Geoffrey Duby

The plant plasma membrane proton pump ATPase: a highly regulated P-type ATPase with multiple physiological roles.

Around 40 P-type ATPases have been identified in Arabidopsis and rice, for which the genomes are known. None seems to exchange sodium and potassium, as does the animal Na+/K+-ATPase. Instead, plants, together with fungi, possess a proton pumping ATPase (H+-ATPase), which couples ATP hydrolysis to proton transport out of the cell, and so establishes an electrochemical gradient across the plasma membrane, which is dissipated by secondary transporters using protons in symport or antiport, as sodium is used in animal cells. Additional functions, such as stomata opening, cell growth, and intracellular pH homeostasis, have been proposed. Crystallographic data and homology modeling suggest that the H+-ATPase has a broadly similar structure to the other P-type ATPases but has an extended C-terminal region, which is involved in enzyme regulation. Phosphorylation of the penultimate residue, a Thr, and the subsequent binding of regulatory 14–3–3 proteins result in the formation of a dodecamer (six H+-ATPase and six 14–3–3 molecules) and enzyme activation. This type of regulation is unique to the P-type ATPase family. However, the recent identification of additional phosphorylated residues suggests further regulatory features.

Mapping the Cellular Response to Small Molecules Using Chemogenomic Fitness Signatures

Yeasty HIPHOP In order to identify how chemical compounds target genes and affect the physiology of the cell, tests of the perturbations that occur when treated with a range of pharmacological chemicals are required. By examining the haploinsufficiency profiling (HIP) and homozygous profiling (HOP) chemogenomic platforms, Lee et al. (p. 208) analyzed the response of yeast to thousands of different small molecules, with genetic, proteomic, and bioinformatic analyses. Over 300 compounds were identified that targeted 121 genes within 45 cellular response signature networks. These networks were used to extrapolate the likely effects of related chemicals, their impact upon genetic pathways, and to identify putative gene functions. Guilt by association helps identify the chemogenomic signatures of compounds targeting yeast genes. Genome-wide characterization of the in vivo cellular response to perturbation is fundamental to understanding how cells survive stress. Identifying the proteins and pathways perturbed by small molecules affects biology and medicine by revealing the mechanisms of drug action. We used a yeast chemogenomics platform that quantifies the requirement for each gene for resistance to a compound in vivo to profile 3250 small molecules in a systematic and unbiased manner. We identified 317 compounds that specifically perturb the function of 121 genes and characterized the mechanism of specific compounds. Global analysis revealed that the cellular response to small molecules is limited and described by a network of 45 major chemogenomic signatures. Our results provide a resource for the discovery of functional interactions among genes, chemicals, and biological processes.

Expression of a Constitutively Activated Plasma Membrane H+-ATPase Alters Plant Development and Increases Salt Tolerance

The plasma membrane proton pump ATPase (H+-ATPase) plays a major role in the activation of ion and nutrient transport and has been suggested to be involved in several physiological processes, such as cell expansion and salt tolerance. Its activity is regulated by a C-terminal autoinhibitory domain that can be displaced by phosphorylation and the binding of regulatory 14-3-3 proteins, resulting in an activated enzyme. To better understand the physiological consequence of this activation, we have analyzed transgenic tobacco (Nicotiana tabacum) plants expressing either wild-type plasma membrane H+-ATPase4 (wtPMA4) or a PMA4 mutant lacking the autoinhibitory domain (ΔPMA4), generating a constitutively activated enzyme. Plants showing 4-fold higher expression of wtPMA4 than untransformed plants did not display any unusual phenotype and their leaf and root external acidification rates were not modified, while their in vitro H+-ATPase activity was markedly increased. This indicates that, in vivo, H+-ATPase overexpression is compensated by down-regulation of H+-ATPase activity. In contrast, plants that expressed ΔPMA4 were characterized by a lower apoplastic and external root pH, abnormal leaf inclination, and twisted stems, suggesting alterations in cell expansion. This was confirmed by in vitro leaf extension and curling assays. These data therefore strongly support a direct role of H+-ATPase in plant development. The ΔPMA4 plants also displayed increased salt tolerance during germination and seedling growth, supporting the hypothesis that H+-ATPase is involved in salt tolerance.

Tobacco VDL Gene Encodes a Plastid DEAD Box RNA Helicase and Is Involved in Chloroplast Differentiation and Plant Morphogenesis

The recessive nuclear vdl (for variegated and distorted leaf) mutant of tobacco was obtained by T-DNA insertion and characterized by variegated leaves and abnormal roots and flowers. Affected leaf tissues were white and distorted, lacked palisadic cells, and contained undifferentiated plastids. The variegation was due to phenotypic, rather than genetic, instability. Genomic and cDNA clones were obtained for both the mutant and wild-type VDL alleles. Three transcripts, resulting from alternate intron splicing or polyadenylation, were found for the wild type. The transcripts potentially encode a set of proteins (53, 19, and 15 kD) sharing the same N-terminal region that contains a chloroplast transit peptide capable of importing the green fluorescent protein into chloroplasts. The predicted 53-kD product belongs to the DEAD box RNA helicase family. In the homozygous vdl mutant, T-DNA insertion resulted in accumulation of the shortest transcript and the absence of the RNA helicase–encoding transcript. Genetic transformation of the homozygous mutant by the 53-kD product–encoding cDNA fully restored the wild-type phenotype. These data suggest that a plastid RNA helicase controls early plastid differentiation and plant morphogenesis.

Targeting of a Nicotiana plumbaginifolia H+-ATPase to the Plasma Membrane Is Not by Default and Requires Cytosolic Structural Determinants

The structural determinants involved in the targeting of multitransmembrane-span proteins to the plasma membrane (PM) remain poorly understood. The plasma membrane H+-ATPase (PMA) from Nicotiana plumbaginifolia, a well-characterized 10 transmembrane–span enzyme, was used as a model to identify structural elements essential for targeting to the PM. When PMA2 and PMA4, representatives of the two main PMA subfamilies, were fused to green fluorescent protein (GFP), the chimeras were shown to be still functional and to be correctly and rapidly targeted to the PM in transgenic tobacco. By contrast, chimeric proteins containing various combinations of PMA transmembrane spanning domains accumulated in the Golgi apparatus and not in the PM and displayed slow traffic properties through the secretory pathway. Individual deletion of three of the four cytosolic domains did not prevent PM targeting, but deletion of the large loop or of its nucleotide binding domain resulted in GFP fluorescence accumulating exclusively in the endoplasmic reticulum. The results show that, at least for this polytopic protein, the PM is not the default pathway and that, in contrast with single-pass membrane proteins, cytosolic structural determinants are required for correct targeting.

Activation of plant plasma membrane H+-ATPase by 14-3-3 proteins is negatively controlled by two phosphorylation sites within the H+-ATPase C-terminal region.

The proton pump ATPase (H+-ATPase) of the plant plasma membrane is regulated by an autoinhibitory C-terminal domain, which can be displaced by phosphorylation of the penultimate Thr residue and the subsequent binding of 14-3-3 proteins. We performed a mass spectrometric analysis of PMA2 (plasma membrane H+-ATPase isoform 2) isolated from Nicotiana tabacum suspension cells and identified two new phosphorylated residues in the enzyme 14-3-3 protein binding site: Thr931 and Ser938. When PMA2 was expressed in Saccharomyces cerevisiae, mutagenesis of each of these two residues into Asp prevented growth of a yeast strain devoid of its own H+-ATPases. When the Asp mutations were individually introduced in a constitutively activated mutant of PMA2 (E14D), they still allowed yeast growth but at a reduced rate. Purification of His-tagged PMA2 showed that the T931D or S938D mutation prevented 14-3-3 protein binding, although the penultimate Thr955 was still phosphorylated, indicating that Thr955 phosphorylation is not sufficient for full enzyme activation. Expression of PMA2 in an N. tabacum cell line also showed an absence of 14-3-3 protein binding resulting from the T931D or S938D mutation. Together, the data show that activation of H+-ATPase by the binding of 14-3-3 proteins is negatively controlled by phosphorylation of two residues in the H+-ATPase 14-3-3 protein binding site. The data also show that phosphorylation of the penultimate Thr and 14-3-3 binding each contribute in part to H+-ATPase activation.

A Phosphorylation in the C-terminal Auto-inhibitory Domain of the Plant Plasma Membrane H+-ATPase Activates the Enzyme with No Requirement for Regulatory 14-3-3 Proteins

The plant plasma membrane H+-ATPase is regulated by an auto-inhibitory C-terminal domain that can be displaced by phosphorylation of the penultimate residue, a Thr, and the subsequent binding of 14-3-3 proteins. By mass spectrometric analysis of plasma membrane H+-ATPase isoform 2 (PMA2) isolated from Nicotiana tabacum plants and suspension cells, we identified a new phosphorylation site, Thr-889, in a region of the C-terminal domain upstream of the 14-3-3 protein binding site. This residue was mutated into aspartate or alanine, and the mutated H+-ATPases expressed in the yeast Saccharomyces cerevisiae. Unlike wild-type PMA2, which could replace the yeast H+-ATPases, the PMA2-Thr889Ala mutant did not allow yeast growth, whereas the PMA2-Thr889Asp mutant resulted in improved growth and increased H+-ATPase activity despite reduced phosphorylation of the PMA2 penultimate residue and reduced 14-3-3 protein binding. To determine whether the regulation taking place at Thr-889 was independent of phosphorylation of the penultimate residue and 14-3-3 protein binding, we examined the effect of combining the PMA2-Thr889Asp mutation with mutations of other residues that impair phosphorylation of the penultimate residue and/or binding of 14-3-3 proteins. The results showed that in yeast, PMA2 Thr-889 phosphorylation could activate H+-ATPase if PMA2 was also phosphorylated at its penultimate residue. However, binding of 14-3-3 proteins was not required, although 14-3-3 binding resulted in further activation. These results were confirmed in N. tabacum suspension cells. These data define a new H+-ATPase activation mechanism that can take place without 14-3-3 proteins.

Two widely expressed plasma membrane H(+)-ATPase isoforms of Nicotiana tabacum, are differentially regulated by phosphorylation of their penultimate threonine.

The plasma membrane H(+)-ATPases PMA2 and PMA4 are the most widely expressed in Nicotiana plumbaginifolia, and belong to two different subfamilies. Both are activated by phosphorylation of a Thr at the penultimate position and the subsequent binding of 14-3-3 proteins. Their expression in Saccharomyces cerevisiae revealed functional and regulatory differences. To determine whether different regulatory properties between PMA2 and PMA4 exist in plants, we generated two monoclonal antibodies able to detect phosphorylation of the penultimate Thr of either PMA2 or PMA4 in a total protein extract. We also raised Nicotiana tabacum transgenic plants expressing 6-His-tagged PMA2 or PMA4, enabling their individual purification. Using these tools we showed that phosphorylation of the penultimate Thr of both PMAs was high during the early exponential growth phase of an N. tabacum cell culture, and then progressively declined. This decline correlated with decreased 14-3-3 binding and decreased plasma membrane ATPase activity. However, the rate and extent of the decrease differed between the two isoforms. Cold stress of culture cells or leaf tissues reduced the Thr phosphorylation of PMA2, whereas no significant changes in Thr phosphorylation of PMA4 were seen. These results strongly suggest that PMA2 and PMA4 are differentially regulated by phosphorylation. Analysis of the H(+)-ATPase phosphorylation status in leaf tissues indicated that no more than 44% (PMA2) or 32% (PMA4) was in the activated state under normal growth conditions. Purification of either isoform showed that, when activated, the two isoforms did not form hetero-oligomers, which is further support for these two H(+)-ATPase subfamilies having different properties.

Mitochondrial protein import machinery and targeting information

During evolution, eukaryotic cells have acquired subcellular compartments, such as nuclei, peroxisomes, mitochondria, and, in the case of plant cells, chloroplasts, this compartmentalization allowing the cell to function more efficiently. Although, mitochondria and chloroplasts possess their own genomes, these have a low coding content and nuclear-encoded proteins have to be imported. Since the organization, content and functioning of organelles are strictly defined, an efficient and rigorous system for importing proteins synthesized in the cytosol is required. Most mitochondrial precursors synthesized in the cytosol are recognized and/or maintained in an unfolded conformation by cytosolic chaperone proteins and are then recognized by translocases of the mitochondrial outer and inner membranes (TOM and TIM, respectively), which transport them across the two membranes. The mechanism of mitochondrial protein import is thought to be well conserved across species. However, while many studies have investigated mitochondrial import in fungi, few have been carried out in plants, although it is clear that differences exist between fungi, mammals, and plants as regards the mitochondrial import system. In plants, for instance, the presence of both chloroplasts and mitochondria, with similar import mechanisms, might have rendered the import system more stringent. We will review the literature concerning the mitochondrial import machinery and the structure of mitochondrial presequences, paying particular attention to those features specific to plants

The proteome complement of Nicotiana tabacum Bright-Yellow-2 culture cells.

The Nicotiana tabacum Bright‐Yellow‐2 (BY2) cell line is one of most commonly used plant suspension cell lines and offers interesting properties, such as fast growth, amenability to genetic transformation, and synchronization of cell division. To build a proteome reference map of BY2 cell proteins, we isolated the soluble proteins from N. tabacum BY2 cells at the end of the exponential growth phase and analyzed them by 2‐DE and MALDI TOF‐TOF. Of the 1422 spots isolated, 795 were identified with a significant score, corresponding to 532 distinct proteins.