Mutant G protein α subunit activated by Gβγ: A model for receptor activation?

How receptors catalyze exchange of GTP for GDP bound to the Gα subunit of trimeric G proteins is not known. One proposal is that the receptor uses the G protein's βγ heterodimer as a lever, tilting it to pull open the guanine nucleotide binding pocket of Gα. To test this possibility, we designed a mutant Gα that would bind to βγ in the tilted conformation. To do so, we excised a helical turn (four residues) from the N-terminal region of αs, the α subunit of GS, the stimulatory regulator of adenylyl cyclase. In the presence, but not in the absence, of transiently expressed β1 and γ2, this mutant (αsΔ), markedly stimulated cAMP accumulation. This effect depended on the ability of the coexpressed β protein to interact normally with the lip of the nucleotide binding pocket of αsΔ. We substituted alanine for an aspartate in β1 that binds to a lysine (K206) in the lip of the α subunit's nucleotide binding pocket. Coexpressed with αsΔ and γ2, this mutant, β1-D228A, elevated cAMP much less than did β1-wild type; it did bind to αsΔ normally, however, as indicated by its unimpaired ability to target αsΔ to the plasma membrane. We conclude that βγ can activate αs and that this effect probably involves both a tilt of βγ relative to αs and interaction of β with the lip of the nucleotide binding pocket. We speculate that receptors use a similar mechanism to activate trimeric G proteins.

Equilibration of the terrestrial water, nitrogen, and carbon cycles

Recent advances in biologically based ecosystem models of the coupled terrestrial, hydrological, carbon, and nutrient cycles have provided new perspectives on the terrestrial biosphere’s behavior globally, over a range of time scales. We used the terrestrial ecosystem model Century to examine relationships between carbon, nitrogen, and water dynamics. The model, run to a quasi-steady-state, shows strong correlations between carbon, water, and nitrogen fluxes that lead to equilibration of water/energy and nitrogen limitation of net primary productivity. This occurs because as the water flux increases, the potentials for carbon uptake (photosynthesis), and inputs and losses of nitrogen, all increase. As the flux of carbon increases, the amount of nitrogen that can be captured into organic matter and then recycled also increases. Because most plant-available nitrogen is derived from internal recycling, this latter process is critical to sustaining high productivity in environments where water and energy are plentiful. At steady-state, water/energy and nitrogen limitation “equilibrate,” but because the water, carbon, and nitrogen cycles have different response times, inclusion of nitrogen cycling into ecosystem models adds behavior at longer time scales than in purely biophysical models. The tight correlations among nitrogen fluxes with evapotranspiration implies that either climate change or changes to nitrogen inputs (from fertilization or air pollution) will have large and long-lived effects on both productivity and nitrogen losses through hydrological and trace gas pathways. Comprehensive analyses of the role of ecosystems in the carbon cycle must consider mechanisms that arise from the interaction of the hydrological, carbon, and nutrient cycles in ecosystems.

Lifespan extension and delayed immune and collagen aging in mutant mice with defects in growth hormone production

Single-gene mutations that extend lifespan provide valuable tools for the exploration of the molecular basis for age-related changes in cell and tissue function and for the pathophysiology of age-dependent diseases. We show here that mice homozygous for loss-of-function mutations at the Pit1 (Snell dwarf) locus show a >40% increase in mean and maximal longevity on the relatively long-lived (C3H/HeJ × DW/J)F1 background. Mutant dwJ/dw animals show delays in age-dependent collagen cross-linking and in six age-sensitive indices of immune system status. These findings thus demonstrate that a single gene can control maximum lifespan and the timing of both cellular and extracellular senescence in a mammal. Pituitary transplantation into dwarf mice does not reverse the lifespan effect, suggesting that the effect is not due to lowered prolactin levels. In contrast, homozygosity for the Ghrhrlit mutation, which like the Pit1dw mutation lowers plasma growth hormone levels, does lead to a significant increase in longevity. Male Snell dwarf mice, unlike calorically restricted mice, become obese and exhibit proportionately high leptin levels in old age, showing that their exceptional longevity is not simply due to alterations in adiposity per se. Further studies of the Pit1dw mutant, and the closely related, long-lived Prop-1df (Ames dwarf) mutant, should provide new insights into the hormonal regulation of senescence, longevity, and late life disease.

Integrality of Tate-cycles

We explain a technical result about p-adic cohomology and apply it to the study of Shimura varieties. The technical result applies to algebraic varieties with torsion-free cohomology, but for simplicity we only treat abelian varieties.

Accumulation of Mutant Huntingtin Fragments in Aggresome-like Inclusion Bodies as a Result of Insufficient Protein Degradation

The huntingtin exon 1 proteins with a polyglutamine repeat in the pathological range (51 or 83 glutamines), but not with a polyglutamine tract in the normal range (20 glutamines), form aggresome-like perinuclear inclusions in human 293 Tet-Off cells. These structures contain aggregated, ubiquitinated huntingtin exon 1 protein with a characteristic fibrillar morphology. Inclusion bodies with truncated huntingtin protein are formed at centrosomes and are surrounded by vimentin filaments. Inhibition of proteasome activity resulted in a twofold increase in the amount of ubiquitinated, SDS-resistant aggregates, indicating that inclusion bodies accumulate when the capacity of the ubiquitin–proteasome system to degrade aggregation-prone huntingtin protein is exhausted. Immunofluorescence and electron microscopy with immunogold labeling revealed that the 20S, 19S, and 11S subunits of the 26S proteasome, the molecular chaperones BiP/GRP78, Hsp70, and Hsp40, as well as the RNA-binding protein TIA-1, the potential chaperone 14–3-3, and α-synuclein colocalize with the perinuclear inclusions. In 293 Tet-Off cells, inclusion body formation also resulted in cell toxicity and dramatic ultrastructural changes such as indentations and disruption of the nuclear envelope. Concentration of mitochondria around the inclusions and cytoplasmic vacuolation were also observed. Together these findings support the hypothesis that the ATP-dependent ubiquitin–proteasome system is a potential target for therapeutic interventions in glutamine repeat disorders.

Root-Growth Behavior of the Arabidopsis Mutant rgr11 : Roles of Gravitropism and Circumnutation in the Waving/Coiling Phenomenon

In this study we investigated the kinetics of the gravitropic response of the Arabidopsis mutant rgr1 (reduced root gravitropism). Although the rate of curvature in rgr1, which is allelic to axr4, was smaller than in the wild type (ecotype Wassilewskija), curvature was initiated in the same region of the root, the distal elongation zone. The time lag for the response was unaffected in the mutant; however, the gravitropic response of rgr1 contained a feature not found in the wild type: when roots growing along the surface of an agar plate were gravistimulated, there was often an upward curvature that initiated in the central elongation zone. Because this response was dependent on the tactile environment of the root, it most likely resulted from the superposition of the waving/coiling phenomenon onto the gravitropic response. We found that the frequency of the waving pattern and circumnutation, a cyclic endogenous pattern of root growth, was the same in rgr1 and in the wild type, so the waving/coiling phenomenon is likely governed by circumnutation patterns. The amplitudes of these oscillations may then be selectively amplified by tactile stimulation to provide a directional preference to the slanting.

petit1, a Conditional Growth Mutant of Arabidopsis Defective in Sucrose-Dependent Elongation Growth1

The hypocotyl of Arabidopsis is well suited for the analysis of cell elongation because it elongates without cell division. We have isolated a new class of recessive mutants, petit1 (pet1), which are defective in aspects of hypocotyl elongation. The short-hypocotyl phenotype of pet1 is caused by shortened cells. The cells of the elongation zone of the hypocotyl are often deformed. pet1 also shows defects in elongation of the roots, flower stalk, leaves, petals, pedicels, and siliques, and these defects cannot be repaired by the application of auxin, gibberellin, brassinolide, or an inhibitor of ethylene biosynthesis. The short-hypocotyl phenotype of pet1 is pronounced only in growth medium supplemented with sucrose, which has promotive effects on hypocotyl elongation. In pet1 this effect is much reduced, causing the sucrose-dependent short-hypocotyl phenotype of pet1. pet1 accumulates more soluble sugars than the wild type and also shows more intensive iodo-starch staining in the cotyledon and hypocotyl. These results indicate that PETIT1 is involved in a sugar-dependent elongation process that may include correct assembly of expanding cell wall architecture.

A Functional Calvin Cycle Is Not Indispensable for the Light Activation of C4 Phosphoenolpyruvate Carboxylase Kinase and Its Target Enzyme in the Maize Mutant bundle sheath defective2-mutable11

We used a pale-green maize (Zea mays L.) mutant that fails to accumulate ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) to test the working hypothesis that the regulatory phosphorylation of C4 phosphoenolpyruvate carboxylase (PEPC) by its Ca2+-insensitive protein-serine/threonine kinase (PEPC kinase) in the C4 mesophyll cytosol depends on cross-talk with a functional Calvin cycle in the bundle sheath. Wild-type (W22) and bundle sheath defective2-mutable1 (bsd2-m1) seeds were grown in a controlled environment chamber at 100 to 130 μmol m−2 s−1 photosynthetic photon flux density, and leaf tissue was harvested 11 d after sowing, following exposure to various light intensities. Immunoblot analysis showed no major difference in the amount of polypeptide present for several mesophyll- and bundle-sheath-specific photosynthetic enzymes apart from Rubisco, which was either completely absent or very much reduced in the mutant. Similarly, leaf net CO2-exchange analysis and in vitro radiometric Rubisco assays showed that no appreciable carbon fixation was occurring in the mutant. In contrast, the sensitivity of PEPC to malate inhibition in bsd2-m1 leaves decreased significantly with an increase in light intensity, and there was a concomitant increase in PEPC kinase activity, similar to that seen in wild-type leaf tissue. Thus, although bsd2-m1 mutant plants lack an operative Calvin cycle, light activation of PEPC kinase and its target enzyme are not grossly perturbed.

wrinkled1: A Novel, Low-Seed-Oil Mutant of Arabidopsis with a Deficiency in the Seed-Specific Regulation of Carbohydrate Metabolism1

During oil deposition in developing seeds of Arabidopsis, photosynthate is imported in the form of carbohydrates into the embryo and converted to triacylglycerols. To identify genes essential for this process and to investigate the molecular basis for the developmental regulation of oil accumulation, mutants producing wrinkled, incompletely filled seeds were isolated. A novel mutant locus, wrinkled1 (wri1), which maps to the bottom of chromosome 3 and causes an 80% reduction in seed oil content, was identified. Wild-type and homozygous wri1 mutant plantlets or mature plants were indistinguishable. However, developing homozygous wri1 seeds were impaired in the incorporation of sucrose and glucose into triacylglycerols, but incorporated pyruvate and acetate at an increased rate. Because the activities of several glycolytic enzymes, in particular hexokinase and pyrophosphate-dependent phosphofructokinase, are reduced in developing homozygous wri1 seeds, it is suggested that WRI1 is involved in the developmental regulation of carbohydrate metabolism during seed filling.

Induction of a Carbon-Starvation-Related Proteolysis in Whole Maize Plants Submitted to Light/Dark Cycles and to Extended Darkness1

Three-week-old maize (Zea mays L.) plants were submitted to light/dark cycles and to prolonged darkness to investigate the occurrence of sugar-limitation effects in different parts of the whole plant. Soluble sugars fluctuated with light/dark cycles and dropped sharply during extended darkness. Significant decreases in protein level were observed after prolonged darkness in mature roots, root tips, and young leaves. Glutamine and asparagine (Asn) changed in opposite ways, with Asn increasing in the dark. After prolonged darkness the increase in Asn accounted for most of the nitrogen released by protein breakdown. Using polyclonal antibodies against a vacuolar root protease previously described (F. James, R. Brouquisse, C. Suire, A. Pradet, P. Raymond [1996] Biochem J 320: 283–292) or the 20S proteasome, we showed that the increase in proteolytic activities was related to an enrichment of roots in the vacuolar protease, with no change in the amount of 20S proteasome in either roots or leaves. Our results show that no significant net proteolysis is induced in any part of the plant during normal light/dark cycles, although changes in metabolism and growth appear soon after the beginning of the dark period, and starvation-related proteolysis probably appears in prolonged darkness earlier in sink than in mature tissues.

The Expression of Light-Regulated Genes in the High-Pigment-1 Mutant of Tomato1

Three light-regulated genes, chlorophyll a/b-binding protein (CAB), ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit, and chalcone synthase (CHS), are demonstrated to be up-regulated in the high-pigment-1 (hp-1) mutant of tomato (Lycopersicon esculentum Mill.) compared with wild type (WT). However, the pattern of up-regulation of the three genes depends on the light conditions, stage of development, and tissue studied. Compared with WT, the hp-1 mutant showed higher CAB gene expression in the dark after a single red-light pulse and in the pericarp of immature fruits. However, in vegetative tissues of light-grown seedlings and adult plants, CAB mRNA accumulation did not differ between WT and the hp-1 mutant. The ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit mRNA accumulated to a higher level in the hp-1 mutant than WT under all light conditions and tissues studied, whereas CHS gene expression was up-regulated in de-etiolated vegetative hp-1-mutant tissues only. The CAB and CHS genes were shown to be phytochrome regulated and both phytochrome A and B1 play a role in CAB gene expression. These observations support the hypothesis that the HP-1 protein plays a general repressive role in phytochrome signal transduction.

The Never ripe Mutant Provides Evidence That Tumor-Induced Ethylene Controls the Morphogenesis of Agrobacterium tumefaciens-Induced Crown Galls on Tomato Stems12

We confirm the hypothesis that Agrobacterium tumefaciens-induced galls produce ethylene that controls vessel differentiation in the host stem of tomato (Lycopersicon esculentum Mill.). Using an ethylene-insensitive mutant, Never ripe (Nr), and its isogenic wild-type parent we show that infection by A. tumefaciens results in high rates of ethylene evolution from the developing crown galls. Ethylene evolution from isolated internodes carrying galls was up to 50-fold greater than from isolated internodes of control plants when measured 21 and 28 d after infection. Tumor-induced ethylene substantially decreased vessel diameter in the host tissues beside the tumor in wild-type stems but had a very limited effect in the Nr stems. Ethylene promoted the typical unorganized callus shape of the gall, which maximized the tumor surface in wild-type stems, whereas the galls on the Nr stems had a smooth surface. The combination of decreased vessel diameter in the host and increased tumor surface ensured water-supply priority to the growing gall over the host shoot. These results indicate that in addition to the well-defined roles of auxin and cytokinin, there is a critical role for ethylene in determining crown-gall morphogenesis.

Correlation between Binding Affinity and Necrosis-Inducing Activity of Mutant AVR9 Peptide Elicitors1

The race-specific peptide elicitor AVR9 of the fungus Cladosporium fulvum induces a hypersensitive response only in tomato (Lycopersicon esculentum) plants carrying the complementary resistance gene Cf-9 (MoneyMaker-Cf9). A binding site for AVR9 is present on the plasma membranes of both resistant and susceptible tomato genotypes. We used mutant AVR9 peptides to determine the relationship between elicitor activity of these peptides and their affinity to the binding site in the membranes of tomato. Mutant AVR9 peptides were purified from tobacco (Nicotiana clevelandii) inoculated with recombinant potato virus X expressing the corresponding avirulence gene Avr9. In addition, several AVR9 peptides were synthesized chemically. Physicochemical techniques revealed that the peptides were correctly folded. Most mutant AVR9 peptides purified from potato virus X::Avr9-infected tobacco contain a single N-acetylglucosamine. These glycosylated AVR9 peptides showed a lower affinity to the binding site than the nonglycosylated AVR9 peptides, whereas their necrosis-inducing activity was hardly changed. For both the nonglycosylated and the glycosylated mutant AVR9 peptides, a positive correlation between their affinity to the membrane-localized binding site and their necrosis-inducing activity in MoneyMaker-Cf9 tomato was found. The perception of AVR9 in resistant and susceptible plants is discussed.

Aluminum Resistance in the Arabidopsis Mutant alr-104 Is Caused by an Aluminum-Induced Increase in Rhizosphere pH1

A mechanism that confers increased Al resistance in the Arabidopsis thaliana mutant alr-104 was investigated. A modified vibrating microelectrode system was used to measure H+ fluxes generated along the surface of small Arabidopsis roots. In the absence of Al, no differences in root H+ fluxes between wild type and alr-104 were detected. However, Al exposure induced a 2-fold increase in net H+ influx in alr-104 localized to the root tip. The increased flux raised the root surface pH of alr-104 by 0.15 unit. A root growth assay was used to assess the Al resistance of alr-104 and wild type in a strongly pH-buffered nutrient solution. Increasing the nutrient solution pH from 4.4 to 4.5 significantly increased Al resistance in wild type, which is consistent with the idea that the increased net H+ influx can account for greater Al resistance in alr-104. Differences in Al resistance between wild type and alr-104 disappeared when roots were grown in pH-buffered medium, suggesting that Al resistance in alr-104 is mediated only by pH changes in the rhizosphere. This mutant provides the first evidence, to our knowledge, for an Al-resistance mechanism based on an Al-induced increase in root surface pH.