Rémy Gibrat

Nitrate Efflux at the Root Plasma Membrane: Identification of an Arabidopsis Excretion Transporter

Root NO3− efflux to the outer medium is a component of NO3− net uptake and can even overcome influx upon various stresses. Its role and molecular basis are unknown. Following a functional biochemical approach, NAXT1 (for NITRATE EXCRETION TRANSPORTER1) was identified by mass spectrometry in the plasma membrane (PM) of Arabidopsis thaliana suspension cells, a localization confirmed using a NAXT1–Green Fluorescent Protein fusion protein. NAXT1 belongs to a subclass of seven NAXT members from the large NITRATE TRANSPORTER1/PEPTIDE TRANSPORTER family and is mainly expressed in the cortex of mature roots. The passive NO3− transport activity (Km = 5 mM) in isolated root PM, electrically coupled to the ATP-dependant H+-pumping activity, is inhibited by anti-NAXT antibodies. In standard culture conditions, NO3− contents were altered in plants expressing NAXT-interfering RNAs but not in naxt1 mutant plants. Upon acid load, unidirectional root NO3− efflux markedly increased in wild-type plants, leading to a prolonged NO3− excretion regime concomitant with a decrease in root NO3− content. In vivo and in vitro mutant phenotypes revealed that this response is mediated by NAXT1, whose expression is upregulated at the posttranscriptional level. Strong medium acidification generated a similar response. In vitro, the passive efflux of NO3− (but not of Cl−) was strongly impaired in naxt1 mutant PM. This identification of NO3− efflux transporters at the PM of plant cells opens the way to molecular studies of the physiological role of NO3− efflux in stressed or unstressed plants.

A procedure for estimating the surface potential of charged or neutral membranes with 8-anilino-1-naphthalenesulphonate probe. Adequacy of the Gouy-Chapman model

Using the fluorescent anion 8-anilino-1-naphthalenesulphonate (ANS) for determining the membrane surface potential necessitates that the intrinsic affinity constant Ki for the ANS sites be known. Two methods are presented which do not rely on a determination of Ki at high ionic strength. They are respectively applied to neutral membranes (egg phosphatidylcholine liposomes) and highly charged natural ones (horse bean microsomes and liposomes from their phospholipids). The value of Ki appears to be insensitive to the level of occupancy of the sites, the KCl concentration and the pH in large ranges. Furthermore, the classical Gouy-Chapman model seems to describe correctly the whole set of data, provided apparent mean molecular areas larger than the published crystallographic ones are admitted.

Effect of pH on the surface charge density of plant membranes. Comparison of microsomes and liposomes

Abstract The surface potential of microsomes of horse bean roots was compared to the one of liposomes prepared from the whole phospholipid extracts. The surface potential was determined from the affinity of the membranes for the anilinonaphthalene sulphonate dye. The effect of pH was studied at two KCl concentrations. It appeared from this comparison that the surface charge density was nearly the same on both materials in the neutral pH range. The isoelectric point was pH 1.7 for the liposomes and pH 4.0 for the microsomes. The implication of these observations is that the surface charge density of microsomes is nearly the same above the lipid and protein components of the membrane. This hypothesis was checked by measuring the activity of a microsomal enzyme with an anionic substrate, while modifying the net surface charge of the membrane. The biological significance of the results is discussed.

The yeast mutant vps5Δ affected in the recycling of Golgi membrane proteins displays an enhanced vacuolar Mg2+/H+ exchange activity

Growth of the yeast vacuolar protein-sorting mutant vps5Δ affected in the endosome-to-Golgi retromer complex was more sensitive to Mg2+-limiting conditions than was the growth of the wild-type (WT) strain. This sensitivity was enhanced at acidic pH. The vps5Δ strain was also sensitive to Al3+, known to inhibit Mg2+ uptake in yeast cells. In contrast, it was found to be resistant to Ni2+ and Co2+, two cytotoxic analogs of Mg2+. Resistance to Ni2+ did not seem to result from the alteration of plasma-membrane transport properties because mutant and WT cells displayed similar Ni2+ uptake. After plasma-membrane permeabilization, intracellular Ni2+ uptake in vps5Δ cells was 3-fold higher than in WT cells, which is consistent with the implication of the vacuole in the observed phenotypes. In reconstituted vacuolar vesicles prepared from vps5Δ, the rates of H+ exchange with Ni2+, Co2+, and Mg2+ were increased (relative to WT) by 170%, 130%, and 50%, respectively. The rates of H+ exchange with Ca2+, Cd2+, and K+ were similar in both strains, as were α-mannosidase and H+-ATPase activities, and SDS/PAGE patterns of vacuolar proteins. Among 14 other vacuolar protein-sorting mutants tested, only the 8 mutants affected in the recycling of trans-Golgi network membrane proteins shared the same Ni2+ resistance phenotype as vps5Δ. It is proposed that a trans-Golgi network Mg2+/H+ exchanger, mislocalized to vps5Δ vacuole, could be responsible for the phenotypes observed in vivo and in vitro.

Measurement of the quantum yield of 8-anilino-1-naphthalene sulphonate bound on plant microsomes. Critical application of the method of Weber and Young

Abstract The method of Weber, G. and Young, L. ((1964) J. Biol. Chem. 239, 1415–1423) allows one to evaluate by graphical means the maximum fluorescence intensity of a fixed amount of anilinoaphthalene sulphonate (ANS) in presence of increasing concentrations of proteic sites for this probe. This value is used to estimate the quantum yield of the fixed ANS. The method is based on the linear extrapolation of the double reciprocal plot of fluorescence intensity vs. protein concentration. We show by experimental means and critical analysis that such an extrapolation is not valid unless two specific conditions are fulfilled: the fractional saturation of the sites at the beginning of the titration must be low and the fractional fixation of the probe at the end of the titration must be high. Due to the low affinity of membranes for ANS, the latter condition is not easily satisfied, which may lead to erroneous interpretations. We propose a procedure which overcomes this drawback. When applied to microsomes of roots from horse bean, it reveals that the quantum yield of bound ANS is insensitive to pH in the pH range 2–8, and to the presence of Ca 2+ .

Spontaneous insertion of plant plasma membrane (H+)ATPase into a preformed bilayer.

SummaryThe purified (H+ATPase from corn roots plasma membrane inserted spontaneously into preformed bilayer from soybean lipids. The yield of the protein insertion, as measured from its H+-pumping activity, increased as a function of lipids and protein concentrations. In optimum conditions, all the (H+)ATPase molecules were closely associated with liposomes, exhibiting a high H+-pumping activity (150,000% quenching· min−1·mg−1 protein of the probe 9-amino-6-chloro-2-methoxyacridine). The insertion was achieved within a few seconds. No latency of the (H+)ATPase hydrolytic activity was revealed when lysophosphatidylcholine was added to permeabilize the vesicles. This indicated that the (H+)ATPase molecules inserted unidirectionally, the catalytic sites being exposed outside the vesicles (“inside-out” orientation), and thus freely accessible to Mg-ATP. The nondelipidated (H+)ATPase could also functionally insert into bilayer from PC∶PE∶PG or PC∶PE∶PI, due to the presence of both hydrophobic defects promoted by PE, and negative phospholipids specifically required by the (H+)ATPase from corn roots. The detergent octylglucoside facilitated the delipidated (H+)ATPase reinsertion probably by promoting both a proper protein conformation and hydrophobic defects in the bilayer. Lysophosphatidylcholine facilitated the delipidated protein insertion only when hydrophobic defects were already present, and thus seemed only capable to ensure a proper protein conformation

In vitro characterization of iron-phytosiderophore interaction with maize root plasma membranes: evidences for slow association kinetics

As an attempt to characterize iron(III)-phytosiderophore transport across plant membranes in vitro, a rapid filtration approach was set up in which plasma membrane vesicles from maize roots were incubated with 55Fe-labelled deoxymugineic acid (DMA). Fe-DMA, and not Fe-EDTA, could associate with plasma membrane vesicles. The rate of Fe-DMA association decreased with a half time of 15 min. The initial Fe-DMA association rate, estimated from the amount of Fe-DMA associated after 10 min incubation, exhibited a saturation curve as a function of Fe-DMA concentration. This curve could be satisfactorily fitted to the Michaelis-Menten model (KM=600 nM, Vmax=2 nmol min-1 mg-1 protein). The association rate of Fe-DMA with control liposomes remained negligible and constant in a pH range from 4 to 8, whereas it strongly increased at acidic pH with plasma membrane vesicles. However, the specific association of Fe-DMA to root plasma membrane could not be explained by a vesicle-filling process because: (i) lowering the vesicle volume by decreasing the osmotic potential of the assay medium with sorbitol did not decrease 55(Fe) labelling of the vesicles, (ii) creating inside-out vesicles by a Brij-58 treatment had almost no effect on Fe-DMA association to vesicles, (iii) 55(Fe) labelling is reversible by EDTA and excess free DMA, and (iv) 55(Fe) labelling was the same using plasmalemma vesicles prepared either from wild type maize or from the ys1 maize mutant deficient in iron-phytosiderophore transport. A model is proposed to account for the observed Fe-DMA association as the result of very slow binding kinetics onto membrane proteins. This model was validated by its ability to describe quantitatively both Fe-DMA association as a function of time and of substrate concentration. A prediction of the model was that association of Fe-DMA to plasma membranes might overcome a high activation energy barrier. Indeed, the Arrhenius plot for the association rate constant was linear with an activation energy of 64 kJ mol-1.

Separation, identification, and profiling of membrane proteins by GFC/IEC/SDS-PAGE and MALDI TOF MS.

Membrane protein identification by matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) requires that proteins be separated prior to MS analysis. After membrane solubilization with the nondenaturing detergent n-dodecyl-beta-D-maltoside, proteins can be separated by ion-exchange chromatography (IEC) and further resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). An additional separation step by gel filtration (GF) before IEC/SDS-PAGE can be required depending on the complexity of the membrane protein mixture. Staining of final SDS-PAGE gels allows one to establish simply the protein expression pattern of a membrane fraction and to profile responses. Moreover, in-gel digestion of hydrophobic integral proteins is valuable. Finally, the resolution capacity of this separation procedure allows identification of proteins by MALDI-TOF MS. The method is illustrated by application to plant and yeast plasma membrane and to plant vacuolar membrane.

Liposomes with Multiple Fluorophores for Measurement of Ionic Fluxes, Selectivity, and Membrane Potential

Publisher Summary This chapter focuses on the rules and principles used for multilabeled liposomes and modern spectrofluorometers fitted with motorized gratings or filter wheels, or diode arrays. Such devices enable investigators to make use of many advantages offered by liposomes, which are formed easily, are stable, and are handled easily for biochemical treatments or reinsertion of purified membrane proteins. They offer a good avenue by which to decipher transport mechanisms of ion carriers, that is, electroenzymes exhibiting large conformational modification along the transport reaction cycle. These transport systems are relatively abundant in biological membranes but display too low a conductance relative to ion channels forming aqueous pores to be easily studied by classic electrophysiological methods, such as the patch-clamp. The chapter presents several practical examples to determine net flux (J K ) and permeability coefficient (P K ) of liposomes to K + using the specific K + dye potassium-binding benzofuran isophthalate (PBFI).

Uncovering pH at both sides of the root plasma membrane interface using noninvasive imaging

Significance The pH on both sides of the plant plasma membrane was accurately measured in vivo using ratiometric fluorescent sensors. This enabled noninvasive access to membrane-associated pH and transmembrane delta pH values from the surface of the root up to the deepest cell layers beyond the Casparian strip barrier. We demonstrate that despite direct contact with the soil, the apoplastic pH close to the plasma membrane was maintained at values ranging from 6.0 to 6.4 in mature root cells, whereas the overall pH in the apoplastic space is far more acidic. Furthermore, we found that the cell wall plays a role in proton homeostasis in mature root. Building a proton gradient across a biological membrane and between different tissues is a matter of great importance for plant development and nutrition. To gain a better understanding of proton distribution in the plant root apoplast as well as across the plasma membrane, we generated Arabidopsis plants expressing stable membrane-anchored ratiometric fluorescent sensors based on pHluorin. These sensors enabled noninvasive pH-specific measurements in mature root cells from the medium–epidermis interface up to the inner cell layers that lie beyond the Casparian strip. The membrane-associated apoplastic pH was much more alkaline than the overall apoplastic space pH. Proton concentration associated with the plasma membrane was very stable, even when the growth medium pH was altered. This is in apparent contradiction with the direct connection between root intercellular space and the external medium. The plasma membrane-associated pH in the stele was the most preserved and displayed the lowest apoplastic pH (6.0 to 6.1) and the highest transmembrane delta pH (1.5 to 2.2). Both pH values also correlated well with optimal activities of channels and transporters involved in ion uptake and redistribution from the root to the aerial part. In growth medium where ionic content is minimized, the root plasma membrane-associated pH was more affected by environmental proton changes, especially for the most external cell layers. Calcium concentration appears to play a major role in apoplastic pH under these restrictive conditions, supporting a role for the cell wall in pH homeostasis of the unstirred surface layer of plasma membrane in mature roots.