Companion cell-specific inhibition of the potato sucrose transporter SUT1

In many plants, translocation of sucrose from mesnsophyll to phloem for long-distance transport is carrier-mediated. The sucrose H⁺-symporter gene SUT1 from potato is expressed at high levels in the phloem of mature, exporting leaves and at lower levels in other organs. Inhibition of SUT1 by expression of an antisense gene in companion cells under control of the rolC promoter leads to accumulation of high amounts of soluble and insoluble carbohydrates in leaves and inhibition of photosynthesis. The distribution of in situ localized starch does not correspond with areas of reduced photosynthesis as shown by fluorescence imaging. Dissection of antisense effects on sink and source organs by reciprocal grafts shows that inhibition of transporter gene expression in leaves is sufficient to produce chlorosis in leaves and reduced tuber yield. In contrast to the arrest of plasmodesmal development found in plants that express yeast invertase in the apoplast, in mature leaves of sucrose transporter antisense plants plasmodesmata are branched and have median cavities. These data strongly support an apoplastic mode of phloem loading in potato, in which the sucrose transporter located at the plasma membrane of the sieve element/companion cell complex represents the primary route for sugar uptake into the long-distance translocation pathway.     

The supply of both CDP-choline and diacylglycerol can regulate the rate of phosphatidylcholine synthesis in HeLa cells

The incorporation of [methyl-14C]CDP-choline into phosphatidylcholine was measured in HeLa cells permeabilized with 0.125 mg digitonin/mL. The rate of phosphatidylcholine formation was influenced by the concentration of CDP-choline in the medium. The CDP-choline:1,2-diacylglycerol cholinephosphotransferase in permeabilized cells showed a Km of 88 microM for CDP-choline. A similar Km value of 104 microM was found for cholinephosphotransferase in microsomes isolated from HeLa cells when assayed in the presence of 2.4 mM dioleoylglycerol. In the absence of added diacylglycerol, the Km for CDP-choline for the microsomal cholinephosphotransferase was only 38 microM. The incorporation of [methyl-14C]CDP-choline into phosphatidylcholine was stimulated by the supply of diacylglycerol in both HeLa cells and isolated microsomes. A 2.4 mM dioleoylglycerol suspension increased cholinephosphotransferase activity fourfold in microsomes. The digitonin-treated cells were impermeable to the dioleoylglycerol suspension. Incubation of permeabilized cells with 150 microM acyl-CoA and 0.8 mM glycero-3-phosphate tripled cellular diacylglycerol levels, causing a doubling in the rate of phosphatidylcholine synthesis. A similar incubation of microsomes with acyl-CoA stimulated phosphatidylcholine synthesis twofold. Furthermore, incubation of microsomes with [3H]diacylglycerol and [14C]CDP-choline showed that both of the substrates were incorporated into phosphatidylcholine at the same rate. This result suggests that the stimulatory effects on cholinephosphotransferase arise from increases in the availability of substrates rather than activation of the enzyme. These results suggest that both in the permeabilized cells and in isolated membranes, the biosynthesis of phosphatidylcholine can be limited by both CDP-choline and diacylglycerol.     

Conformation and Orientation of Gramicidin a in Oriented Phospholipid Bilayers Measured by Solid State Carbon-13 NMR

Three analogues of the helical ionophore gramicidin A have been synthesized with ¹³C-labeled carbonyls (¹³C=O) incorporated at either Gly², Ala³, or Val⁷. A fourth compound incorporated ¹³C at both the carbonyl and α-carbon of Gly² within the same molecule. These labels were studied using solid-state, proton-enhanced, ¹³C nuclear magnetic resonance (NMR) in hydrated dispersions of dimyristoylphosphatidylcholine (DMPC)-gramicidin A. The dispersions were aligned on glass coverslips whose orientation to the magnetic field could be varied through 180°. The orientation dependence of the NMR spectrum was used to obtain an accurate measurement of the ¹³C chemical shift anisotropy (CSA), and in the case of the fourth compound, the ¹³C—¹³C dipolar coupling constant. From the measured CSA and estimates of the orientation of the ¹³C shielding tensor, we are able to determine the direction of the ¹³C=O bonds and to compare these with the predictions of the various reported models for the configuration of gramicidin A in phospholipid bilayers. Our results are consistent with the left-handed ππ^6.3_LD single-stranded helix (Urry, D. W., J. T. Walker, and T. L. Trapane. 1982. J. Membr. Biol. 69:225-231). The right-handed ππ^6.3_LD single-stranded helix observed for gramicidin A in sodium dodecyl sulfate micelles (Arseniev, A. S., I. L. Barsukov, V. F. Bystrov, A. L. Loize, and Yu A. Ovchinnikov. 1985. FEBS (Fed. Eur. Biochem. Soc.) Lett. 186:168-174) yields a poorer fit to the data. However, the width of the carbonyl resonances suggests a distribution of molecular geometries possibly resulting from a spread in the helix pitch and handedness. Double-stranded helices and β sheet structures are excluded. In dispersions in which the lipid is in the L_α phase, the gramicidin A undergoes rapid reorientation about an axis which is centered on the normal to the plane of the coverslips. When the supporting lipid is in the L_β′ phase the helices are rigid on the timescale of ¹³C-NMR. The configuration of gramicidin A is unaltered by L_α-L_β′ phase transition of the bilayer lipid.     

Ablation of the 14‐3‐3gamma Protein Results in Neuronal Migration Delay and Morphological Defects in the Developing Cerebral Cortex

14‐3‐3 proteins are ubiquitously‐expressed and multifunctional proteins. There are seven isoforms in mammals with a high level of homology, suggesting potential functional redundancy. We previously found that two of seven isoforms, 14‐3‐3epsilon and 14‐3‐3zeta, are important for brain development, in particular, radial migration of pyramidal neurons in the developing cerebral cortex. In this work, we analyzed the function of another isoform, the protein 14‐3‐3gamma, with respect to neuronal migration in the developing cortex. We found that in utero 14‐3‐3gamma‐deficiency resulted in delays in neuronal migration as well as morphological defects. Migrating neurons deficient in 14‐3‐3gamma displayed a thicker leading process stem, and the basal ends of neurons were not able to reach the boundary between the cortical plate and the marginal zone. Consistent with the results obtained from in utero electroporation, time‐lapse live imaging of brain slices revealed that the ablation of the 14‐3‐3gamma proteins in pyramidal neurons slowed down their migration. In addition, the 14‐3‐3gamma deficient neurons showed morphological abnormalities, including increased multipolar neurons with a thicker leading processes stem during migration. These results indicate that the 14‐3‐3gamma proteins play an important role in radial migration by regulating the morphology of migrating neurons in the cerebral cortex. The findings underscore the pathological phenotypes of brain development associated with the disruption of different 14‐3‐3 proteins and will advance the preclinical data regarding disorders caused by neuronal migration defects. © 2015 Wiley Periodicals, Inc. Develop Neurobiol 76: 600–614, 2016     

Spin-label studies on the sulfhydryl environment in bovine plasma albumin. 2. The neutral transition and the A isomer.

Since we were able to demonstrate that the sulfhydryl group is located in the crevice which opens during the N--F transition (Cornell, C. N., & Kaplan, L. J. (1978) Biochemistry 17 (preceding paper in this issue), the investigation was extended by characterizing the environment during the N--B transition and in the A isomer. The results indicate that the N--B and N--F transitions are very similar in that the sulfhydryl group moves from a restricted to unhindered environment during both. The use of molecular dipstick technique further demonstrated the similarity between the F and A forms. However, since A is a covalently stabilized form of albumin after a pH dependent transition, it retains its properties during subsequent pH changes rather than reverting to the N form. We were thus able to titrate spin-labeled A through the pH range of the acidic transitions without detecting the N--F transition. Isoelectric focusing analysis of A generated during alkaline aging and purified by SP-Sephadex chromatography indicates that it is a mixture of a small number of albumin forms rather than the large number of components once thought to be formed during aging.     

An Engineered Membrane to Measure Electroporation: Effect of Tethers and Bioelectronic Interface

This article reports on the construction and predictive models for a platform comprised of an engineered tethered membrane. The platform provides a controllable and physiologically relevant environment for the study of the electroporation process. The mixed self-assembled membrane is formed via a rapid solvent exchange technique. The membrane is tethered to the gold electrode and includes an ionic reservoir separating the membrane and gold surface. Above the membrane, there is an electrolyte solution, and a gold counterelectrode. A voltage is applied between the gold electrodes and the current measured. The current is dependent on the energy required to form aqueous pores and the conductance of each pore. A two-level predictive model, consisting of a macroscopic and a continuum model, is developed to relate the pore dynamics to the measured current. The macroscopic model consists of an equivalent circuit model of the tethered membrane, and asymptotic approximations to the Smoluchowski-Einstein equation of electroporation that is dependent on the pore conductance and the energy required to form aqueous pores. The continuum model is a generalized Poisson-Nernst-Planck (GPNP) system where an activity coefficient to account for steric effects of ions is added to the standard PNP system. The GPNP is used to evaluate the conductance of aqueous pores, and the electrical energy required to form the pores. As an outcome of the setup of the device and the two-level model, biologically important variables can be estimated from experimental measurements. To validate the accuracy of the two-level model, the predicted current is compared with experimentally measured current for different tethering densities.     

A 22-mer Segment in the Structurally Pliable Regulatory Domain of Metazoan CTP: Phosphocholine Cytidylyltransferase Facilitates Both Silencing and Activating Functions

CTP:phosphocholine cytidylyltransferase (CCT), an amphitropic enzyme that regulates phosphatidylcholine synthesis, is composed of a catalytic head domain and a regulatory tail. The tail region has dual functions as a regulator of membrane binding/enzyme activation and as an inhibitor of catalysis in the unbound form of the enzyme, suggesting conformational plasticity. These functions are well conserved in CCTs across diverse phyla, although the sequences of the tail regions are not. CCT regulatory tails of diverse origins are composed of a long membrane lipid-inducible amphipathic helix (m-AH) followed by a highly disordered segment, reminiscent of the Parkinson disease-linked protein, α-synuclein, which we show shares a novel sequence motif with vertebrate CCTs. To unravel features required for silencing, we created chimeric enzymes by fusing the catalytic domain of rat CCTα to the regulatory tail of CCTs from Drosophila, Caenorhabditis elegans, or Saccharomyces cerevisiae or to α-synuclein. Only the tail domains of the two invertebrate CCTs were competent for both suppression of catalytic activity and for activation by lipid vesicles. Thus, both silencing and activating functions of the m-AH can tolerate significant changes in length and sequence. We identified a highly amphipathic 22-residue segment in the m-AH with features conserved among animal CCTs but not yeast CCT or α-synuclein. Deletion of this segment from rat CCT increased the lipid-independent V_max by 10-fold, equivalent to the effect of deleting the entire tail, and severely weakened membrane binding affinity. However, membrane binding was required for additional increases in catalytic efficiency. Thus, full activation of CCT may require not only loss of a silencing conformation in the m-AH but a gain of an activating conformation, promoted by membrane binding.     

Increased nuclear size in BHK21 cells after treatment with non-toxic levels of calf thymus histones.

The effect of treatment with either whole calf thymus histones, or individually isolated histones, or polyarginine, or polylysine, on the nuclear size of BHK21 cells has been investigated. Incubation of the cells with mixed histones (12.5--44 microgram/ml) for 1 h considerably increased nuclear size. Increasing the histone concentration and/or the incubation time resulted in a decrease in the effect and could result in no change in nuclear size. Treatment of the cells with polyarginine or polylysine did not affect nuclear size. Experiments with individually isolated histones showed that the nuclear size effect was almost exclusively due to the histone H4. It is argued that the changes observed most likely resulted from interaction of H4 with the nucleus, and could reflect the properties of this particular histone molecule.     

A Cl and Cl NMR study of chloride binding to the erythrocyte anion transport protein

Band 3, the erythrocyte anion transport protein, mediates the one-for-one exchange of bicarbonate and chloride ions across the membrane and consequently plays an important role in respiration. Binding to the protein forms the first step in the translocation of the chloride across the membrane. ³⁵Cl and ³⁷Cl NMR relaxation measurements at various field strengths were used to study chloride binding to the protein in the presence and absence of the transport inhibitor 4,4′-dinitrostilbene-2,2′-disulfonate. Significant differences occurred in the NMR relaxation rates depending on whether the inhibitor was present or not. The results indicate that the rate of chloride association and dissociation at each external binding site occurs on a time scale of 5 μs. This implies that the transmembrane flux is not limited by the rate of chloride binding to the external chloride binding site of band 3. The rotational correlation-time of chloride bound to band 3 was found to be 20 ns with a quadrupole coupling constant of 3 MHz.