of protein translocons at the outer and inner envelope membrane of chloroplasts (Toc and Tic complexes, respectively) are a subject of intensive research. One of the proteins that have been ascribed to the Tic complex is Tic62. This protein was proposed as a redox sensor protein and may possibly act as a regulator during the translocation process. Tic62 is a bimodular protein that comprises an N-terminal module, responsible for binding to pyridine nucleotides, and a C-terminal module which serves as a docking site for ferredoxin-NAD(P)-oxido-reductase (FNR). This work focuses on evolutionary analysis of the Tic62-NAD(P)-related protein family, derived from the comparison of all available sequences, and discusses the structure of Tic62.ResultsWhereas the N-terminal module of Tic62 is highly conserved among all oxyphototrophs, the C-terminal region (FNR-binding module) is only found in vascular plants. Phylogenetic analyses classify four Tic62-NAD(P)-related protein subfamilies in land plants, closely related to members from cyanobacteria and green sulphur bacteria. Although most of the Tic62-NAD(P)-related eukaryotic proteins are localized in the chloroplast, one subgroup consists of proteins without a predicted transit peptide. The N-terminal module of Tic62 contains the structurally conserved Rossman fold and probably belongs to the extended family of short-chain dehydrogenases-reductases. Key residues involved in NADP-binding and residues that may attach the protein to the inner envelope membrane of chloroplasts or to the Tic complex are proposed.ConclusionThe Tic62-NAD(P)-related proteins are of ancient origin since they are not only found in cyanobacteria but also in green sulphur bacteria. The FNR-binding module at the C-terminal region of the Tic62 proteins is probably a recent acquisition in vascular plants, with no sequence similarity to any other known motifs. The presence of the FNR-binding domain in vascular plants might be essential for the function of the protein as a Tic component and/or for its regulation.

Redox extends its regulatory reach to chloroplast protein import

The import of chloroplast proteins synthesized in the cytosol of a plant cell is mediated by two multiprotein complexes or translocons located at the outer and inner membranes of the chloroplast envelope, respectively, TOC and TIC. These complexes integrate different signals to assure the timely transport of proteins into the chloroplast in accordance with the metabolic and developmental needs of the cell. The past few years have witnessed the emergence of redox as a regulator of the protein transport process. Here, we discuss evidence that the metabolic redox state of the chloroplast regulates the import of preproteins by altering either the activity or composition of participating transport components. It appears that, through these redox changes, chloroplasts communicate with other compartments of the plant cell.

Thioredoxin targets fundamental processes in a methane-producing archaeon, Methanocaldococcus jannaschii

Significance This study extends thioredoxin (Trx)-based oxidative redox regulation to the archaea, the third domain of life. Our study suggests that Trx is nearly ubiquitous in anaerobic methanogens, enabling them to recover from oxidative stress and synchronize cellular processes, including methane biogenesis, with the availability of reductants. As methane is a valuable fuel, an end product of anaerobic biodegradation and a potent greenhouse gas, Trx may now be considered a critical participant in the global carbon cycle, climate change, and bioenergy production. Because methanogenesis developed before the oxygenation of the earth, our work raises the possibility that Trx functioned in a complex redox regulatory network in anaerobic prokaryotes at least 2.5 billion years ago. Thioredoxin (Trx), a small redox protein, controls multiple processes in eukaryotes and bacteria by changing the thiol redox status of selected proteins. The function of Trx in archaea is, however, unexplored. To help fill this gap, we have investigated this aspect in methanarchaea—strict anaerobes that produce methane, a fuel and greenhouse gas. Bioinformatic analyses suggested that Trx is nearly universal in methanogens. Ancient methanogens that produce methane almost exclusively from H2 plus CO2 carried approximately two Trx homologs, whereas nutritionally versatile members possessed four to eight. Due to its simplicity, we studied the Trx system of Methanocaldococcus jannaschii—a deeply rooted hyperthermophilic methanogen growing only on H2 plus CO2. The organism carried two Trx homologs, canonical Trx1 that reduced insulin and accepted electrons from Escherichia coli thioredoxin reductase and atypical Trx2. Proteomic analyses with air-oxidized extracts treated with reduced Trx1 revealed 152 potential targets representing a range of processes—including methanogenesis, biosynthesis, transcription, translation, and oxidative response. In enzyme assays, Trx1 activated two selected targets following partial deactivation by O2, validating proteomics observations: methylenetetrahydromethanopterin dehydrogenase, a methanogenesis enzyme, and sulfite reductase, a detoxification enzyme. The results suggest that Trx assists methanogens in combating oxidative stress and synchronizing metabolic activities with availability of reductant, making it a critical factor in the global carbon cycle and methane emission. Because methanogenesis developed before the oxygenation of Earth, it seems possible that Trx functioned originally in metabolic regulation independently of O2, thus raising the question whether a complex biological system of this type evolved at least 2.5 billion years ago.

Guanine nucleotide binding to the Bateman domain mediates the allosteric inhibition of eukaryotic IMP dehydrogenases.

Inosine-5′-monophosphate dehydrogenase (IMPDH) plays key roles in purine nucleotide metabolism and cell proliferation. Although IMPDH is a widely studied therapeutic target, there is limited information about its physiological regulation. Using Ashbya gossypii as a model, we describe the molecular mechanism and the structural basis for the allosteric regulation of IMPDH by guanine nucleotides. We report that GTP and GDP bind to the regulatory Bateman domain, inducing octamers with compromised catalytic activity. Our data suggest that eukaryotic and prokaryotic IMPDHs might have developed different regulatory mechanisms, with GTP/GDP inhibiting only eukaryotic IMPDHs. Interestingly, mutations associated with human retinopathies map into the guanine nucleotide-binding sites including a previously undescribed non-canonical site and disrupt allosteric inhibition. Together, our results shed light on the mechanisms of the allosteric regulation of enzymes mediated by Bateman domains and provide a molecular basis for certain retinopathies, opening the door to new therapeutic approaches.

Quaternary structure of the oxaloacetate decarboxylase membrane complex and mechanistic relationships to pyruvate carboxylases.

The oxaloacetate decarboxylase primary Na+ pump (OAD) is an essential membrane protein complex that functions in the citrate fermentation pathway of some pathogenic bacteria under anaerobic conditions. OAD contains three different subunits: Oad-α, a biotinylated extrinsic protein that catalyzes the α-ketodecarboxylation of oxaloacetate; Oad-γ, a structural bitopic membrane protein whose cytosolic tail (named as Oad-γ′) binds tightly to Oad-α; and Oad-β, a multispan transmembrane α-helical protein that constitutes the Na+ channel. How OAD is organized structurally at the membrane and what the molecular determinants are that lead to an efficient energy coupling mechanism remain elusive. In the present work, we elucidate the stoichiometry of the native complex as well as the low resolution structure of the peripheral components of OAD (Oad-α and Oad-γ′) by small angle x-ray scattering. Our results point to a quaternary assembly similar to the pyruvate carboxylase complex organization. Herein, we propose a model in which the association in pairs of Oad-α dimers, mediated by Oad-γ, results in the acquisition of a functional oligomeric state at the bacterial membrane. New structural insights for the conformational rearrangements associated with the carboxylbiotin transfer reaction within OAD are provided.

An Arabidopsis soluble chloroplast proteomic analysis reveals the participation of the Executer pathway in response to increased light conditions

Highlight Executer proteins have emerged as participants in singlet oxygen-mediated retrograde signalling in plants. This study relates the function of Executer in chloroplasts with the acclimation response to increased light.

A New Member of the Thioredoxin Reductase Family from Early Oxygenic Photosynthetic Organisms.

B.B.B. acknowledges support from an Alexander von Humboldt Research Award that catalyzed the launching of this project at the Ludwig-Maximilians-Universitat in Munich. R.M.B. is supported by a Ramon y Cajal contract from the Spanish Ministerio de Economia y Competitividad. L.L.-M. is supported by a postdoctoral contract from Universidad de Sevilla.