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The assembly of iron-sulfur (Fe-S) clusters involves several pathways and in prokaryotes the mobilization of the sulfur (SUF) system is paramount for Fe-S biogenesis and repair during oxidative stress. The prokaryotic SUF system consists of six proteins: SufC is an ABC/ATPase that forms a complex with SufB and SufD, SufA acts as a scaffold protein, and SufE and SufS are involved in sulfur mobilization from cysteine. Despite the importance of Fe-S proteins in higher plant plastids, little is known regarding plastidic Fe-S cluster assembly. We have recently shown that Arabidopsis harbors an evolutionary conserved plastidic SufC protein (AtNAP7) capable of hydrolyzing ATP and interacting with the SufD homolog AtNAP6. Based on this and the prokaryotic SUF system we speculated that a SufB-like protein may exist in plastids. Here we demonstrate that the Arabidopsis plastid-localized SufB homolog AtNAP1 can complement SufB deficiency in Escherichia coli during oxidative stress. Furthermore, we demonstrate that AtNAP1 can interact with AtNAP7 inside living chloroplasts suggesting the presence of a plastidic AtNAP1.AtNAP6.AtNAP7 complex and remarkable evolutionary conservation of the SUF system. However, in contrast to prokaryotic SufB proteins with no associated ATPase activity we show that AtNAP1 is an iron-stimulated ATPase and that AtNAP1 is capable of forming homodimers. Our results suggest that AtNAP1 represents an atypical plastidic SufB-like protein important for Fe-S cluster assembly and for regulating iron homeostasis in Arabidopsis.

U-box proteins, as well as other proteins involved in regulated protein degradation, are apparently over-represented in Arabidopsis compared with other model eukaryotes. The Arabidopsis protein AtPUB14 contains a typical U-box domain followed by an Armadillo repeat region, a domain organization that is frequently found in plant U-box proteins. In vitro ubiquitination assays demonstrated that AtPUB14 functions as an E3 ubiquitin ligase with specific E2 ubiquitin-conjugating enzymes. The structure of the AtPUB14 U-box domain was determined by NMR spectroscopy. It adopts the betabetaalphabeta fold of the Prp19p U-box and RING finger domains. In these proteins, conserved hydrophobic residues form a putative E2-binding cleft. By contrast, they contain no common polar E2 binding site motif. Two hydrophobic cores stabilize the AtPUB14 U-box fold, and hydrogen bonds and salt bridges interconnect the residues corresponding to zinc ion-coordinating residues in RING domains. Residues from a C-terminal alpha-helix interact with the core domain and contribute to stabilization. The Prp19p U-box lacks a corresponding C-terminal alpha-helix. Chemical shift analysis suggested that aromatic residues exposed at the N terminus and the C-terminal alpha-helix of the AtPUB14 U-box participate in dimerization. Thus, AtPUB14 may form a biologically relevant dimer. This is the first plant U-box structure to be determined, and it provides a model for studies of the many plant U-box proteins and their interactions. Structural insight into these interactions is important, because ubiquitin-dependent protein degradation is a prevalent regulatory mechanism in plants.

The thermodynamics and kinetics of actin interaction with Arabidopsis thaliana actin-depolymerizing factor (ADF)1, human ADF, and S6D mutant ADF1 protein mimicking phosphorylated (inactive) ADF are examined comparatively. ADFs interact with ADP.G-actin in rapid equilibrium (k+ = 155 microM-1.s-1 and k- = 16 s-1 at 4 degreesC under physiological ionic conditions). The kinetics of interaction of plant and human ADFs with F-actin are slower and exhibit kinetic cooperativity, consistent with a scheme in which the initial binding of ADF to two adjacent subunits of the filament nucleates a structural change that propagates along the filament, allowing faster binding of ADF in a "zipper" mode. ADF binds in a non-cooperative faster process to gelsolin-capped filaments or to subtilisin-cleaved F-actin, which are structurally different from standard filaments (Orlova, A., Prochniewicz, E., and Egelman, E. H. (1995) J. Mol. Biol. 245, 598-607). In contrast, the binding of phalloidin to F-actin cooperatively inhibits its interaction with ADF. The ADF-facilitated nucleation of ADP.actin self-assembly indicates that ADF stabilizes lateral interactions in the filament. Plant and human ADFs cause only partial depolymerization of F-actin at pH 8, consistent with identical functions in enhancing F-actin dynamics. Phosphorylation does not affect ADF activity per se, but decreases its affinity for actin by 20-fold.

Profilin, an essential G-actin-binding protein, has two opposite regulatory functions in actin filament assembly. It facilitates assembly at the barbed ends by lowering the critical concentration (Pantaloni, D., and Carlier, M.-F. (1993) Cell 75, 1007-1014); in contrast it contributes to the pool of unassembled actin when barbed ends are capped. We proposed that the first of these functions required an input of energy. How profilin uses the ATP hydrolysis that accompanies actin polymerization and whether the acceleration of nucleotide exchange on G-actin by profilin participates in its function in filament assembly are the issues addressed here. We show that 1) profilin increases the treadmilling rate of actin filaments in the presence of Mg2+ ions; 2) when filaments are assembled from CaATP-actin, which polymerizes in a quasireversible fashion, profilin does not promote assembly at the barbed ends and has only a G-actin-sequestering function; 3) plant profilins do not accelerate nucleotide exchange on G-actin, yet they promote assembly at the barbed end. The enhancement of nucleotide exchange by profilin is therefore not involved in its promotion of actin assembly, and the productive growth of filaments from profilin-actin complex requires the coupling of ATP hydrolysis to profilin-actin assembly, a condition fulfilled by Mg-actin, and not by Ca-actin.

Phosphorylation of proteins on serine, threonine, or tyrosine residues represents an important biochemical mechanism to regulate the activity of enzymes and is used in many cellular processes. In animals, protein serine/threonine and protein tyrosine kinases are known to perform essential roles in many pathways that transmit external stimuli from the cell surface to the cell inferior and the nucleus. In plants, although an increasing number of protein serine/threonine kinases have been cloned, the existence of protein tyrosine kinases remains yet to be demonstrated. Here, we report the cloning and biochemical characterization of a plant protein kinase, Arabidopsis dual specificity kinase 1 (ADK1), using a functional screening method, namely by screening an Arabidopsis expression library with antiphosphotyrosine antibodies. Four independent cDNA clones that define a polypeptide of 319 amino acids length with homology to protein kinases were identified in this screen. Phosphoamino acid analysis of the autophosphorylated kinase shows that ADK1 phosphorylates serine, threonine, and tyrosine. Using poly (Glu/Tyr) as a substrate, we confirm that ADK1 is capable of phosphorylating tyrosine residues.

A conserved 21-base pair element, designated as hex-1, located between -180 and -160 of the wheat histone H3 promoter, is known to interact with two tobacco nuclear factors, activating sequence factor 1 and hex-1-specific binding factor. We have shown previously that a mutant sequence (hex-3), which differs from hex-1 by three base pairs, can no longer bind these two factors significantly. In the present work, we examined the functional characteristics of these two sequences in transgenic tobacco. Surprisingly, we found that a tetramer of hex-3, but not of hex-1, confers high level expression in mature seeds. Expression of this synthetic promoter rapidly diminishes upon germination but can be reactivated in young seedlings and mature leaves by desiccation, NaCl, or the phytohormone abscisic acid (ABA). Treatment with auxin or cytokinin has no apparent effect on the expression. Since the endogenous ABA level of plant cells is known to increase upon water stress, our data suggest that hex-3, the mutated hex-1 sequence, is an abscisic acid-responsive element (abre). We propose that a tobacco nuclear factor, distinct from activating sequence factor 1 and hex-1-specific binding factor, interacts with this sequence and is involved in mediating the effects of ABA and water stress on gene expression.

We have characterized a tobacco nuclear factor that binds to the -118 region of the nopaline synthase (nos) promoter from the Ti plasmid of Agrobacterium tumefaciens. The binding site for this factor, identified by DNase I footprinting, encompasses the region from -138 to -103 of the nos promoter. This region, which contains a potential Z-DNA-forming sequence, was previously shown to be essential for nos promoter activity in transgenic tobacco. A synthetic 21-base pair sequence from the protected region (from -131 to -111), designated as nos-1, was sufficient for factor recognition in vitro. In transgenic tobacco, a tetramer of nos-1 can confer leaf and root expression when fused upstream of a truncated 35 S promoter from the cauliflower mosaic virus. Mutations at the two TGACG-like motifs in nos-1 abolish factor binding while preserving the potential for Z-DNA formation. A tetramer of the nos-1 mutant sequence has no significant activity above background when tested in transgenic tobacco. Competition experiments with activation sequence factor (ASF)-1 binding sites from the 35 S promoter of cauliflower mosaic virus (as-1) and the wheat histone H3 promoter (hex-1) demonstrate that ASF-1 is the factor that binds to nos-1.

The genes coding for the light-harvesting chlorophyll a/b-binding protein of photosystem II (cab) in wheat are regulated by phytochrome and an endogenous circadian clock. The regulation by these factors has been shown to be at the transcriptional level. Here we show that the phase of cab gene expression is reset by light to dark transition of the previous day. The time of light to dark transition is linearly related to the time of maximal cab gene expression under free running conditions in darkness. We propose that in nature the onset of darkness resets the phase of the circadian clock, which regulates the timing of cab gene expression, so as to anticipate the time of sunrise in the following day.

Two major chloroplast proteins are encoded by nuclear genes and synthesized on free cytoplasmic ribosomes: the small subunit of ribulose 1,5-bisphosphate carboxylase and the apoprotein components of the chlorophyll a/b light harvesting complex. We have recently reported the isolation of two cDNA clones from pea which encode both the small subunit of ribulose 1,5-bisphosphate carboxylase (pSS15) and the polypeptide 15 (pAB96), the major chlorophyll a/b binding protein (Broglie, R., Bellemare, G., Bartlett, S., Chua, N.-H., and Cashmore, A. R. (1981) Proc. Natl. Acad. Sci. U.S.A. 78, 7304-7308). To further characterize these clones, we determined their nucleotide sequence. Clone pSS15 contains a 691-base pair cDNA insert which encodes the entire 123 amino acids of the mature small subunit protein. In addition, this clone also encodes 33 amino acids of the NH2-terminal transit peptide extension and 148 nucleotides of the 3' noncoding region preceding the poly(A)tail. A second cDNA clone (pAB96) contains an 833-nucleotide insert which encodes most of polypeptide 15. The DNA sequence of this cloned cDNA was used to deduce the previously undetermined amino acid sequence of this integral thylakoid membrane protein. The nucleotide sequence of the cDNA clone, pSS15, should provide information concerning the role of the transit sequence in the transport of cytoplasmically synthesized chloroplast proteins. Similarly, the deduced amino acid sequence of polypeptide 15 will provide information for predicting its orientation in thylakoid membranes as well as its role in binding chlorophyll.

The large subunit of ribulose 1,5-bisphosphate carboxylase (rbcL) and the beta subunit of chloroplast ATP synthase (atpB) are encoded by divergently transcribed genes on the plastid genome. We have identified DNA binding factors specific for sequences located in the intergenic region between these two genes. Soluble plastid extracts from pea or whole cell extracts from maize protected a maize chloroplast DNA probe containing the 160-base pair region between the 5' ends of rbcL and atpB genes from exonuclease III digestion between positions -16 and -101 relative to the rbcL gene transcription start site. Competition assay with partial sequences from this intergenic region demonstrated that specific sequence(s) are required for the protection. The borders of the binding domain are conserved among the homologous regions of maize, tobacco, spinach, and pea chloroplast genomes. Gel filtration chromatography revealed a molecular weight of about 115,000 for the active complex involved in DNA binding. Using the exonuclease III protection assay, we have also shown that purified Escherichia coli RNA polymerase protects from +25 to -20 of the rbcL gene and from +21 to -23 of the atpB gene relative to their respective transcription start sites. These regions are analogous to open complexes found when E. coli RNA polymerase interacts with the prokaryotic promoters and are consistent with the ability of E. coli RNA polymerase to initiate transcription correctly on linear templates containing these chloroplast promoters. Possible role(s) for the chloroplast DNA binding factor in chloroplast gene expression and its regulation are discussed.

Many polypeptides translated in the cytosol enter the chloroplast where they assemble into macromolecular complexes. The transport of these polypeptides into the plastid can be examined in vitro by mixing isolated chloroplasts with pea poly(A) RNA translation products. Following optimization of both translation in the wheat germ system and the conditions during in vitro uptake, we observe the post-translational transport of over 100 polypeptides; many remain in the soluble phase of the organelle while others integrate into the thylakoid membranes. Most products transported in vitro co-migrate with in vivo products on sodium dodecyl sulfate-polyacrylamide gels. Furthermore, with the improved conditions, we demonstrate the transport of plastocyanin, ferredoxin-NADP+ oxidoreductase, and fructose-1,6-bisphosphatase into isolated plastids. While we have not been able to detect any cell-free translation product that is immunologically related to fructose-1,6-bisphosphatase, both plastocyanin and ferredoxin-NADP+ oxidoreductase are synthesized as precursors in vitro. These precursors are imported into the organelle where they are processed to the size of their mature counterparts. As determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the molecular weight of the precursor to plastocyanin is 15,000 larger than the mature product and the precursor to ferredoxin-NADP+ oxidoreductase is 8,000 larger than the mature product.

Nonpenetrating proteolytic enzymes (such as thermolysin) were used to probe the cytosolic surface of the outer envelope membrane from spinach chloroplasts. Up to 20 different envelope polypeptides were susceptible to a mild digestion of isolated intact chloroplasts by thermolysin. Most of the thermolysin-sensitive envelope polypeptides were not extracted by a mixture of chloroform/methanol (2:1, v/v). A clear exception was E10 which is hydrophobic and, in addition, is an integral membrane polypeptide. Using antibodies to envelope polypeptides sensitive (E10 and E24) and insensitive (E30 and E37) to thermolysin, we demonstrated that only antibodies to E10 and E24, but not antibodies to E30 and E37, induced agglutination of intact chloroplasts. In addition, immunofluorescence experiments demonstrated that only antibodies to E10 and E24, but not antibodies to E30 and E37, gave a green fluorescence at the outer surface of intact chloroplasts. These experiments demonstrate that E10 and E24, and probably all the thermolysin-sensitive envelope polypeptides, are accessible from the cytosolic side of the outer membrane of the chloroplast envelope.