Nitrite Transport in Chloroplast Inner Envelope Vesicles (I. Direct Measurement of Proton-Linked Transport)

Chloroplast inner envelope membrane vesicles that are loaded with the pH-sensitive fluorophore, pyranine, show rapid internal acidification when nitrite is added. Acidification is dependent upon [delta]pH, with the inside of vesicles being alkaline with respect to the outside. The rate of vesicle acidification was directly proportional to the concentration of nitrite that was added and the imposed pH difference across the membrane. In contrast, added nitrate had no effect on vesicle acidification. Nitrite also caused acidification of asolectin vesicles. The extent of vesicle acidification is dependent on the internal volume of vesicles. Inner envelope and asolectin vesicles that were prepared by extrusion were approximately the same size, allowing them to be compared when the final extent of acidification, measured after the pH gradient had collapsed, was similar. The rate of nitrite-dependent acidification was similar in these two preparations at any single nitrite concentration. These results indicate that nitrite movement occurs by rapid diffusion across membranes as nitrous acid, and this movement is dependent on a proton gradient across the lipid bilayer. Under conditions approximating those in vivo, the rate of diffusion of nitrous acid far exceeds that of nitrite reduction within chloroplasts.     

Gravity-Induced Polar Transport of Calcium across Root Tips of Maize

Calcium movement across primary roots of maize (Zea mays, L.) was determined by application of ⁴⁵Ca²⁺ to one side of the root and collection of radioactivity in an agar receiver block on the opposite side. Ca movement across the root tip was found to be at least 20 times greater than movement across the elongation zone. The rapid movement of Ca across the tip was severely inhibited in roots from which the root cap had been removed. Ca movement across the tip was also strongly retarded in roots pretreated with 2,4-dinitrophenol or potassium cyanide. Orientation of roots horizontally had no effect on Ca movement across the elongation zone but caused a strong asymmetry in the pattern of Ca movement across the tip. In gravistimulated roots, the movement of Ca from top to bottom increased while movement from bottom to top decreased. The data indicate that gravistimulation induces polar movement of Ca toward the lower side of the root cap. An earlier report (Lee, Mulkey, Evans 1983 Science 220: 1375-1376) from this laboratory showed that artificial establishment of calcium gradients at the root tip can cause gravitropic-like curvature. Together, the two studies indicate that Ca plays a key role in linking gravistimulation to the gravitropic growth response in roots.     

Direct Measurement of Nitrite Transport Across Erythrocyte Membrane Vesicles Using the Fluorescent Probe, 6-Methoxy-N-(3-sulfopropyl) quinolinium

Nitrite was shown to quench the fluorescence of 6-methoxy-N-(3-sulfopropyl) quinolinium (SPQ) almost twofold more than chloride. SPQ loaded inside vesicles prepared from asolectin and isolated erythrocyte ghosts allowed for the direct measurement of nitrite movement across these membranes. Movement of nitrite across asolectin occurred by diffusion as HNO₂ in a pH-dependent manner. By contrast, erythrocyte ghosts had very low diffusion rates for nitrous acid. Erythrocyte ghosts preloaded with 50 mM nitrite to quench SPQ fluorescence were utilized to study heteroexchange with externally added anions. SPQ fluorescence increases (becomes unquenched) with added bicarbonate and nitrate, indicating that nitrite is moving out of the preloaded vesicles. The pH optimum for this exchange was approximately 7.6 and exchange was inhibited by N-ethylmaleimide (NEM) and dihydro-4,4-diisothiocyanostilbene-2,2-disulfonic acid (DIDS). These data indicate that nitrite moves across erythrocyte plasma membranes as NO₂- by a heteroexchange mechanism with other monovalent anions.     

Water Transport across Yeast Vacuolar and Plasma Membrane-Targeted Secretory Vesicles Occurs by Passive Diffusion

To determine whether solute transport across yeast membranes was facilitated, we measured the water and solute permeations of vacuole-derived and late secretory vesicles in Saccharomyces cerevisiae; all permeations were consistent with passive diffusive flow. We also overexpressed Fps1p, the putative glycerol facilitator in S. cerevisiae, in secretory vesicles but observed no effect on water, glycerol, formamide, or urea permeations. However, spheroplasts prepared from the strain overexpressing Fps1p showed enhanced glycerol uptake, suggesting that Fps1p becomes active only upon insertion in the plasma membrane.     

Calcium Transport in Membrane Vesicles Isolated from Maize Coleoptiles (Effect of Indoleacetic Acid and Fusicoccin)

Maize (Zea mays L.) coleoptile segments loaded with 45Ca released about 50% of the ion after 1 h when treated with indoleacetic acid (IAA). In contrast, fusicoccin (FC) had no effect. The same relation was found when ATP-dependent Ca2+ transport, measured as 45Ca uptake, was determined in a plasmalemma-rich membrane vesicle fraction isolated from coleoptiles treated or untreated for 1 h with IAA or FC. In fact, IAA-treated membranes showed an increase in ATP-dependent 45Ca uptake by more than 30% with respect to the control and the FC treatment. Ca2+ uptake in IAA-treated membranes was only slightly affected (+27%) by supplying calmodulin (Cam) exogenously. However, Ca2+ uptake in membranes from the control and FC-treated coleoptiles were stimulated (+80%) by exogenous Cam. Calmidazolium, a Cam antagonist, inhibited Ca2+ uptake in the IAA treatment (-48%) to a greater extent with respect to the control and FC treatment (-33 and -29%, respectively). A possible relationship between the effect of IAA on the ATP-dependent Ca2+ transport activity, the involvement of Cam, and their effect on growth are discussed.     

Polar Transport of Auxin across Gravistimulated Roots of Maize and Its Enhancement by Calcium

The effect of Ca on the polar movement of [³H]indoleacetic acid ([³H] IAA) in gravistimulated roots was examined using 3-day-old seedlings of maize (Zea mays L.). Transport of label was measured by placing an agar donor block containing [³H]IAA on one side of the elongation zone and measuring movement of label across the root into an agar receiver block on the opposite side. In vertically oriented roots, movement of label across the elongation zone into the receiver was slight and was not enhanced by incorporating 10 millimolar CaCl₂ into the receiver block. In horizontally oriented roots, movement of label across the root was readily detectable and movement to a receiver on the bottom was about 3-fold greater than movement in the opposite direction. This polarity was abolished in roots from which the caps were removed prior to gravistimulation. When CaCl₂ was incorporated into the receivers, movement of label across horizontally oriented intact roots was increased about 3-fold in both the downward and upward direction. The ability of Ca to enhance the movement of label from [³H]IAA increased with increasing Ca concentration in the receiver up to 5 to 10 millimolar CaCl₂. With the inclusion of CaCl₂ in the receiver blocks, gravity-induced polar movement of label into receiver blocks from applied [³H]IAA was detectable within 30 minutes, and asymmetric distribution of label within the tissue was detectable within 20 minutes. The results indicate that gravistimulation induces a physiological asymmetry in the auxin transport system of maize roots and that Ca increases the total transport of auxin across the root.     

Kinetics of Water Transport in Eel Intestinal Vesicles

Brush border membrane vesicles, BBMV, from eel intestinal cells or kidney proximal tubule cells were prepared in a low osmolarity cellobiose buffer. The osmotic water permeability coefficient P _f for eel vesicles was not affected by pCMBS and was measured at 1.6 × 10⁻³ cm sec⁻¹ at 23°C, a value lower than 3.6 × 10⁻³ cm sec⁻¹ exhibited by the kidney vesicles and similar to published values for lipid bilayers. An activation energy E _a of 14.7 Kcal mol⁻¹ for water transport was obtained for eel intestine, contrasting with 4.8 Kcal mol⁻¹ determined for rabbit kidney proximal tubule vesicles using the same method of analysis. The high value of E _a , as well as the low P _f for the eel intestine is compatible with the absence of water channels in these membrane vesicles and is consistent with the view that water permeates by dissolution and diffusion in the membrane. Further, the initial transient observed in the osmotic response of kidney vesicles, which is presumed to reflect the inhibition of water channels by membrane stress, could not be observed in the eel intestinal vesicles. The P _f dependence on the tonicity of the osmotic shock, described for kidney vesicles and related to the dissipation of pressure and stress at low tonicity shocks, was not seen with eel vesicles. These results indicate that the membranes from two volume transporter epithelia have different mechanisms of water permeation. Presumably the functional water channels observed in kidney vesicles are not present in eel intestine vesicles. The elastic modulus of the membrane was estimated by analysis of swelling kinetics of eel vesicles following hypotonic shock. The value obtained, 0.79 × 10⁻³ N cm⁻¹, compares favorably with the corresponding value, 0.87 × 10⁻³ N cm⁻¹, estimated from measurements at osmotic equilibrium. Received: 28 January 1999/Revised: 15 June 1999     

Calcium Transport in Sealed Vesicles from Red Beet (Beta vulgaris L.) Storage Tissue : II. Characterization of Ca Uptake into Plasma Membrane Vesicles

Calcium uptake was examined in sealed plasma membrane vesicles isolated from red beet (Beta vulgaris L.) storage tissue using (45)Ca(2+). Uptake of (45)Ca(2+) by the vesicles was ATP-dependent and radiotracer accumulated by the vesicles could be released by the addition of the calcium ionophore A23187. The uptake was stimulated by gramicidin D but slightly inhibited by carbonylcyanide m-chlorophenylhydrazone. Although the latter result might suggest some degree of indirect coupling of (45)Ca(2+) uptake to ATP utilization via deltamuH(+), no evidence for a secondary H(+)/Ca(2+) antiport in this vesicle system could be found. Following the imposition of an acid-interior pH gradient, proton efflux from the vesicle was not enhanced by the addition of Ca(2+) and an imposed pH gradient could not drive (45)Ca(2+) uptake. Optimal uptake of (45)Ca(2+) occurred broadly between pH 7.0 and 7.5 and the transport was inhibited by orthovanadate, N,N'-dicyclohexylcarbodiimide, and diethylstilbestrol but insensitive to nitrate and azide. The dependence of (45)Ca(2+) uptake on both calcium and Mg:ATP concentration demonstrated saturation kinetics with K(m) values of 6 micromolar and 0.37 millimolar, respectively. While ATP was the preferred substrate for driving (45)Ca(2+) uptake, GTP could drive transport at about 50% of the level observed for ATP. The results of this study demonstrate the presence of a unique primary calcium transport system associated with the plasma membrane which could drive calcium efflux from the plant cell.     

Direct Interaction of Otoferlin with Syntaxin 1A, SNAP-25, and the L-type Voltage-gated Calcium Channel CaV1.3

The molecular mechanisms underlying synaptic exocytosis in the hair cell, the auditory and vestibular receptor cell, are not well understood. Otoferlin, a C2 domain-containing Ca²⁺-binding protein, has been implicated as having a role in vesicular release. Mutations in the OTOF gene cause nonsyndromic deafness in humans, and OTOF knock-out mice are deaf. In the present study, we generated otoferlin fusion proteins containing two of the same amino acid substitutions detected in DFNB9 patients (P1825A in C2F and L1011P in C2D). The native otoferlin C2F domain bound syntaxin 1A and SNAP-25 in a Ca²⁺-dependent manner (with optimal 61 μm free Ca²⁺ required for binding). These interactions were greatly diminished for C2F with the P1825A mutation, possibly because of a reduction in tertiary structural change, induced by Ca²⁺, for the mutated C2F compared with the native C2F. The otoferlin C2D domain also bound syntaxin 1A, but with weaker affinity (K_d = 1.7 × 10^–5 m) than for the C2F interaction (K_d = 2.6 × 10^–9 m). In contrast, it was the otoferlin C2D domain that bound the Ca_v1.3 II-III loop, in a Ca²⁺-dependent manner. The L1011P mutation in C2D rendered this binding insensitive to Ca²⁺ and considerably diminished. Overall, we demonstrated that otoferlin interacts with two main target-SNARE proteins of the hair-cell synaptic complex, syntaxin 1A and SNAP-25, as well as the calcium channel, with the otoferlin C2F and C2D domains of central importance for binding. Because mutations in the otoferlin C2 domains that cause deafness in humans impair the ability of otoferlin to bind syntaxin, SNAP-25, and the Ca_v1.3 calcium channel, it is these interactions that may mediate regulation by otoferlin of hair cell synaptic exocytosis critical to inner ear hair cell function.Calcium is a key regulator of synaptic vesicle fusion (reviewed in Ref. 1). In mechanosensory hair cells, calcium microdomains (2) and possibly nanodomains (3) are formed when voltage-gated calcium channels open upon depolarization. Calcium at these sites is thought to activate protein interactions, leading to vesicle fusion. Some of the key players in this process are the target-SNARE² proteins, syntaxin 1A and SNAP-25, and the vesicle-SNARE, synaptobrevin (4). Vesicle-SNARE synaptotagmin 1 plays a crucial role as a calcium sensor at the neuronal synapse, modulating calcium channels and vesicle release by a Ca²⁺-dependent interaction with other SNARE proteins in the presence of lipid molecules (4–6). However, in vertebrate mechanosensory hair cells, synaptotagmin 1 is not detected (7). Instead, fast neurotransmitter release in auditory and vestibular hair cells, facilitated largely by an L-type voltagegated calcium channel, Ca_v1.3 (8, 9), is thought to be modulated by a newly discovered protein, otoferlin, acting as the Ca²⁺ sensor and vesicle-binding protein. When mutated, otoferlin causes DFNB9 nonsyndromic deafness (10). Gene sequences of different deaf families show that the OTOF gene can undergo mutation at multiple locations (11–13). Recently, it has been demonstrated that otoferlin is necessary for synaptic exocytosis from hair cells (14). Further, an engineered mutation in the C2B domain of otoferlin has been shown to cause deafness in mice (15). However, the precise function of otoferlin as a synaptic protein is not well understood.Specific mutations in the otoferlin C2F (P1825A) or C2D (L1011P) domains in humans have been documented to cause DFNB9 deafness (11, 12). Previous studies suggested that a region of otoferlin containing all three C2 domains, D, E, and F, binds directly to the t-SNARE molecules syntaxin 1A and SNAP-25 in response to an increase in Ca²⁺ concentration (14). However, it is not understood how a single amino acid substitution in one domain of otoferlin, such as C2F (11) or C2D (12), might independently lead to deafness. Here, we examine the role of otoferlin as a Ca²⁺ sensor as well as a facilitator of vesicle fusion, as indicated by protein-protein interactions and their [Ca²⁺] dependence.     

Direct Measurement of Potential Difference across the Human Red Blood Cell Membrane

The electrical potential difference across the human red cell membrane has been measured directly. A biological amplifier with neutralized input capacity was used. Human red cells in modified Ringer solution were impaled individually with 3 M KCl-filled glass microelectrodes. Movements of the microelectrodes were effected by Leitz micromanipulators. Results showed a potential difference of -8.0 ± 0.21 (SEM) mv, the inside being negative with respect to the outside. This value is approximately that calculated by using the Nernst equation considering the intracellular and extracellular chloride concentrations.

As a control, similar measurements were made on nylon microcapsules containing hemoglobin. The measured potential of -0.52 ± 0.02 (SEM) mv, which agreed very well with the value calculated on the basis of Donnan equilibrium, was much smaller in magnitude as compared to the results for the red cell, and there was evidence of fixed charges on the microcapsule membrane. There was no evidence of this in the case of the red cell.

     

Aluminum Interactions with Voltage-Dependent Calcium Transport in Plasma Membrane Vesicles Isolated from Roots of Aluminum-Sensitive and -Resistant Wheat Cultivars

The role of Al interactions with root-cell plasma membrane (PM) Ca2+ channels in Al toxicity and resistance was studied. The experimental approach involved the imposition of a transmembrane electrical potential (via K+ diffusion) in right-side-out PM vesicles derived from roots of two wheat (Triticum aestivum L.) cultivars (Al-sensitive Scout 66 and Al-resistant Atlas 66). We previously used this technique to characterize a voltage-dependent Ca2+ channel in the wheat root PM (J.W. Huang, D.L. Grunes, L.V. Kochian [1994] Proc Natl Acad Sci USA 91: 3473-3477). We found that Al3+ effectively blocked this PM Ca2+ channel; however, Al3+ blocked this Ca2+ channel equally well in both the Al-sensitive and -resistant cultivars. It was found that the differential genotypic sensitivity of this Ca2+ transport system to Al in intact roots versus isolated PM vesicles was due to Al-induced malate exudation localized to the root apex in Al-resistant Atlas but not in Al-sensitive Scout. Because malate can effectively chelate Al3+ in the rhizosphere and exclude it from the root apex, the differential sensitivity of Ca2+ influx to Al in intact roots of Al-resistant versus Al-sensitive wheat cultivars is probably due to the maintenance of lower Al3+ activities in the root apical rhizosphere of the resistant cultivar.     

Three Mechanisms for the Calcium Alleviation of Mineral Toxicities

Ca²⁺ in rooting medium is essential for root elongation, even in the absence of added toxicants. In the presence of rhizotoxic levels of Al³⁺, H⁺, or Na⁺ (or other cationic toxicants), supplementation of the medium with higher levels of Ca²⁺ alleviates growth inhibition. Experiments to determine the mechanisms of alleviation entailed measurements of root elongation in wheat (Triticum aestivum L. cv Scout 66) seedlings in controlled medium. A Gouy-Chapman-Stern model was used to compute the electrical potentials and the activities of ions at the root-cell plasma membrane surfaces. Analysis of root elongation relative to the computed surface activities of ions revealed three separate mechanisms of Ca²⁺ alleviation. Mechanism I is the displacement of cell-surface toxicant by the Ca²⁺-induced reduction in cell-surface negativity. Mechanism II is the restoration of Ca²⁺ at the cell surface if the surface Ca²⁺ has been reduced by the toxicant to growth-limiting levels. Mechanism III is the collective ameliorative effect of Ca²⁺ beyond mechanisms I and II, and may involve Ca²⁺-toxicant interactions at the cell surface other than the displacement interactions of mechanisms I and II. Mechanism I operated in the alleviation of all of the tested toxicities; mechanism II was generally a minor component of alleviation; and mechanism III was toxicant specific and operated strongly in the alleviation of Na⁺ toxicity, moderately in the alleviation of H⁺ toxicity, and not at all in the alleviation of Al³⁺ toxicity.     

Folate Transport by Prawn Hepatopancreas Brush-Border Membrane Vesicles

The transport system of folic acid (Pte-Glu) by brush-border membrane vesicles (BBMV) isolated from prawn (Penaeus japonicm) hepatopancreas, was studied by measuring the uptake of Pte-Glu. This uptake was found to have two components, intravesicular transport and membrane binding. Membrane binding was not affected by the presence of a transmembrane pH-gradient at a short incubation period. However, a transmembrane pH-gradient increased membrane binding at 60 min. The transport of Pte-Glu appeared to be carrier-mediated, was stimulated by an inwardly proton gradient (pH 5.5 outside, 7.4 inside) and was unaffected by a sodium-gradient. The relationship between pH gradient-driven Pte-Glu uptake and medium Pte-Glu concentration followed saturating Michaelis–Menten kinetics. Eadie–Hofstee representation of the pH gradient-driven Pte-Glu uptake indicated a single transport system with a Km of 0.37 M and Vmax of 1.06 pmol/mg protein/15 s. These findings indicate that BBMV isolated from prawn hepatopancreas possesses a Pte-Glu transport system similar to that described in mammalian intestine.     

Functional UDP-xylose Transport across the Endoplasmic Reticulum/Golgi Membrane in a Chinese Hamster Ovary Cell Mutant Defective in UDP-xylose Synthase

In mammals, xylose is found as the first sugar residue of the tetrasaccharide GlcAβ1-3Galβ1-3Galβ1-4Xylβ1-O-Ser, initiating the formation of the glycosaminoglycans heparin/heparan sulfate and chondroitin/dermatan sulfate. It is also found in the trisaccharide Xylα1-3Xylα1-3Glcβ1-O-Ser on epidermal growth factor repeats of proteins, such as Notch. UDP-xylose synthase (UXS), which catalyzes the formation of the UDP-xylose substrate for the different xylosyltransferases through decarboxylation of UDP-glucuronic acid, resides in the endoplasmic reticulum and/or Golgi lumen. Since xylosylation takes place in these organelles, no obvious requirement exists for membrane transport of UDP-xylose. However, UDP-xylose transport across isolated Golgi membranes has been documented, and we recently succeeded with the cloning of a human UDP-xylose transporter (SLC25B4). Here we provide new evidence for a functional role of UDP-xylose transport by characterization of a new Chinese hamster ovary cell mutant, designated pgsI-208, that lacks UXS activity. The mutant fails to initiate glycosaminoglycan synthesis and is not capable of xylosylating Notch. Complementation was achieved by expression of a cytoplasmic variant of UXS, which proves the existence of a functional Golgi UDP-xylose transporter. A ∼200 fold increase of UDP-glucuronic acid occurred in pgsI-208 cells, demonstrating a lack of UDP-xylose-mediated control of the cytoplasmically localized UDP-glucose dehydrogenase in the mutant. The data presented in this study suggest the bidirectional transport of UDP-xylose across endoplasmic reticulum/Golgi membranes and its role in controlling homeostasis of UDP-glucuronic acid and UDP-xylose production.Xylose is only known to occur in two different mammalian glycans. First, xylose is the starting sugar residue of the common tetrasaccharide, GlcAβ1,3Galβ1,3Galβ1,4Xylβ1-O-Ser, attached to proteoglycan core proteins to initiate the biosynthesis of glycosaminoglycans (GAGs)² (1). Second, xylose is found in the trisaccharide Xylα1,3Xylα1,3Glcβ1-O-Ser in epidermal growth factor (EGF)-like repeats of proteins, such as blood coagulation factors VII and IX (2) and Notch (3) (Fig. 1). Two variants of O-xylosyltransferases (XylT1 and XylT2) are responsible for the initiation of glycosaminoglycan biosynthesis, which differ in terms of acceptor specificity and tissue distribution (4-7), and two different enzymatic activities have been identified that catalyze xylosylation of O-glucose residues added to EGF repeats (8-10). On Notch, O-glucose occurs on EGF repeats in a similar fashion as O-fucose, which modifications have been shown to influence ligand-mediated Notch signaling (11-16). Recently, rumi, the gene encoding the Notch O-glucosyltransferase in Drosophila, has been identified, and inactivation of the gene was found to cause a temperature-sensitive Notch phenotype (17). Although this finding clearly demonstrated that O-glucosylation is essential for Notch signaling, the importance of xylosylation for Notch functions remains ambiguous.Open in a separate windowFIGURE 1.UDP-xylose metabolism in mammalian cells. A, UDP-Xyl is synthesized in two steps from UDP-Glc by the enzymes UGDH, forming UDP-GlcA, and UXS, also referred to as UDP-glucuronic acid decarboxylase. UGDH is inhibited by the product of the second enzyme, UDP-Xyl (42). B, in mammals, UDP-Xyl is synthesized within the lumen of the ER/Golgi, where it is substrate for different xylosyltransferases incorporating xylose in the glycosaminoglycan core (XylT1 and XylT2) or in O-glucose-linked glycans. The nucleotide sugar transporter SLC35D1 (52) has been shown to transport UDP-GlcA over the ER membrane and SLC35B4 (29) to transport UDP-Xyl over the Golgi membrane. The function of this latter transporter is unclear.Several different Chinese hamster ovary (CHO) cell lines with defects in GAG biosynthesis have been isolated by screening for reduced incorporation of sulfate (18) and reduced binding of fibroblast growth factor 2 (FGF-2) (19, 20) and by direct selection with FGF-2 conjugated to the plant cytotoxin saporin (21). Isolated cells (called pgs, for proteoglycan synthesis mutants) (21) exhibited defects in various stages of GAG biosynthesis, ranging from the initiating xylosyltransferase to specific sulfation reactions (18, 19, 21-25). Mutants that affect overall GAG biosynthesis were shown to have a defect in the assembly of the common core tetrasaccharide. Interestingly, these latter mutants could be separated into clones in which GAG biosynthesis can be restored by the external addition of xylosides as artificial primers and those that cannot (18). The two mutants belonging to the first group are pgsA-745 and pgsB-761. Although pgs-745 is defective in XylT2 (4-6, 18), pgsB-761 exhibits a defect in galactosyltransferase I (B4GalT7), the enzyme that catalyzes the first step in the elongation of the xylosylated protein (25 (see Fig. 1B). Restoration of GAG biosynthesis in the latter mutant presumably occurs through a second β1-4-galactosyltransferase, able to act on xylosides when provided at high concentration but not on the endogenous protein-linked xylose.Here we describe the isolation of a third CHO cell line (pgsI-208) with the xyloside-correctable phenotype. The mutant is deficient in UDP-xylose synthase (UXS), also known as UDP-glucuronic acid decarboxylase. This enzyme catalyzes the synthesis of UDP-Xyl, the common donor substrate for the different xylosyltransferases, by decarboxylation of UDP-glucuronic acid. Importantly, UXS in the animal cell is localized in the lumen of the ER and/or Golgi (26-28), superseding at first sight the need for the Golgi UDP-xylose transporter, which has been recently cloned and characterized (29). Using this cell variant, experiments were designed that establish the functional significance of UDP-Xyl transport with respect to UDP-glucuronic acid production and xylosylation.     

Transport of Sterols to the Plasma Membrane of Leek Seedlings

To investigate the intracellular transport of sterols in etiolated leek (Allium porrum L.) seedlings, in vivo pulse-chase experiments with [1-¹⁴C]acetate were performed. Then, endoplasmic reticulum-, Golgi-, and plasma membrane (PM)-enriched fractions were prepared and analyzed for the radioactivity incorporated into free sterols. In leek seedlings sterols are present as a mixture in which (24R)-24-ethylcholest-5-en-3β-ol is by far the major compound (around 60%). The other sterols are represented by cholest-5-en-3β-ol, 24-methyl-cholest-5-en-3β-ol, (24S)-24-ethylcholesta-5,22E-dien-3β-ol, and stigmasta-5,24(24¹)Z-dien-3β-ol. These compounds are shown to reside mainly in the PM. Our results clearly indicate that free sterols are actively transported from the endoplasmic reticulum to the PM during the first 60 min of chase, with kinetics very similar to that of phosphatidylserine. Such a transport was found to be decreased at low temperature (12°C) and following treatment with monensin and brefeldin A. These data are consistent with a membrane-mediated process for the intracellular transport of sterols to the PM, which likely involves the Golgi apparatus.     

Characterization of Nucleotide Transport into Rat Brain Synaptic Vesicles

ATP transport to synaptic vesicles from rat brain has been studied using the fluorescent substrate analogue 1,N6-ethenoadenosine 5'-triphosphate (epsilon-ATP). The increase in intravesicular concentration was time dependent for the first 30 min, epsilon-ATP being the most abundant nucleotide. The complexity of the saturation curve indicates the existence of kinetic and allosteric cooperativity in the nucleotide transport, which exhibits various affinity states with K0.5 values of 0.39 +/- 0.06 and 3.8 +/- 0.1 mM with epsilon-ATP as substrate. The Vmax values obtained were 13.5 +/- 1.4 pmol x min(-1) x mg of protein(-1) for the first curve and 28.3 +/- 1.6 pmol x min(-1) x mg of protein(-1) considering both components. This kinetic behavior can be explained on the basis of a mnemonic model. The nonhydrolyzable adenine nucleotide analogues adenosine 5'-O-3-(thiotriphosphate), adenosine 5'-O-2-(thiodiphosphate), and adenosine 5'-(beta,gamma-imino)triphosphate and the diadenosine polyphosphates P1,P3-di(adenosine)triphosphate, P1,P4-di(adenosine)tetraphosphate, and P1,P5-di(adenosine)pentaphosphate inhibited the nucleotide transport. The mitochondrial ATP/ADP exchange inhibitor atractyloside, N-ethylmaleimide, and polysulfonic aromatic compounds such as Evans blue and 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid also inhibit epsilon-ATP vesicular transport.     

The Mechanics of Calcium Transport

With the recent atomic models for the sarcoplasmic reticulum Ca²⁺-ATPase in the Ca²⁺-bound state, the Ca²⁺-free, thapsigargin-inhibited state, and the Ca²⁺-free, vanadate-inhibited state, we are that much closer to understanding and animating the Ca²⁺-transport cycle. These snapshots of the Ca²⁺-transport cycle reveal an impressive breadth and complexity of conformational change. The cytoplasmic domains undergo rigid-body movements that couple the energy of ATP to the transport of Ca²⁺ across the membrane. Large-scale rearrangements in the transmembrane domain suggest that the Ca²⁺-binding sites may alternately cease to exist and reform during the transport cycle. Of the three cytoplasmic domains, the actuator (A) domain undergoes the largest movement, namely a 110° rotation normal to the membrane. This domain is linked to transmembrane segments M1–M3, which undergo large rearrangements in the membrane domain. Together, these movements are a main event in Ca²⁺ transport, yet their significance is poorly understood. Nonetheless, inhibition or modulation of Ca²⁺-ATPase activity appears to target these conformational changes. Thapsigargin is a high-affinity inhibitor that binds to the M3 helix near Phe²⁵⁶, and phospholamban is a modulator of Ca²⁺-ATPase activity that has been cross-linked to M2 and M4. The purpose of this review is to postulate roles for the A domain and M1–M3 in Ca²⁺ transport and inhibition.