A Novel Ras-interacting Protein Required for Chemotaxis and Cyclic Adenosine Monophosphate Signal Relay in Dictyostelium

We have identified a novel Ras-interacting protein from Dictyostelium, RIP3, whose function is required for both chemotaxis and the synthesis and relay of the cyclic AMP (cAMP) chemoattractant signal. rip3 null cells are unable to aggregate and lack receptor activation of adenylyl cyclase but are able, in response to cAMP, to induce aggregation-stage, postaggregative, and cell-type-specific gene expression in suspension culture. In addition, rip3 null cells are unable to properly polarize in a cAMP gradient and chemotaxis is highly impaired. We demonstrate that cAMP stimulation of guanylyl cyclase, which is required for chemotaxis, is reduced ∼60% in rip3 null cells. This reduced activation of guanylyl cyclase may account, in part, for the defect in chemotaxis. When cells are pulsed with cAMP for 5 h to mimic the endogenous cAMP oscillations that occur in wild-type strains, the cells will form aggregates, most of which, however, arrest at the mound stage. Unlike the response seen in wild-type strains, the rip3 null cell aggregates that form under these experimental conditions are very small, which is probably due to the rip3 null cell chemotaxis defect. Many of the phenotypes of the rip3 null cell, including the inability to activate adenylyl cyclase in response to cAMP and defects in chemotaxis, are very similar to those of strains carrying a disruption of the gene encoding the putative Ras exchange factor AleA. We demonstrate that aleA null cells also exhibit a defect in cAMP-mediated activation of guanylyl cyclase similar to that of rip3 null cells. A double-knockout mutant (rip3/aleA null cells) exhibits a further reduction in receptor activation of guanylyl cyclase, and these cells display almost no cell polarization or movement in cAMP gradients. As RIP3 preferentially interacts with an activated form of the Dictyostelium Ras protein RasG, which itself is important for cell movement, we propose that RIP3 and AleA are components of a Ras-regulated pathway involved in integrating chemotaxis and signal relay pathways that are essential for aggregation.

Galectin-4 and Small Intestinal Brush Border Enzymes Form Clusters

Detergent-insoluble complexes prepared from pig small intestine are highly enriched in several transmembrane brush border enzymes including aminopeptidase N and sucrase-isomaltase, indicating that they reside in a glycolipid-rich environment in vivo. In the present work galectin-4, an animal lectin lacking a N-terminal signal peptide for membrane translocation, was discovered in these complexes as well, and in gradient centrifugation brush border enzymes and galectin-4 formed distinct soluble high molecular weight clusters. Immunoperoxidase cytochemistry and immunogold electron microscopy showed that galectin-4 is indeed an intestinal brush border protein; we also localized galectin-4 throughout the cell, mainly associated with membraneous structures, including small vesicles, and to the rootlets of microvillar actin filaments. This was confirmed by subcellular fractionation, showing about half the amount of galectin-4 to be in the microvillar fraction, the rest being associated with insoluble intracellular structures. A direct association between the lectin and aminopeptidase N was evidenced by a colocalization along microvilli in double immunogold labeling and by the ability of an antibody to galectin-4 to coimmunoprecipitate aminopeptidase N and sucrase-isomaltase. Furthermore, galectin-4 was released from microvillar, right-side-out vesicles as well as from mucosal explants by a brief wash with 100 mM lactose, confirming its extracellular localization. Galectin-4 is therefore secreted by a nonclassical pathway, and the brush border enzymes represent a novel class of natural ligands for a member of the galectin family. Newly synthesized galectin-4 is rapidly “trapped” by association with intracellular structures prior to its apical secretion, but once externalized, association with brush border enzymes prevents it from being released from the enterocyte into the intestinal lumen.

Replication Factor C3 of Schizosaccharomyces pombe, a Small Subunit of Replication Factor C Complex, Plays a Role in Both Replication and Damage Checkpoints

We report here the isolation and functional analysis of the rfc3+ gene of Schizosaccharomyces pombe, which encodes the third subunit of replication factor C (RFC3). Because the rfc3+ gene was essential for growth, we isolated temperature-sensitive mutants. One of the mutants, rfc3-1, showed aberrant mitosis with fragmented or unevenly separated chromosomes at the restrictive temperature. In this mutant protein, arginine 216 was replaced by tryptophan. Pulsed-field gel electrophoresis suggested that rfc3-1 cells had defects in DNA replication. rfc3-1 cells were sensitive to hydroxyurea, methanesulfonate (MMS), and gamma and UV irradiation even at the permissive temperature, and the viabilities after these treatments were decreased. Using cells synchronized in early G2 by centrifugal elutriation, we found that the replication checkpoint triggered by hydroxyurea and the DNA damage checkpoint caused by MMS and gamma irradiation were impaired in rfc3-1 cells. Association of Rfc3 and Rad17 in vivo and a significant reduction of the phosphorylated form of Chk1 in rfc3-1 cells after treatments with MMS and gamma or UV irradiation suggested that the checkpoint signal emitted by Rfc3 is linked to the downstream checkpoint machinery via Rad17 and Chk1. From these results, we conclude that rfc3+ is required not only for DNA replication but also for replication and damage checkpoint controls, probably functioning as a checkpoint sensor.

Direct nitric oxide signal transduction via nitrosylation of iron-sulfur centers in the SoxR transcription activator

Nitric oxide (NO) has diverse roles in intercellular communication and (at higher levels) in immune-mediated cell killing. NO reacts with many cellular targets, with cell-killing effects correlated to inactivation of key enzymes through nitrosylation of their iron-sulfur centers. SoxR protein, a redox-sensitive transcription activator dependent on the oxidation state of its binuclear iron-sulfur ([2Fe-2S]) centers, is also activated in Escherichia coli on exposure to macrophage-generated NO. We show here that SoxR activation by NO occurs through direct modification of the [2Fe-2S] centers to form protein-bound dinitrosyl-iron-dithiol adducts, which we have observed both in intact bacterial cells and in purified SoxR after NO treatment. Functional activation through nitrosylation of iron-sulfur centers contrasts with the inactivation typically caused by this modification. Purified, nitrosylated SoxR has transcriptional activity similar to that of oxidized SoxR and is relatively stable. In contrast, nitrosylated SoxR is short-lived in intact cells, indicative of mechanisms that actively dispose of nitrosylated iron-sulfur centers.

Down-regulation of transcripts for Na channel α-SNS in spinal sensory neurons following axotomy

Spinal sensory (dorsal root ganglion; DRG) neurons display slowly inactivating, tetrodotoxin-resistant (TTX-R), and rapidly inactivating, TTX-sensitive (TTX-S) Na currents. Attenuation of the TTX-R Na current and enhancement of TTX-S Na current have been demonstrated in cutaneous afferent DRG neurons in the adult rat after axotomy and may underlie abnormal bursting. We show here that steady-state levels of transcripts encoding the α-SNS subunit, which is associated with a slowly inactivating, TTX-R current when expressed in oocytes, are reduced significantly 5 days following axotomy of DRG neurons, and continue to be expressed at reduced levels, even after 210 days. Steady-state levels of α-III transcripts, which are present at low levels in control DRG neurons, show a pattern of transiently increased expression. In situ hybridization using α-SNS- and α-III-specific riboprobes showed a decreased signal for α-SNS, and an increased signal for α-III, in both large and small DRG neurons following axotomy. Reduced levels of α-SNS may explain the selective loss of slowly inactivating, TTX-R current. The abnormal electrophysiological properties of DRG neurons following axonal injury thus appear to reflect a switch in Na channel gene expression.

The photoisomerization of retinal in bacteriorhodopsin: Experimental evidence for a three-state model

The primary events in the all-trans to 13-cis photoisomerization of retinal in bacteriorhodopsin have been investigated with femtosecond time-resolved absorbance spectroscopy. Spectra measured over a broad range extending from 7000 to 22,400 cm−1 reveal features whose dynamics are inconsistent with a model proposed earlier to account for the highly efficient photoisomerization process. Emerging from this work is a new three-state model. Photoexcitation of retinal with visible light accesses a shallow well on the excited state potential energy surface. This well is bounded by a small barrier, arising from an avoided crossing that separates the Franck–Condon region from the nearby reactive region of the photoisomerization coordinate. At ambient temperatures, the reactive region is accessed with a time constant of ≈500 fs, whereupon the retinal rapidly twists and encounters a second avoided crossing region. The protein mediates the passage into the second avoided crossing region and thereby exerts control over the quantum yield for forming 13-cis retinal. The driving force for photoisomerization resides in the retinal, not in the surrounding protein. This view contrasts with an earlier model where photoexcitation was thought to access directly a reactive region of the excited-state potential and thereby drive the retinal to a twisted conformation within 100–200 fs.

Transition-state structure as a unifying basis in protein-folding mechanisms: Contact order, chain topology, stability, and the extended nucleus mechanism

I attempt to reconcile apparently conflicting factors and mechanisms that have been proposed to determine the rate constant for two-state folding of small proteins, on the basis of general features of the structures of transition states. Φ-Value analysis implies a transition state for folding that resembles an expanded and distorted native structure, which is built around an extended nucleus. The nucleus is composed predominantly of elements of partly or well-formed native secondary structure that are stabilized by local and long-range tertiary interactions. These long-range interactions give rise to connecting loops, frequently containing the native loops that are poorly structured. I derive an equation that relates differences in the contact order of a protein to changes in the length of linking loops, which, in turn, is directly related to the unfavorable free energy of the loops in the transition state. Kinetic data on loop extension mutants of CI2 and α-spectrin SH3 domain fit the equation qualitatively. The rate of folding depends primarily on the interactions that directly stabilize the nucleus, especially those in native-like secondary structure and those resulting from the entropy loss from the connecting loops, which vary with contact order. This partitioning of energy accounts for the success of some algorithms that predict folding rates, because they use these principles either explicitly or implicitly. The extended nucleus model thus unifies the observations of rate depending on both stability and topology.

Denaturant-induced movement of the transition state of protein folding revealed by high-pressure stopped-flow measurements

The small all-β protein tendamistat folds and unfolds with two-state kinetics. We determined the volume changes associated with the folding process by performing kinetic and equilibrium measurements at variable pressure between 0.1 and 100 MPa (1 to 1,000 bar). GdmCl-induced equilibrium unfolding transitions reveal that the volume of the native state is increased by 41.4 ± 2.0 cm3/mol relative to the unfolded state. This value is virtually independent of denaturant concentration. The use of a high-pressure stopped-flow instrument enabled us to measure the activation volumes for the refolding (ΔVf0‡) and unfolding reaction (ΔVu0‡) over a broad range of GdmCl concentrations. The volume of the transition state is 60% native-like (ΔVf0‡ = 25.0 ± 1.2 cm3/mol) in the absence of denaturant, indicating partial solvent accessibility of the core residues. The volume of the transition state increases linearly with denaturant concentration and exceeds the volume of the native state above 6 M GdmCl. This result argues for a largely desolvated transition state with packing deficiencies at high denaturant concentrations and shows that the structure of the transition state depends strongly on the experimental conditions.

Signal recognition particle components in the nucleolus

The signal recognition particle (SRP) is a ribonucleoprotein composed of an Alu domain and an S domain. The S domain contains unique sequence SRP RNA and four SRP proteins: SRP19, SRP54, SRP68, and SRP72. SRP interacts with ribosomes to bring translating membrane and secreted proteins to the endoplasmic reticulum (ER) for proper processing. Additionally, SRP RNA is a member of a family of small nonribosomal RNAs found recently in the nucleolus, suggesting that the nucleolus is more plurifunctional than previously realized. It was therefore of interest to determine whether other SRP components localize to this intranuclear site. In transfected rat fibroblasts, green fluorescent protein fusions of SRP19, SRP68, and SRP72 localized to the nucleolus, as well as to the cytoplasm, as expected. SRP68 also accumulated in the ER, consistent with its affinity for the ER-bound SRP receptor. SRP54 was detected in the cytoplasm as a green fluorescent protein fusion and in immunofluorescence studies, but was not detected in the nucleolus. In situ hybridization experiments also revealed endogenous SRP RNA in the nucleolus. These results demonstrate that SRP RNA and three SRP proteins visit the nucleolus, suggesting that partial SRP assembly, or another unidentified activity of the SRP components, occurs at the nucleolus. SRP54 apparently interacts with nascent SRP beyond the nucleolus, consistent with in vitro reconstitution experiments showing that SRP19 must bind to SRP RNA before SRP54 binds. Our findings support the notion that the nucleolus is the site of assembly and/or interaction between the family of ribonucleoproteins involved in protein synthesis, in addition to ribosomes themselves.

NMR investigation of ferricytochrome c unfolding: Detection of an equilibrium unfolding intermediate and residual structure in the denatured state

Horse ferricytochrome c (cyt c) undergoes exchange of one of its axial heme ligands (Met-80) for one or more non-native ligands under denaturing conditions. We have used 1H NMR spectroscopy to detect two conformations of paramagnetic cyt c with non-native heme ligation through a range of urea concentrations. One non-native form is an equilibrium unfolding intermediate observed under partially denaturing conditions and is attributed to replacement of Met-80 with one or more Lys side chains. The second non-native form, in which the native Met ligand is replaced by a His, is observed under strongly denaturing conditions. Thermodynamic analysis of these data indicates a relatively small ΔG (17 kJ/mol) for the transition from native to the Lys-ligated intermediate and a significantly larger ΔG (47 kJ/mol) for the transition from native to the His-ligated species. Although CD and fluorescence data indicate that the equilibrium unfolding of cyt c is a two-state process, these NMR results implicate an intermediate with His-Lys ligation.

Inositol hexakisphosphate is a physiological signal regulating the K+-inward rectifying conductance in guard cells

(RS)-2-cis, 4-trans-abscisic acid (ABA), a naturally occurring plant stress hormone, elicited rapid agonist-specific changes in myo-inositol hexakisphosphate (InsP6) measured in intact guard cells of Solanum tuberosum (n = 5); these changes were not reproduced by (RS)-2-trans, 4-trans-abscisic acid, an inactive stereoisomer of ABA (n = 4). The electrophysiological effects of InsP6 were assessed on both S. tuberosum (n = 14) and Vicia faba (n = 6) guard cell protoplasts. In both species, submicromolar concentrations of InsP6, delivered through the patch electrode, mimicked the inhibitory effects of ABA and internal calcium (Cai2+) on the inward rectifying K+ current, IK,in, in a dose-dependent manner. Steady state block of IK,in by InsP6 was reached much more quickly in Vicia (3 min at ≈1 μM) than Solanum (20–30 min). The effects of InsP6 on IK,in were specific to the myo-inositol isomer and were not elicited by other conformers of InsP6 (e.g., scyllo- or neo-). Chelation of Ca2+ by inclusion of 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid or EGTA in the patch pipette together with InsP6 prevented the inhibition of IK,in, suggesting that the effect is Ca2+ dependent. InsP6 was ≈100-fold more potent than Ins(1,4,5)P3 in modulating IK,in. Thus ABA increases InsP6 in guard cells, and InsP6 is a potent Ca2+-dependent inhibitor of IK,in. Taken together, these results suggest that InsP6 may play a major role in the physiological response of guard cells to ABA.

Nucleocytoplasmic translocation of Stat1 is regulated by a leucine-rich export signal in the coiled-coil domain

Signal transducer and activator of transcription (Stat) proteins are latent transcription factors that reside in the cytoplasm before activation. On cytokine-induced tyrosine phosphorylation, these molecules dimerize and accumulate transiently in the nucleus. No specific signals mediating these processes have been identified to date. In this report, we examine the nuclear export of Stat1. We find that treatment of cells with the export inhibitor leptomycin B does not affect steady-state localization of Stat1 but impedes nuclear export after IFNγ-induced nuclear accumulation. We identify a conserved leucine-rich helical segment in the coiled-coil domain of Stat1, which is responsible for the efficient nuclear export of this protein. Mutation of two hallmark leucines within this segment greatly attenuate the back transport of Stat1 in the cytoplasm. When fused to a carrier protein, the Stat1 export sequence can mediate nuclear export after intranuclear microinjection. We show that prolonging the nuclear presence of Stat1 by inhibiting nuclear export reduces the transcriptional response to stimulation with IFNγ. These data suggest that Stats are actively exported from the nucleus via several separate pathways and link this activity to transcriptional activation.

Photochemically induced nuclear spin polarization in reaction centers of photosystem II observed by 13C-solid-state NMR reveals a strongly asymmetric electronic structure of the P680.+ primary donor chlorophyll

We report 13C magic angle spinning NMR observation of photochemically induced dynamic nuclear spin polarization (photo- CIDNP) in the reaction center (RC) of photosystem II (PS2). The light-enhanced NMR signals of the natural abundance 13C provide information on the electronic structure of the primary electron donor P680 (chlorophyll a molecules absorbing around 680 nm) and on the pz spin density pattern in its oxidized form, P680⨥. Most centerband signals can be attributed to a single chlorophyll a (Chl a) cofactor that has little interaction with other pigments. The chemical shift anisotropy of the most intense signals is characteristic for aromatic carbon atoms. The data reveal a pronounced asymmetry of the electronic spin density distribution within the P680⨥. PS2 shows only a single broad and intense emissive signal, which is assigned to both the C-10 and C-15 methine carbon atoms. The spin density appears shifted toward ring III. This shift is remarkable, because, for monomeric Chl a radical cations in solution, the region of highest spin density is around ring II. It leads to a first hypothesis as to how the planet can provide itself with the chemical potential to split water and generate an oxygen atmosphere using the Chl a macroaromatic cycle. A local electrostatic field close to ring III can polarize the electronic charge and associated spin density and increase the redox potential of P680 by stabilizing the highest occupied molecular orbital, without a major change of color. This field could be produced, e.g., by protonation of the keto group of ring V. Finally, the radical cation electronic structure in PS2 is different from that in the bacterial RC, which shows at least four emissive centerbands, indicating a symmetric spin density distribution over the entire bacteriochlorophyll macrocycle.

Structure of a biological oxygen sensor: A new mechanism for heme-driven signal transduction

The FixL proteins are biological oxygen sensors that restrict the expression of specific genes to hypoxic conditions. FixL’s oxygen-detecting domain is a heme binding region that controls the activity of an attached histidine kinase. The FixL switch is regulated by binding of oxygen and other strong-field ligands. In the absence of bound ligand, the heme domain permits kinase activity. In the presence of bound ligand, this domain turns off kinase activity. Comparison of the structures of two forms of the Bradyrhizobium japonicum FixL heme domain, one in the “on” state without bound ligand and one in the “off” state with bound cyanide, reveals a mechanism of regulation by a heme that is distinct from the classical hemoglobin models. The close structural resemblance of the FixL heme domain to the photoactive yellow protein confirms the existence of a PAS structural motif but reveals the presence of an alternative regulatory gateway.

Ubc9 interacts with a nuclear localization signal and mediates nuclear localization of the paired-like homeobox protein Vsx-1 independent of SUMO-1 modification

Vsx-1 is a paired-like:CVC homeobox gene whose expression is linked to bipolar cell differentiation during zebrafish retinogenesis. We used a yeast two-hybrid screen to identify proteins interacting with Vsx-1 and isolated Ubc9, an enzyme that conjugates the small ubiquitin-like modifier SUMO-1. Despite its interaction with Ubc9, we show that Vsx-1 is not a substrate for SUMO-1 in COS-7 cells or in vitro. When a yeast two-hybrid assay is used, deletion analysis of the interacting domain on Vsx-1 shows that Ubc9 binds to a nuclear localization signal (NLS) at the NH2 terminus of the homeodomain. In SW13 cells, Vsx-1 localizes to the nucleus and is excluded from nucleoli. Deletion of the NLS disrupts this nuclear localization, resulting in a diffuse cytoplasmic distribution of Vsx-1. In SW13 AK1 cells that express low levels of endogenous Ubc9, Vsx-1 accumulates in a perinuclear ring and colocalizes with an endoplasmic reticulum marker. However, NLS-tagged STAT1 protein exhibits normal nuclear localization in both SW13 and SW13 AK1 cells, suggesting that nuclear import is not globally disrupted. Cotransfection of Vsx-1 with Ubc9 restores Vsx-1 nuclear localization in SW3 AK1 cells and demonstrates that Ubc9 is required for the nuclear localization of Vsx-1. Ubc9 continues to restore nuclear localization even after a C93S active site mutation has eliminated its SUMO-1-conjugating ability. These results suggest that Ubc9 mediates the nuclear localization of Vsx-1, and possibly other proteins, through a nonenzymatic mechanism that is independent of SUMO-1 conjugation.