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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.

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.

Summary PMA2 and PMA4 are the most widely expressed plasma membrane H(+)-ATPases 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 N. tabacum transgenic plants expressing 6-His-tagged PMA2 or PMA4, allowing their individual purification. Using these tools, we showed that phosphorylation of the penultimate Thr of both PMA 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 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 one-third of PMA2 or 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, further support for these two H(+)-ATPase subfamilies having different properties.

The plant plasma membrane H+-ATPase couples ATP hydrolysis to the transport of protons out of the cell, thus generating an electrochemical gradient used by secondary transporters to move ions and metabolites through the membrane. In addition, H+-ATPase is involved in several physiological roles such as stomata opening and cell elongation. This enzyme is highly regulated by (de)phosphorylation of several residues and binding of regulatory proteins. A well characterized example is the enzyme activation by phosphorylation of its penultimate residue, a Thr, and the subsequent binding of 14-3-3 regulatory proteins. Mass spectroscopy analysis of purified PMA4 and PMA2, the most broadly expressed Nicotiana plumbaginifolia plasma membrane H+-ATPases, led to the identification of new phosphorylated residues. Two of them were studied in more details. PMA4 Ser448 and PMA2 Thr889 were both mutated into aspartate or alanine and the mutant H+-ATPases were expressed in the yeast Saccharomyces cerevisae as well as in Nicotiana tabacum suspension cells. Both mutations of PMA4 Ser448 resulted, in BY2 cells as in yeast, in higher penultilmate residue phosphorylation than in wild-type. However, unlike in yeast, the ATPase activity was lower in BY2 cells expressing the PMA4 Ser448 mutants compared to that expressing wild–type PMA4. The PMA4-S448D mutant conferred higher proton pumping activity than the wild–type PMA4 and the PMA4-S448A mutant, suggesting that the former has a better H+ pumping/ATP hydrolysis coupling. Concerning PMA2, the PMA2-T889A mutation did not allow yeast growth while PMA2-T889D resulted in improved growth and increased H+-ATPase activity in spite of reduced PMA2 penultimate Thr phosphorylation and 14-3-3 protein binding. Combining PMA2-T889D with other mutations indicated that PMA2 Thr889 phosphorylation can activate the H+-ATPase, provided that PMA2 penultimate residue is phosphorylated. Interestingly, 14-3-3 protein binding was not required but brought addition, (AGRO 3) -- UCL, 2010

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 2D gel electrophoresis and MALDI TOFTOF. Of the 1422 spots isolated, 795 were identified with a significant score, corresponding to 532 distinct proteins.

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.

The proton pump ATPase (H+-ATPase) of the plant plasma membrane is regulated by an auto-inhibitory 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 plasma membrane H+-ATPase isoform 2 (PMA2) 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 (Glu14Asp), they still allowed yeast growth, but at a reduced rate. Purification of His-tagged PMA2 showed that the Thr931Asp or Ser938Asp 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 absence of 14-3-3 protein binding resulting from the Thr931Asp or Ser938Asp 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.

Amongst the nine voltage-gated K(+) channel (Kv) subunits expressed in Arabidopsis, AtKC1 does not seem to form functional Kv channels on its own, and is therefore said to be silent. It has been proposed to be a regulatory subunit, and to significantly influence the functional properties of heteromeric channels in which it participates, along with other Kv channel subunits. The mechanisms underlying these properties of AtKC1 remain unknown. Here, the transient (co-)expression of AtKC1, AKT1 and/or KAT1 genes was obtained in tobacco mesophyll protoplasts, which lack endogenous inward Kv channel activity. Our experimental conditions allowed both localization of expressed polypeptides (GFP-tagging) and recording of heterologously expressed Kv channel activity (untagged polypeptides). It is shown that AtKC1 remains in the endoplasmic reticulum unless it is co-expressed with AKT1. In these conditions heteromeric AtKC1-AKT1 channels are obtained, and display functional properties different from those of homomeric AKT1 channels in the same context. In particular, the activation threshold voltage of the former channels is more negative than that of the latter ones. Also, it is proposed that AtKC1-AKT1 heterodimers are preferred to AKT1-AKT1 homodimers during the process of tetramer assembly. Similar results are obtained upon co-expression of AtKC1 with KAT1. The whole set of data provides evidence that AtKC1 is a conditionally-targeted Kv subunit, which probably downregulates the physiological activity of other Kv channel subunits in Arabidopsis.

Potassium translocation in plants is accomplished by a large variety of transport systems. Most of the available molecular information on these proteins concerns voltage-gated potassium channels (Kv channels). The Arabidopsis genome comprises nine genes encoding alpha-subunits of Kv channels. Based on knowledge of their animal homologues, and on biochemical investigations, it is broadly admitted that four such polypeptides must assemble to yield a functional Kv channel. The intrinsic functional properties of Kv channel alpha-subunits have been described by expressing them in suitable heterologous contexts where homo-tetrameric channels could be characterized. However, due to the high similarity of both the polypeptidic sequence and the structural scheme of Kv channel alpha-subunits, formation of heteromeric Kv channels by at least two types of alpha-subunits is conceivable. Several examples of such heteromeric plant Kv channels have been studied in heterologous expression systems and evidence that heteromerization actually occurs in planta has now been published. It is therefore challenging to uncover the physiological role of this heteromerization. Fine tuning of Kv channels by heteromerisation could be relevant not only to potassium transport but also to electrical signaling within the plant.

Regulatory 14-3-3 proteins activate the plant plasma membrane H+-ATPase by binding to its C-terminal autoinhibitory domain. This interaction requires phosphorylation of a C-terminal, mode III, recognition motif as well as an adjacent span of approximately 50 amino acids. Here we report the X-ray crystal structure of 14-3-3 in complex with the entire binding motif, revealing a previously unidentified mode of interaction. A 14-3-3 dimer simultaneously binds two H+-ATPase peptides, each of which forms a loop within the typical 14-3-3 binding groove and therefore exits from the center of the dimer. Several H+-ATPase mutants support this structure determination. Accordingly, 14-3-3 binding could result in H+-ATPase oligomerization. Indeed, by using single-particle electron cryomicroscopy, the 3D reconstruction of the purified H+-ATPase/14-3-3 complex demonstrates a hexameric arrangement. Fitting of 14-3-3 and H+-ATPase atomic structures into the 3D reconstruction map suggests the spatial arrangement of the holocomplex.

Friedreich's ataxia is caused by a deficit in frataxin, a small mitochondrial protein of unknown function that has been conserved during evolution. Previous studies have pointed out a role for frataxin in mitochondrial iron-sulfur (Fe-S) metabolism. Here, we have analyzed the incorporation of Fe-S clusters into yeast ferredoxin imported into isolated energized mitochondria from cells grown in the presence of glycerol, an obligatory respiratory carbon source. Similar amounts of apo-ferredoxin precursor were imported into mitochondria and processed in wild-type and yfh1-deleted (delta YF111) strains. However, the incorporation of Fe-S clusters into apo-ferredoxin was significantly reduced in delta YFH1 mitochondria. The newly assembled ferredoxin was stable, excluding the possibility that the decreased incorporation was a result of increased oxidative damage. When delta YFH1 cells were grown in raffinose medium, the formation of holo-ferredoxin was low, as a consequence of the decrease in ferredoxin precursor import into mitochondria. However, the decrease in the conversion rate of apo- into holo-ferredoxin was in the same range as for glycerol-grown cells, indicating that the extent of the defect in Fe-S protein assembly is similar under different physiological conditions. These data show that frataxin is not essential for Fe-S protein assembly, but improves the efficiency of the process. The large variations observed in the activity of Fe-S cluster proteins under different physiological conditions result from secondary defects in the physiology of delta YFH1 cells.

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. (C) 2002 Elsevier Science Ireland Ltd. All rights reserved.

We sought to determine the structural features involved in the processing of the mitochondrial F1-ATPase beta-subunit (F1beta) presequence (54 residues) from Nicotiana plumbaginifolia. The cleavage efficiency of F1beta presequence mutants linked to the green fluorescent protein (GFP) was evaluated in vivo in tobacco by in situ microscopy and Western blotting. The residue at position -1 (Tyr) was required to be an aromatic residue and the residue at position +2 (Thr) was found to be important for F1beta processing, while, unexpectedly, changing the distal (Arg-15) and proximal (Arg-5) arginine residues did not strongly reduce processing. In addition, results also supported the requirement of a helical structure around the cleavage position. Sequencing of the mature form of a precursor containing the first 30 residues of the F1beta presequence linked to GFP revealed the presence of a cryptic cleavage site between residues 26 and 27, which showed the features of a classical mitochondrial processing site, suggesting dual processing of the F1beta presequence. In vitro processing confirmed these data and showed that processing was sensitive to o-phenanthroline, thus catalyzed by mitochondrial processing peptidase.

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.