Characterization of Mutants with Alterations of the Phosphorylation Site in the D2 Photosystem II Polypeptide of Chlamydomonas reinhardtii1

We have changed the potential phosphorylation site, a threonine residue at position 2 of the D2 polypeptide of the photosystem II complex of Chlamydomonas reinhardtii, to alanine, valine, aspartate, proline, glycine, or glutamate. Mutants with neutral amino acid changes did not display any phenotype with regard to photoautotrophic growth, light sensitivity, fluorescence transients, or photoinhibition. Pulse labeling of these mutants with 32P indicated that a phosphorylated protein of the same size as D2 is absent in these mutants, suggesting that threonine-2 is indeed the unique phosphorylation site of D2. In contrast, mutants in which threonine-2 has been replaced with acidic residues are deficient in photosystem II. Use of chimeric genes containing the psbD 5′-untranslated region revealed that the initiation of translation was not affected in these mutants, but the mutations interfered with a later step of D2 synthesis and accumulation.

Yeast Rab GTPase-activating Protein Gyp1p Localizes to the Golgi Apparatus and Is a Negative Regulator of Ypt1p

A family of related proteins in yeast Saccharomyces cerevisiae is known to have in vitro GTPase-activating protein activity on the Rab GTPases. However, their in vivo function remains obscure. One of them, Gyp1p, acts on Sec4p, Ypt1p, Ypt7p, and Ypt51p in vitro. Here, we present data to reveal its in vivo substrate and the role that it plays in the function of the Rab GTPase. Red fluorescent protein-tagged Gyp1p is concentrated on cytoplasmic punctate structures that largely colocalize with a cis-Golgi marker. Subcellular fractionation of a yeast lysate confirmed that Gyp1p is peripherally associated with membranes and that it cofractionates with Golgi markers. This localization suggests that Gyp1p may only act on Rab GTPases on the Golgi. A gyp1Δ strain displays a growth defect on synthetic medium at 37°C. Overexpression of Ypt1p, but not other Rab GTPases, strongly inhibits the growth of gyp1Δ cells. Conversely, a partial loss-of-function allele of YPT1, ypt1-2, can suppress the growth defect of gyp1Δ cells. Furthermore, deletion of GYP1 can partially suppress growth defects associated with mutants in subunits of transport protein particle complex, a complex that catalyzes nucleotide exchange on Ypt1p. These results establish that Gyp1p functions on the Golgi as a negative regulator of Ypt1p.

Timing of metamorphosis and the onset of the negative feedback loop between the thyroid gland and the pituitary is controlled by type II iodothyronine deiodinase in Xenopus laevis

Two important features of amphibian metamorphosis are the sequential response of tissues to different concentrations of thyroid hormone (TH) and the development of the negative feedback loop between the pituitary and the thyroid gland that regulates TH synthesis by the thyroid gland. At the climax of metamorphosis in Xenopus laevis (when the TH level is highest), the ratio of the circulating precursor thyroxine (T4) to the active form 3,5,3′-triiodothyronine (T3) in the blood is many times higher than it is in tissues. This difference is because of the conversion of T4 to T3 in target cells of the tadpole catalyzed by the enzyme type II iodothyronine deiodinase (D2) and the local effect (cell autonomy) of this activity. Limb buds and tails express D2 early and late in metamorphosis, respectively, correlating with the time that these organs undergo TH-induced change. T3 is required to complete metamorphosis because the peak concentration of T4 that is reached at metamorphic climax cannot induce the final morphological changes. At the climax of metamorphosis, D2 expression is activated specifically in the anterior pituitary cells that express the genes for thyroid-stimulating hormone but not in the cells that express proopiomelanocortin. Physiological concentrations of T3 but not T4 can suppress thyrotropin subunit β gene expression. The timing and the remarkable specificity of D2 expression in the thyrotrophs of the anterior pituitary coupled with the requirement for locally synthesized T3 strongly support a role for D2 in the onset of the negative feedback loop at the climax of metamorphosis.

Adenylate kinase phosphotransfer communicates cellular energetic signals to ATP-sensitive potassium channels

Transduction of energetic signals into membrane electrical events governs vital cellular functions, ranging from hormone secretion and cytoprotection to appetite control and hair growth. Central to the regulation of such diverse cellular processes are the metabolism sensing ATP-sensitive K+ (KATP) channels. However, the mechanism that communicates metabolic signals and integrates cellular energetics with KATP channel-dependent membrane excitability remains elusive. Here, we identify that the response of KATP channels to metabolic challenge is regulated by adenylate kinase phosphotransfer. Adenylate kinase associates with the KATP channel complex, anchoring cellular phosphotransfer networks and facilitating delivery of mitochondrial signals to the membrane environment. Deletion of the adenylate kinase gene compromised nucleotide exchange at the channel site and impeded communication between mitochondria and KATP channels, rendering cellular metabolic sensing defective. Assigning a signal processing role to adenylate kinase identifies a phosphorelay mechanism essential for efficient coupling of cellular energetics with KATP channels and associated functions.

Isolation and Characterization of a Histidine Biosynthetic Gene in Arabidopsis Encoding a Polypeptide with Two Separate Domains for Phosphoribosyl-ATP Pyrophosphohydrolase and Phosphoribosyl-AMP Cyclohydrolase

Phosphoribosyl-ATP pyrophosphohydrolase (PRA-PH) and phosphoribosyl-AMP cyclohydrolase (PRA-CH) are encoded by HIS4 in yeast and by hisIE in bacteria and catalyze the second and the third step, respectively, in the histidine biosynthetic pathway. By complementing a hisI mutation of Escherichia coli with an Arabidopsis cDNA library, we isolated an Arabidopsis cDNA (At-IE) that possesses these two enzyme activities. The At-IE cDNA encodes a bifunctional protein of 281 amino acids with a calculated molecular mass of 31,666 D. Genomic DNA-blot analysis with the At-IE cDNA as a probe revealed a single-copy gene in Arabidopsis, and RNA-blot analysis showed that the At-IE gene was expressed ubiquitously throughout development. Sequence comparison suggested that the At-IE protein has an N-terminal extension of about 50 amino acids with the properties of a chloroplast transit peptide. We demonstrated through heterologous expression studies in E. coli that the functional domains for the PRA-CH (hisI) and PRA-PH (hisE) resided in the N-terminal and the C-terminal halves, respectively, of the At-IE protein.

Brittle-1, an Adenylate Translocator, Facilitates Transfer of Extraplastidial Synthesized ADP-Glucose into Amyloplasts of Maize Endosperms1

Amyloplasts of starchy tissues such as those of maize (Zea mays L.) function in the synthesis and accumulation of starch during kernel development. ADP-glucose pyrophosphorylase (AGPase) is known to be located in chloroplasts, and for many years it was generally accepted that AGPase was also localized in amyloplasts of starchy tissues. Recent aqueous fractionation of young maize endosperm led to the conclusion that 95% of the cellular AGPase was extraplastidial, but immunolocalization studies at the electron- and light-microscopic levels supported the conclusion that maize endosperm AGPase was localized in the amyloplasts. We report the results of two nonaqueous procedures that provide evidence that in maize endosperms in the linear phase of starch accumulation, 90% or more of the cellular AGPase is extraplastidial. We also provide evidence that the brittle-1 protein (BT1), an adenylate translocator with a KTGGL motif common to the ADP-glucose-binding site of starch synthases and bacterial glycogen synthases, functions in the transfer of ADP-glucose into the amyloplast stroma. The importance of the BT1 translocator in starch accumulation in maize endosperms is demonstrated by the severely reduced starch content in bt1 mutant kernels.

Protein Changes in Response to Progressive Water Deficit in Maize1 : Quantitative Variation and Polypeptide Identification

Three-week-old plants of two unrelated lines of maize (Zea mays L.) and their hybrid were submitted to progressive water stress for 10 d. Changes induced in leaf proteins were studied by two-dimensional electrophoresis and quantitatively analyzed using image analysis. Seventy-eight proteins out of a total of 413 showed a significant quantitative variation (increase or decrease), with 38 of them exhibiting a different expression in the two genotypes. Eleven proteins that increased by a factor of 1.3 to 5 in stressed plants and 8 proteins detected only in stressed plants were selected for internal amino acid microsequencing, and by similarity search 16 were found to be closely related to previously reported proteins. In addition to proteins already known to be involved in the response to water stress (e.g. RAB17 [Responsive to ABA]), several enzymes involved in basic metabolic cellular pathways such as glycolysis and the Krebs cycle (e.g. enolase and triose phosphate isomerase) were identified, as well as several others, including caffeate O-methyltransferase, the induction of which could be related to lignification.

Transcription from Heterologous rRNA Operon Promoters in Chloroplasts Reveals Requirement for Specific Activating Factors1

The plastid rRNA (rrn) operon in chloroplasts of tobacco (Nicotiana tabacum), maize, and pea is transcribed by the plastid-encoded plastid RNA polymerase from a ς70-type promoter (P1). In contrast, the rrn operon in spinach (Spinacia oleracea) and mustard chloroplasts is transcribed from the distinct Pc promoter, probably also by the plastid-encoded plastid RNA polymerase. Primer-extension analysis reported here indicates that in Arabidopsis both promoters may be active. To understand promoter selection in the plastid rrn operon in the different species, we have tested transcription from the spinach rrn promoter in transplastomic tobacco and from the tobacco rrn promoter in transplastomic Arabidopsis. Our data suggest that transcription of the rrn operon depends on species-specific factors that facilitate transcription initiation by the general transcription machinery.

Stepwise Photoinhibition of Photosystem II1 : Studies with Synechocystis Species PCC 6803 Mutants with a Modified D-E Loop of the Reaction Center Polypeptide D1

Several mutant strains of Synechocystis sp. PCC 6803 with large deletions in the D-E loop of the photosystem II (PSII) reaction center polypeptide D1 were subjected to high light to investigate the role of this hydrophilic loop in the photoinhibition cascade of PSII. The tolerance of PSII to photoinhibition in the autotrophic mutant ΔR225-F239 (PD), when oxygen evolution was monitored with 2,6-dichloro-p-benzoquinone and the equal susceptibility compared with control when monitored with bicarbonate, suggested an inactivation of the QB-binding niche as the first event in the photoinhibition cascade in vivo. This step in PD was largely reversible at low light without the need for protein synthesis. Only the next event, inactivation of QA reduction, was irreversible and gave a signal for D1 polypeptide degradation. The heterotrophic deletion mutants ΔG240-V249 and ΔR225-V249 had severely modified QB pockets, yet exhibited high rates of 2,6-dichloro-p-benzoquinone-mediated oxygen evolution and less tolerance to photoinhibition than PD. Moreover, the protein-synthesis-dependent recovery of PSII from photoinhibition was impaired in the ΔG240-V249 and ΔR225-V249 mutants because of the effects of the mutations on the expression of the psbA-2 gene. No specific sequences in the D-E loop were found to be essential for high rates of D1 polypeptide degradation.

Mutation of Residue Threonine-2 of the D2 Polypeptide and Its Effect on Photosystem II Function in Chlamydomonas reinhardtii1

The D2 polypeptide of the photosystem II (PSII) complex in the green alga Chlamydomonas reinhardtii is thought to be reversibly phosphorylated. By analogy to higher plants, the phosphorylation site is likely to be at residue threonine-2 (Thr-2). We have investigated the role of D2 phosphorylation by constructing two mutants in which residue Thr-2 has been replaced by either alanine or serine. Both mutants grew photoautotrophically at wild-type rates, and noninvasive biophysical measurements, including the decay of chlorophyll fluorescence, the peak temperature of thermoluminescence bands, and rates of oxygen evolution, indicate little perturbation to electron transfer through the PSII complex. The susceptibility of mutant PSII to photoinactivation as measured by the light-induced loss of PSII activity in whole cells in the presence of the protein-synthesis inhibitors chloramphenicol or lincomycin was similar to that of wild type. These results indicate that phosphorylation at Thr-2 is not required for PSII function or for protection from photoinactivation. In control experiments the phosphorylation of D2 in wild-type C. reinhardtii was examined by 32P labeling in vivo and in vitro. No evidence for the phosphorylation of D2 in the wild type could be obtained. [14C]Acetate-labeling experiments in the presence of an inhibitor of cytoplasmic protein synthesis also failed to identify phosphorylated (D2.1) and nonphosphorylated (D2.2) forms of D2 upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Our results suggest that the existence of D2 phosphorylation in C. reinhardtii is still in question.

Guanylyl cyclase C agonists regulate progression through the cell cycle of human colon carcinoma cells

The effects of Escherichia coli heat-stable enterotoxin (ST) and uroguanylin were examined on the proliferation of T84 and Caco2 human colon carcinoma cells that express guanylyl cyclase C (GC-C) and SW480 human colon carcinoma cells that do not express this receptor. ST or uroguanylin inhibited proliferation of T84 and Caco2 cells, but not SW480 cells, in a concentration-dependent fashion, assessed by quantifying cell number, cell protein, and [3H]thymidine incorporation into DNA. These agonists did not inhibit proliferation by induction of apoptosis, assessed by TUNEL (terminal deoxynucleotidyl transferase-mediated dNTP-biotin nick end labeling of DNA fragments) assay and DNA laddering, or necrosis, assessed by trypan blue exclusion and lactate dehydrogenase release. Rather, ST prolonged the cell cycle, assessed by flow cytometry and [3H]thymidine incorporation into DNA. The cytostatic effects of GC-C agonists were associated with accumulation of intracellular cGMP, mimicked by the cell-permeant analog 8-Br-cGMP, and reproduced and potentiated by the cGMP-specific phosphodiesterase inhibitor zaprinast but not the inactive ST analog TJU 1-103. Thus, GC-C agonists regulate the proliferation of intestinal cells through cGMP-dependent mechanisms by delaying progression of the cell cycle. These data suggest that endogenous agonists of GC-C, such as uroguanylin, may play a role in regulating the balance between epithelial proliferation and differentiation in normal intestinal physiology. Therefore, GC-C ligands may be novel therapeutic agents for the treatment of patients with colorectal cancer.

Catalytic consumption of nitric oxide by 12/15- lipoxygenase: Inhibition of monocyte soluble guanylate cyclase activation

12/15-Lipoxygenase (LOX) activity is elevated in vascular diseases associated with impaired nitric oxide (⋅NO) bioactivity, such as hypertension and atherosclerosis. In this study, primary porcine monocytes expressing 12/15-LOX, rat A10 smooth muscle cells transfected with murine 12/15-LOX, and purified porcine 12/15-LOX all consumed ⋅NO in the presence of lipid substrate. Suppression of LOX diene conjugation by ⋅NO was also found, although the lipid product profile was unchanged. ⋅NO consumption by porcine monocytes was inhibited by the LOX inhibitor, eicosatetraynoic acid. Rates of arachidonate (AA)- or linoleate (LA)-dependent ⋅NO depletion by porcine monocytes (2.68 ± 0.03 nmol ⋅ min−1 ⋅ 106 cells−1 and 1.5 ± 0.25 nmol ⋅ min−1 ⋅ 106 cells−1, respectively) were several-fold greater than rates of ⋅NO generation by cytokine-activated macrophages (0.1–0.2 nmol ⋅ min−1 ⋅ 106 cells−1) and LA-dependent ⋅NO consumption by primary porcine monocytes inhibited ⋅NO activation of soluble guanylate cyclase. These data indicate that catalytic ⋅NO consumption by 12/15-LOX modulates monocyte ⋅NO signaling and suggest that LOXs may contribute to vascular dysfunction not only by the bioactivity of their lipid products, but also by serving as catalytic sinks for ⋅NO in the vasculature.

Folliculostellate cell network: A route for long-distance communication in the anterior pituitary

All higher life forms critically depend on hormones being rhythmically released by the anterior pituitary. The proper functioning of this master gland is dynamically controlled by a complex set of regulatory mechanisms that ultimately determine the fine tuning of the excitable endocrine cells, all of them heterogeneously distributed throughout the gland. Here, we provide evidence for an intrapituitary communication system by which information is transferred via the network of nonendocrine folliculostellate (FS) cells. Local electrical stimulation of FS cells in acute pituitary slices triggered cytosolic calcium waves, which propagated to other FS cells by signaling through gap junctions. Calcium wave initiation was because of the membrane excitability of FS cells, hitherto classified as silent cells. FS cell coupling could relay information between opposite regions of the gland. Because FS cells respond to central and peripheral stimuli and dialogue with endocrine cells, the form of large-scale intrapituitary communication described here may provide an efficient mechanism that orchestrates anterior pituitary functioning in response to physiological needs.

Polypeptide signaling for plant defensive genes exhibits analogies to defense signaling in animals.

The activation of plant defensive genes in leaves of tomato plants in response to herbivore damage or mechanical wounding is mediated by a mobile 18-amino acid polypeptide signal called systemin. Systemin is derived from a larger, 200-amino acid precursor called prosystemin, similar to polypeptide hormones and soluble growth factors in animals. Systemin activates a lipid-based signaling cascade, also analogous to signaling systems found in animals. In plants, linolenic acid is released from membranes and is converted to the oxylipins phytodienoic acid and jasmonic acid through the octadecanoid pathway. Plant oxylipins are structural analogs of animal prostaglandins which are derived from arachidonic acid in response to various signals, including polypeptide factors. Constitutive overexpression of the prosystemin gene in transgenic tomato plants resulted in the overproduction of prosystemin and the abnormal release of systemin, conferring a constitutive overproduction of several systemic wound-response proteins (SWRPs). The data indicate that systemin is a master signal for defense against attacking herbivores. The same defensive proteins induced by wounding are synthesized in response to oligosaccharide elicitors that are generated in leaf cells in response to pathogen attacks. Inhibitors of the octadecanoid pathway, and a mutation that interrupts this pathway, block the induction of SWRPs by wounding, systemin, and oligosaccharide elicitors, indicating that the octadecanoid pathway is essential for the activation of defense genes by all of these signals. The tomato mutant line that is functionally deficient in the octadecanoid pathway is highly susceptible to attacks by Manduca sexta larvae. The similarities between the defense signaling pathway in tomato leaves and those of the defense signaling pathways of macrophages and mast cells of animals suggests that both the plant and animal pathways may have evolved from a common ancestral origin.