Phosducin facilitates light-driven transducin translocation in rod photoreceptors. Evidence from the phosducin knockout mouse

Phosducin is a photoreceptor-specific protein known to interact with the beta gamma subunits of G proteins. In pursuit of the function of phosducin, we tested the hypothesis that it regulates the light-driven translocation of G protein transducin from the outer segments of rod photoreceptors to other compartments of the rod cell. Transducin translocation has been previously shown to contribute to rod adaptation to bright illumination, yet the molecular mechanisms underlying the translocation phenomenon remain unknown. In this study we provide two major lines of evidence in support of the role of phosducin in transducin translocation. First, we have demonstrated that transducin beta gamma subunits interact with phosducin along their entire intracellular translocation route, as evident from their co-precipitation in serial tangential sections from light-adapted but not dark-adapted retinas. Second, we generated a phosducin knockout mouse and found that the degree of light-driven transducin translocation in the rods of these mice was significantly reduced as compared with that observed in the rods of wild type animals. In knockout animals the translocation of transducin beta gamma subunits was affected to a larger degree than the translocation of the alpha subunit. We also found that the amount of phosducin in rods is sufficient to interact with practically all of the transducin present in these cells and that the subcellular distribution of phosducin is consistent with that of a soluble protein evenly distributed throughout the entire rod cytoplasm. Together, these data indicate that phosducin binding to transducin beta gamma subunits facilitates transducin translocation. We suggest that the mechanism of phosducin action is based on the reduction of transducin affinity to the membranes of rod outer segments, achieved by keeping the transducin beta gamma subunits apart from the alpha subunit. This increased solubility of transducin would make it more susceptible to translocation from the outer segments.     

Farnesylation of retinal transducin underlies its translocation during light adaptation

G proteins are posttranslationally modified by isoprenylation: either farnesylation or geranylgeranylation. The gamma subunit of retinal transducin (Talpha/Tbetagamma) is selectively farnesylated, and the farnesylation is required for light signaling mediated by transducin in rod cells. However, whether and how this selective isoprenylation regulates cellular functions remain poorly understood. Here we report that knockin mice expressing geranylgeranylated Tgamma showed normal rod responses to dim flashes under dark-adapted conditions but exhibited impaired properties in light adaptation. Of note, geranylgeranylation of Tgamma suppressed light-induced transition of Tbetagamma from membrane to cytosol, and also attenuated its light-dependent translocation from the outer segment to the inner region, an event contributing to retinal light adaptation. These results indicate that, while the farnesylation of transducin is interchangeable with the geranylgeranylation in terms of the light signaling, the selective farnesylation is important for visual sensitivity regulation by providing sufficient but not excessive membrane anchoring of Tbetagamma.     

Effect of light on phosphatidate phosphohydrolase activity of retina rod outer segments: the role of transducin

The aim of the present paper is to evaluate the modulation of phosphatidate phosphohydrolase (PAPase) and diacylglyceride lipase (DGL) activities in bovine rod outer segment (ROS) under dark and light conditions and to evaluate the role of transducin (T) in this phenomenon. In dark-adapted ROS membranes exposed to light, PAPase activity is inhibited by 20% with respect to the activity found under dark conditions. To determine whether the retinal G protein, T, participates in the regulation of PAPase activity in these membranes, the effects of GTPgammaS and GDPbetaS on enzyme activity were examined. Under dark conditions in the presence of GTPgammaS, which stabilizes T in its active form (Talpha + Tbetagamma), enzyme activity was inhibited and approached control values under light conditions. GDPbetaS, on the other hand, which stabilizes the inactive state of T (Talphabetagamma), stimulated PAPase activity by 36% with respect to control light conditions. ADP-ribosylation by cholera and pertussis toxin was also studied. In ADP-rybosilated ROS membranes with pertussis toxin under dark conditions, PAPase activity was 36% higher than the activity found under control light conditions. ADP-ribosylation by CTx, on the other hand, inhibited PAPase activity by 22%, with respect to dark control conditions, mimicking light effect. The effects of GTPgammaS and GDPbetaS and conditions of ADP-ribosylation by PTx and CTx on DGL activity were similar to those of PAPase activities. Based on NEM sensitivity we have also demonstrated that the PAPase present in ROS is the PAP 2 isoform. Our findings therefore suggest that light inhibition of PAP 2 in ROS is a transducin-mediated mechanism.     

A novel magnesium-dependent mechanism for the activation of transducin by fluoride

Activation of transducin-GDP by NaF is mainly mediated by aluminofluorde or beryllofluoride complexes acting as GTP gamma-phosphate analogs. In millimolar magnesium, NaF at concentrations above 3 mM is active even in the absence of aluminium or beryllium. This activation has a Hill coefficient of 3 with respect to F-, and its rate is linear with respect to Mg2+ concentrations above 2 mM. Upon fluoride dilution, inactivation rate is hundreds of times faster than for aluminofluoride-activated T alpha GDP. We propose that at high NaF concentrations, 3 hydrogen-bonded fluorides in the gamma-phosphate site of T alpha GDP entrap a magnesium counterion and this induces the transconformation to the T alpha GTP form.     

Helical inchworming: a novel translocation mechanism for a ring ATPase

Ring ATPases perform a variety of tasks in the cell. Their function involves complex communication and coordination among the often identical subunits. Translocases in this group are of particular interest as they involve both chemical and mechanical actions in their operation. We study the DNA packaging motor of bacteriophage φ29, and using single-molecule optical tweezers and single-particle cryo-electron microscopy, have discovered a novel translocation mechanism for a molecular motor.     

Phosducin regulates the expression of transducin betagamma subunits in rod photoreceptors and does not contribute to phototransduction adaptation

For over a decade, phosducin's interaction with the βγ subunits of the G protein, transducin, has been thought to contribute to light adaptation by dynamically controlling the amount of transducin heterotrimer available for activation by photoexcited rhodopsin. In this study we directly tested this hypothesis by characterizing the dark- and light-adapted response properties of phosducin knockout (Pd−/−) rods. Pd−/− rods were notably less sensitive to light than wild-type (WT) rods. The gain of transduction, as measured by the amplification constant using the Lamb-Pugh model of activation, was 32% lower in Pd−/− rods than in WT rods. This reduced amplification correlated with a 36% reduction in the level of transducin βγ-subunit expression, and thus available heterotrimer in Pd−/− rods. However, commonly studied forms of light adaptation were normal in the absence of phosducin. Thus, phosducin does not appear to contribute to adaptation mechanisms of the outer segment by dynamically controlling heterotrimer availability, but rather is necessary for maintaining normal transducin expression and therefore normal flash sensitivity in rods.     

Interactions between the subunits of transducin and cyclic GMP phosphodiesterase in Rana catesbiana rod photoreceptors

In bullfrog (Rana catesbiana) rods the activity of cyclic GMP (cGMP) phosphodiesterase was stimulated 10 times by washing disc membranes with an isotonic, GTP-containing buffer. This stimulation was maintained following hydrolysis of GTP and after removal of guanine nucleotides. At least 60-70% of the inhibitory gamma subunit of cGMP phosphodiesterase (P gamma) was physically released from membranes by these washing procedures. When cGMP phosphodiesterase was activated by a hydrolysis-resistant GTP analogue, P gamma was found in the supernatant complexed with the transducin alpha subunit (T alpha) using three chromatography systems. When GTP was used to activate cGMP phosphodiesterase, P gamma was also found in the supernatant complexed with GDP.T alpha. This complex was also isolated using the same three chromatography systems, indicating that P gamma remained tightly bound to T alpha even after bound GTP was hydrolyzed. Interaction with the beta,gamma subunits of transducin, which remained associated with disc membranes, was required for the release of P gamma from the GDP.T alpha complex, which resulted in the deactivation of active cGMP phosphodiesterase. We conclude that during activation of cGMP phosphodiesterase, P gamma is complexed with T alpha (both GTP and GDP forms) in the supernatant and that, following GTP hydrolysis, beta,gamma subunits of transducin are necessary for the release of P gamma from the complex and the resulting inactivation of cGMP phosphodiesterase in frog photoreceptors.     

Interaction of transducin with uncoordinated 119 protein (UNC119): implications for the model of transducin trafficking in rod photoreceptors

The key visual G protein, transducin undergoes bi-directional translocations between the outer segment (OS) and inner compartments of rod photoreceptors in a light-dependent manner thereby contributing to adaptation and neuroprotection of rods. A mammalian uncoordinated 119 protein (UNC119), also known as Retina Gene 4 protein (RG4), has been recently implicated in transducin transport to the OS in the dark through its interaction with the N-acylated GTP-bound transducin-α subunit (Gα(t1)). Here, we demonstrate that the interaction of human UNC119 (HRG4) with transducin is dependent on the N-acylation, but does not require the GTP-bound form of Gα(t1). The lipid specificity of UNC119 is unique: UNC119 bound the myristoylated N terminus of Gα(t1) with much higher affinity than a prenylated substrate, whereas the homologous prenyl-binding protein PrBP/δ did not interact with the myristoylated peptide. UNC119 was capable of interacting with Gα(t1)GDP as well as with heterotrimeric transducin (G(t)). This interaction of UNC119 with G(t) led to displacement of Gβ(1)γ(1) from the heterotrimer. Furthermore, UNC119 facilitated solubilization of G(t) from dark-adapted rod OS membranes. Consistent with these observations, UNC119 inhibited rhodopsin-dependent activation of G(t), but had no effect on the GTP-hydrolysis by Gα(t1). A model for the role of UNC119 in the IS→OS translocation of G(t) is proposed based on the UNC119 ability to dissociate G(t) subunits from each other and the membrane. We also found that UNC119 inhibited activation of G(o) by D2 dopamine receptor in cultured cells. Thus, UNC119 may play conserved inhibitory role in regulation of GPCR-G protein signaling in non-visual tissues.     

Isolation and characterization of the two major intracellular Glut4 storage compartments.

In rat adipose cells, intracellular Glut4 resides in two distinct vesicular populations one of which contains cellugyrin whereas another lacks this protein (Kupriyanova, T. A., and Kandror, K. V. (2000) J. Biol. Chem. 275, 36263--36268). Cell surface biotinylated MPR and (125)I-labeled transferrin are accumulated in cellugyrin-positive vesicles and to a lesser extent in cellugyrin-negative vesicles. An average cellugyrin-positive vesicle carries not more than one molecule of either Glut4, insulin-responsive aminopeptidase (IRAP), or transferrin receptor (TfR), whereas cellugyrin-negative vesicles contain five to six molecules of Glut4, more than 10 molecules of IRAP, and still one molecule of TfR per vesicle. Cellugyrin-negative vesicles are translocated to the cell surface after insulin stimulation, whereas cellugyrin-positive vesicles maintain intracellular localization both in the absence and in the presence of insulin and, therefore, may be involved in interendosomal protein transport. Both cellugyrin-positive and cellugyrin-negative vesicles are present in extracts of non-homogenized cells and therefore may represent the major form of Glut4 storage in vivo.     

Fluorescence induction in whole leaves: Differentiation between the two leaf sides and adaptation to different light regimes

In a variety of plants, the induction kinetics of chlorophyll fluorescence vary substantially depending on whether measured on the upper or lower side of the same leaf. The responses are comparable to those of plants grown under sun and shade conditions. Leaf morphology appears not to be the primary cause of the differences since inversion of the leaves can lead to reversed fluorescence responses. Fluorescence induction was analyzed in control and inverted leaves, and in one case, in chloroplasts from sun and shade leaves. It is concluded from the data that the major differences between the chloroplasts of the upper and lower leaf side reflect ionic and thylakoidmembrane conformational factors, rather than structural differences. Mg²⁺ flux probably plays a significant role in the adjustment of the thylakoid membrane to high or low light conditions.     

Functional redundancy between two Caenorhabditis elegans nucleotide sugar transporters with a novel transport mechanism

Transporters of nucleotide sugars regulate the availability of these substrates required for glycosylation reactions in the lumen of the Golgi apparatus and play an important role in the development of multicellular organisms. Caenorhabditis elegans has seven different sugars in its glycoconjugates, although 18 putative nucleotide sugar transporters are encoded in the genome. Among these, SQV-7, SRF-3, and CO3H5.2 exhibit partially overlapping substrate specificity and expression patterns. We now report evidence of functional redundancy between transporters CO3H5.2 and SRF-3. Reducing the activity of the CO3H5.2 gene product by RNA interference (RNAi) in SRF-3 mutants results in oocyte accumulation and abnormal gonad morphology, whereas comparable RNAi treatment of wild type or RNAi hypersensitive C. elegans strains does not cause detectable defects. We hypothesize this genetic enhancement to be a mechanism to ensure adequate glycoconjugate biosynthesis required for normal tissue development in multicellular organisms. Furthermore, we show that transporters SRF-3 and CO3H5.2, which are closely related in the phylogenetic tree, share a simultaneous and independent substrate transport mechanism that is different from the competitive one previously demonstrated for transporter SQV-7, which shares a lower amino acid sequence identity with CO3H5.2 and SRF-3. Therefore, different mechanisms for transporting multiple nucleotide sugars may have evolved parallel to transporter amino acid divergence.     

Inhibition of glutamine synthetase in the mouse kidney: a novel mechanism of adaptation to metabolic acidosis

As part of a study on the regulation of renal ammoniagenesis in the mouse kidney, we investigated the effect of chronic metabolic acidosis on glutamine synthesis by isolated mouse renal proximal tubules. The results obtained reveal that, in tubules from control mice, glutamine synthesis occurred at high rates from glutamate and proline and, to a lesser extent, from ornithine, alanine, and aspartate. A 48 h, metabolic acidosis caused a marked inhibition of glutamine synthesis from near-physiological concentrations of both alanine and proline that were avidly metabolized by the tubules; metabolic acidosis also greatly stimulated glutamine utilization and metabolism. These effects were accompanied by a large increase (i) in alanine, proline, and glutamine gluconeogenesis and (ii) in ammonia accumulation from proline and glutamine. In the renal cortex of acidotic mice, the activity of phosphoenolpyruvate carboxykinase increased 4-fold, but that of glutamate dehydrogenase did not change; in contrast with what is known in the rat renal cortex, metabolic acidosis markedly diminished the glutamine synthetase activity and protein level, but not the glutamine synthetase mRNA level in the mouse renal cortex. These results strongly suggest that, in the mouse kidney, glutamine synthetase is an important regulatory component of the availability of the ammonium ions to be excreted for defending systemic acid-base balance. Furthermore, they show that, in rodents, the regulation of renal glutamine synthetase is species-specific.     

Activation of retinal rod cyclic GMP-phosphodiesterase by transducin: Characterization of the complex formed by phosphodiesterase inhibitor and transducin α-subunit

The GTP-binding subunit of transducin (Tα) activates the cGMP phosphodiesterase (PDE) of bovine retinal rods by relieving the constraint imposed by the inhibitory subunit PDEγ. We have isolated and characterized the complex Tα.GTPγS-PDEγ formed when Tα is activated by the nonhydrolyzable analog GTPγS. Sedimentation and light-scattering techniques demonstrate that, in contrast to free Tγ.GTPγS, which is soluble, the Tα.GTPγS-PDEγ complex, as well as Tα.GTP-PDEγ, is membrane bound at cytosolic ionic strength. It is eluted from the membrane at low ionic strength as a monomeric and 1:1 stoichiometric complex. The relative affinities of PDEγ for PDEαβ and for Tα.GTP are discussed.     

Escherichia coli alpha-haemolysin induces focal leaks in colonic epithelium: a novel mechanism of bacterial translocation

Extraintestinal pathogenic Escherichia coli (ExPEC) are usually harmless colonizer of the intestinal microflora. However, they are capable to translocate and cause life-threatening disease. Translocation of ExPEC isolates was quantified in colonic monolayers. Transepithelial resistance (R(t)) was monitored and local changes in conductivity analysed with conductance scanning. Confocal microscopy visualized the translocation route. Corroboratory experiments were performed on native rat colon. One translocating strain E. coli O4 was identified. This translocation process was associated with an R(t) decrease (36 +/- 1% of initial resistance) beginning only 2 h after inoculation. The sites of translocation were small defects in epithelial integrity (focal leaks) exhibiting highly increased local ion permeability. Translocation was enhanced by preincubation of monolayers with tumour necrosis factor-alpha or interleukin-13. Mutant strains lacking alpha-haemolysin lost the ability to induce focal leaks, while this effect could be restored by re-introducing the haemolysin determinant. Filtrate of a laboratory strain carrying the alpha-haemolysin operon was sufficient for focal leak induction. In native rat colon, E. coli O4 decreased R(t) and immunohistology demonstrated focal leaks resembling those in cell monolayers. E. coli alpha-haemolysin is able to induce focal leaks in colonic cell cultures as well as in native colon. This process represents a novel route of bacterial translocation facilitated by pro-inflammatory cytokines.     

Nuclear export of actin: a novel mechanism regulating the subcellular localization of a major cytoskeletal protein.

Actin is a highly conserved, ubiquitous cytoskeletal protein, which is essential for multiple cellular functions. Despite its small size (Mr = 42 000), unpolymerized forms of actin, as well as polymerized forms, exist primarily in the cytoplasm, excluded from the nucleus. Although spatial control of actin is crucially important, the molecular mechanisms ensuring the cytoplasmic localization of unpolymerized actin have not been revealed so far. In this paper we report that actin contains two leucine-rich type nuclear export signal (NES) sequences in the middle part of the molecule, which are both shown to be functional. Monomeric actin, when injected into the nucleus, was rapidly exported in a manner which was sensitive to leptomycin B (LMB), a specific inhibitor of NES-dependent nuclear export. LMB treatment of cells prevented nuclear exclusion of endogenous actin, inducing its nuclear accumulation. Furthermore, actin mutants with disrupted NESs accumulated in the nucleus. Expression of these NES-disrupted actin mutants, but not of wild-type actin, induced a decrease in the proliferative potential of the cell. These results reveal a novel molecular mechanism controlling the subcellular distribution of actin.     

A model for the mechanism of light and dark adaptation of vertebrate cones

A model is proposed for the mechanism of light and dark adaptation of vertebrate cones, especially for the one of operating curves shifting during light and dark adaptation, on the basis of physiological results. The mechanism is modeled in terms of bleaching levels and background effects through horizontal cell feedback loops. Furthermore, the spectral sensitivity of vertebrate cones is examined with the model. Simulations of the model are made and the results of the simulations extremely coincide with experimental results.