Biosynthesis of Xyloglucan in Suspension-Cultured Soybean Cells. Synthesis of Xyloglucan from UDP-Glucose and UDP-Xylose in the Cell-Free System

When UDP-[¹⁴C]glucose or UDP-[¹⁴C]xylose was incubated witha particulate fraction from soybean cells, radioactive polymerswere synthesized. On digestion with Aspergillus oryzae enzymes,these polymers gave ¹⁴C-monosaccharides and a ¹⁴C-disaccharidewith chromatographic and electrophoretic mobilities indistinguishablefrom those of authentic isoprimeverose (6-O--D-xylopyranosyl-D-glucopyranose).The disaccharide consisted of xylose and glucose, and the latterwas located at the reducing end. Evidence that the disaccharideis isoprimeverose was provided by methylation analysis. Hydrolysisof the methylated disaccharide yielded 2,3,4-tri-O-methyl-D-xyloseand 2,3,4-tri-O-methyl-D-glucose. Thus, incorporation of radioactivityinto isoprimeverose, the smallest structural unit of xyloglucan,suggests that xyloglucan is synthesized in vitro from UDP-glucoseand UDP-xylose. (Received November 20, 1980; Accepted February 14, 1981)     

Incorporation of UDP-[C]Glucose into Xyloglucan by Pea Membranes

The water-insoluble 1,4-β-linked products formed from UDP-[¹⁴C]glucose by pea membranes were dissolved in hot dimethyl-sulfoxide/paraformaldehyde and fractionated on columns of controlled pore glass beads calibrated with dextran standards. The products eluted with a peak size close to 70 kilodaltons in dextran equivalents. Similar elution profiles were obtained for products formed in brief or extended incubations and at high or low substrate concentrations. Methylation analysis indicated that only a few [¹⁴C]glucose units had been added to an endogenous acceptor to form this product. In the presence of UDP-xylose at concentrations equal to or less than UDP-[¹⁴C]glucose, incorporation from the latter was enhanced and the products elongated with time to a size range where the major components eluted between dextran 264 and 500 kilodaltons. Treatment with endo-1,4-β-glucanase resulted in a mixture of oligosaccharides, including the xyloglucan subunit Glc₄Xyl₃, which were hydrolyzed further by mixed glycosidases to labeled glucose and isoprimeverose (xylosyl-1,6-α-d-glucose). In pulse-chase experiments, the low molecular weight product formed from UDP-[¹⁴C]glucose alone was clearly a precursor for high molecular weight products formed subsequently in the presence of both UDP-glucose and UDP-xylose. It is concluded that the 1,4-β-transglucosylation activity detected in these tests was due to an enzyme that is required for biosynthesis of the backbone of xyloglucan.     

Degradation of Xyloglucan by Wall-bound Enzymes from Soybean Tissue I. Occurrence of Xyloglucan-degrading Enzymes in Soybean Cell Wall

An enzyme preparation that catalyzes the degradation of xyloglucanwas obtained by extraction of the cell walls of soybean hypocotylswith a buffer containing 1.0 M NaCl. The enzyme preparationwas shown to catalyze two-step degradation of xyloglucan. Thepolysaccharide was first degraded into comparatively large fragments,which were then further degraded into monosaccharides. In orderto elucidate the mode of degradation of the xyloglucan duringcell growth, the activities of xyloglucandegrading enzymes ofsoybean-hypocotyl segments were assayed at different stagesof elongation. The total activities of the degrading enzymeswere lower in the elongating regions than in the non-elongatingregions. However, high levels of endo-ß-l,4-glucanasewere found in the elongating regions. These results suggestthat xyloglucan is hydrolyzed by endo-ß-1,4-glucanaseinto comparatively large fragments at the initial stage of growthand the resulting fragments are further degraded into monosaccharidesduring cell elongation. (Received May 20, 1981; Accepted August 8, 1981)     

A Role for Giantin in Docking COPI Vesicles to Golgi Membranes

We have previously shown that p115, a vesicle docking protein, binds to two proteins (p130 and p400) in detergent extracts of Golgi membranes. p130 was identified as GM130, a Golgi matrix protein, and was shown to act as a membrane receptor for p115. p400 has now been identified as giantin, a Golgi membrane protein with most of its mass projecting into the cytoplasm. Giantin is found on COPI vesicles and pretreatment with antibodies inhibits both the binding of p115 and the docking of these vesicles with Golgi membranes. In contrast, GM130 is depleted from COPI vesicles and inhibition of the GM130 on Golgi membranes, using either antibodies or an NH₂-terminal GM130 peptide, inhibits p115 binding and vesicle docking. Together these results suggest that COPI vesicles are docked by giantin on the COPI vesicles and GM130 on Golgi membranes with p115 providing a bridge.     

Formation and Ultrastructure of Extra Membranes in Escherichia coli

A temperature-sensitive strain of Escherichia coli (strain 0111a(1)) was shown to accumulate membranous structures at 40 C. These "extra membranes" appeared as vesicles or whorls (or both), depending on the time of growth at 40 C. After 2 hr of growth at 40 C, only vesicles were observed in E. coli 0111a(1) cells; both vesicles and whorls were apparent after 6 hr. The number of cells which contained both types of extra membrane reached a maximum value (75%) after 10 hr of growth at 40 C. Extra membrane production was also studied by using temperature shifts. In shift-up experiments, cells grown at 30 C into early stationary phase accumulated extra membrane after a shift to 40 C. The percentage of E. coli 0111a(1) cells containing extra membrane decreased significantly after a shift from 40 to 30 C. Phase- and electron-microscopic observations indicated that E. coli 0111a(1) cells grown at 40 C were larger than E. coli 0111: B(4) cells grown at either temperature. The ratio of optical density per cell and cell measurements obtained from quantitative electron microscopy confirmed that E. coli 0111a(1) cells grown at 40 C were about twice as large. Microdensitometer traces indicated that the dimension of a single membrane of either whorls or vesicles was 5.4 nm in peak-to-peak distance (8.8 nm total thickness).     

Xyloglucan Endotransglucosylase-Hydrolase17 Interacts with Xyloglucan Endotransglucosylase-Hydrolase31 to Confer Xyloglucan Endotransglucosylase Action and Affect Aluminum Sensitivity in Arabidopsis

Previously, we reported that although the Arabidopsis (Arabidopsis thaliana) Xyloglucan Endotransglucosylase-Hydrolase31 (XTH31) has predominately xyloglucan endohydrolase activity in vitro, loss of XTH31 results in remarkably reduced in vivo xyloglucan endotransglucosylase (XET) action and enhanced Al resistance. Here, we report that XTH17, predicted to have XET activity, binds XTH31 in yeast (Saccharomyces cerevisiae) two-hybrid and coimmunoprecipitations assays and that this interaction may be required for XTH17 XET activity in planta. XTH17 and XTH31 may be colocalized in plant cells because tagged XTH17 fusion proteins, like XTH31 fusion proteins, appear to target to the plasma membrane. XTH17 expression, like that of XTH31, was substantially reduced in the presence of aluminum (Al), even at concentrations as low as 10 µm for 24 h or 25 µm for just 30 min. Agrobacterium tumefaciens-mediated transfer DNA insertion mutant of XTH17, xth17, showed low XET action and had moderately shorter roots than the wild type but was more Al resistant than the wild type. Similar to xth31, xth17 had low hemicellulose content and retained less Al in the cell wall. These data suggest a model whereby XTH17 and XTH31 may exist as a dimer at the plasma membrane to confer in vivo XET action, which modulates cell wall Al-binding capacity and thereby affects Al sensitivity in Arabidopsis.Soil acidity (pH < 5.5) affects about 40% of the world’s arable land (von Uexküll and Mutert, 1995) and more than 50% of land that is potentially arable, particularly in the tropics and subtropics (Kochian et al., 2004; Eticha et al., 2010). Al is the most growth-limiting factor for crop production on acid soils worldwide (Foy, 1988; Kochian, 1995), especially when the pH drops below 5 (Eswaran et al., 1997).To survive in an Al-toxic environment, Al-resistant plants have evolved two mechanisms to cope with Al toxicity. One is to restrict Al uptake from the root, while the other is to cope with internalized Al (Taylor, 1991; Kochian et al., 2004). The latter is usually employed by Al-accumulating species such as Hydrangea macrophylla (Ma et al., 1997a) and buckwheat (Fagopyrum esculentum; Ma et al., 1997b). In most cases, secretion of the organic acid anions is the most important Al exclusion mechanism (Kochian, 1995; Ryan et al., 2001; Ma and Furukawa, 2003), although it does not explain all the Al resistance in some plants such as signalgrass (Brachiaria decumbens Stapf cv Basilisk; Wenzl et al., 2001), maize (Zea mays; Piñeros et al., 2005), buckwheat (Zheng et al., 2005), rice (Oryza sativa; Ma et al., 2005; Yang et al., 2008), or Fagopyrum tataricum (Yang et al., 2011a). Therefore, it is possible that for some plant species (such as buckwheat), Al tolerance is a combination of mechanisms including organic anion efflux.Recently, evidence has accumulated that the cell wall, especially the hemicellulose component, may impact Al resistance. For example, Al induces significant changes in the hemicellulose fraction of wheat (Triticum aestivum; Tabuchi and Matsumoto, 2001), triticale (× Triticosecale Wittmack; Liu et al., 2008), rice (Yang et al., 2008), and Arabidopsis (Arabidopsis thaliana; Zhu et al., 2012), especially the Al-sensitive cultivars. Moreover, we found that Arabidopsis hemicellulose is not only very sensitive to Al stress (the content of hemicellulose increased quickly under Al stress), but is also the principal binding site for Al (Yang et al., 2011b). Furthermore, loss of Xyloglucan Endotransglucosylase-Hydrolase31 (XTH31) function resulted in lower xyloglucan content and cell wall Al-binding capacity in Arabidopsis (Zhu et al., 2012). Thus, xyloglucan may be a major Al-binding site in Arabidopsis, and any effects leading to xyloglucan modifications may regulate Al sensitivity.XTHs are a family of enzymes that play principal roles in the construction and restructuring of the load-bearing cross links among cellulose microfibrils (Osato et al., 2006) through catalyzing the molecular grafting or hydrolyzing of the xyloglucans to form the framework (Fry et al., 1992; Nishitani and Tominaga, 1992; Okazawa et al., 1993; Rose et al., 2002). There are 33 identified XTH genes in the Arabidopsis genome, and one-third occur as clusters resulting from genome duplication (Blanc et al., 2000; Yokoyama and Nishitani, 2001); XTH1-11 are classified in subgroup 1, XTH12-26 are in subgroup 2, and XTH27-33 are in subgroup 3 (Rose et al., 2002). Each member of the XTH gene family is likely regulated by specific cues and committed to cell wall dynamics specific to certain tissues or cell types (Nishitani, 2002; Becnel et al., 2006; Osato et al., 2006). For example, XTH27 is involved in the cell wall modification of tracheary elements at a specific stage of rosette leaf development and is essential for tertiary vein development (Matsui et al., 2005), whereas XTH31 is involved in cell wall modification and cell elongation through modulating xyloglucan endotransglucosylase (XET) action under Al stress (Zhu et al., 2012). However, XTH31 is an XTH for which xyloglucan endohydrolase (XEH) activity has been predicted (Baumann et al., 2007), and in our previous report, we demonstrated that XTH31 produced heterologously in Pichia pastoris has high XEH activity but low XET activity in vitro (Zhu et al., 2012), which is in accordance with Kaewthai et al. (2013), who reported that XTH31 is a predominant hydrolase using the in vitro activity assays and enzyme product analysis, as well as the use of a fluorogenic substrate in vivo. Unexpectedly, however, the xth31 mutant has very low XET action and activity (Zhu et al., 2012). One possible explanation for this result is that XTH31 may interact with and be required for activity of XET-active XTHs.In this study, we demonstrate that XTH17 can bind to XTH31 in vitro and in vivo and that a transfer DNA (T-DNA) insertional mutant of XTH17 has elevated Al resistance and exhibits a phenotype very similar to xth31. Together, these data are consistent with the interpretation that XTH17 and XTH31 may interact with each other and thereby contribute to Al-inhibited XET action in Arabidopsis.     

Localization of Xyloglucan in the Macromolecular Complex Composed of Xyloglucan and Cellulose in Pea Stems

A macromolecular complex composed of xyloglucan and cellulosewas isolated from elongating regions of stems of etiolated pea(Pisum sativum L. var Alaska) seedlings and binding of a xyloglucan-specificantibody was examined after treatment of the complex with endo-1,4-ß-glucanaseor 24% KOH. The antibody bound to the complex but the extentof binding was reduced after treatment of the complex with endo-1,4-ß-glucanaseand was hardly detectable after treatment with 24% KOH. Themolecular weight of the xyloglucan that remained (5%) in theß-glucanase-treated complexes was less than 9,200.Pea xyloglucan was allowed to bind to enzymeand alkali-treatedcomplexes to generaly reconstituted complexes. The amount ofthe antibody that bound to each type of reconstituted complexwas similar but was much lower than that bound to the nativecomplex. Immunogold labeling indicated that most of the antigenwas widely distributed between microfibrils in the native complex,whereas the antigen appeared to be confined to the microfibrilsin the reconstituted complexes. These findings suggest thata part of each xyloglucan molecule is strongly associated withcellulose microfibrils while the rest is free of the microfibrilsin the native complex. ¹This work was supported in part by a grant from the YamadaScience Foundation.     

Auxin-Modulated Protein Disulfide-Thiol-Interchange Activity from Soybean Plasma Membranes

The renaturation of scrambled (oxidized and inactive) RNase A is catalyzed by soybean (Glycine max cv Williams 82) plasma membranes. The catalysis is stimulated by the auxin herbicide 2,4-dichlorophenoxyacetic acid or by the natural auxin indole-3-acetic acid. The inactive auxin analog, 2,3-dichlorophenoxyacetic acid, is without effect. The activity occurs in the absence of external electron acceptors or donors and therefore appears to be a true disulfide-thiol-interchange activity between protein disulfides and thiols of RNase A and those of plasma membrane proteins. The activity is not affected by a mixture of reduced and oxidized glutathione. However, no auxin-stimulated activity was observed in the presence of either oxidized glutathione or reduced glutathione alone, a response characteristic of the previously described auxin-stimulated NADH oxidase activity of soybean plasma membranes. Taken together, the results suggest the operation in the plant plasma membrane of a protein disulfide-thiol-interchange activity that is stimulated by auxins. The auxin stimulations of the interchange activity are prevented by glutathione, reduced glutathione, and brefeldin A at concentrations that also prevent auxin stimulation of NADH oxidation by isolated plasma membranes and inhibit, as well, the auxin-stimulated elongation of excised segments of soybean hypocotyls.     

Molecular Motors and a Spectrin Matrix Associate with Golgi Membranes In Vitro

Cytoplasmic dynein is a microtubule minus-end–directed motor that is thought to power the transport of vesicles from the TGN to the apical cortex in polarized epithelial cells. Trans-Golgi enriched membranes, which were isolated from primary polarized intestinal epithelial cells, contain both the actin-based motor myosin-I and dynein, whereas isolated Golgi stacks lack dynein but contain myosin-I (Fath, K.R., G.M. Trimbur, and D.R. Burgess. 1994. J. Cell Biol. 126:661–675). We show now that Golgi stacks in vitro bind dynein supplied from cytosol in the absence of ATP, and bud small membranes when incubated with cytosol and ATP. Cytosolic dynein binds to regions of stacks that are destined to bud because dynein is present in budded membranes, but absent from stacks after budding. Budded membranes move exclusively towards microtubule minus-ends in in vitro motility assays. Extraction studies suggest that dynein binds to a Golgi peripheral membrane protein(s) that resists extraction by ice-cold Triton X-100. In the presence of cytosol, these membrane ghosts can move towards the minus-ends of microtubules. Detergent-extracted Golgi stacks and TGN-containing membranes are closely associated with an amorphous matrix composed in part of spectrin and ankyrin. Although spectrin has been proposed to help link dynein to organellar membranes, we found that functional dynein may bind to extracted membranes independently of spectrin and ankyrin.     

Nitrogen Fixation and Allantoin Formation in Soybean Plants

The activity of nitrogenase and the concentration of ammonia and allantoin (+ allantoic acid) in root nodules were measured throughout the growth period of soybean plants. Nitrogenase activity measured by acetylene reduction increased with plant growth and reached a maximum level at the flowering period. The level of ammonia and allantoin concentration in nodules was parallel with increased nitrogenase activity. At the late reproductive stage (pod-forming period), nitrogenase activity showed a marked decrease, but the ammonia and allantoin in the nodules remained at a constant level. Detached nodules from 56 day-old soybean plants were exposed to ¹⁵N₂ gas, and the distribution of ¹⁵N among nitrogen compounds was investigated. Enrichment of ¹⁵N in allantoin and allantoic acid reached a fairly high level after 90 min of nitrogen fixation; ca. 22% of ¹⁵N in acid-soluble nitrogen compounds was incorporated into allantoin + allantoic acid. In contrast, enrichment of ¹⁵N in amide nitrogen was relatively low. No significant ¹⁵N was detected in the RNA fraction. The data suggested that ureide formation in nitrogen-fixing root nodules did not take place through the breakdown of nucleic acids, but directly associated with the assimilating system of biologically fixed nitrogen.     

Identification and Characterization of a Golgi-Localized UDP-Xylose Transporter Family from Arabidopsis

Most glycosylation reactions require activated glycosyl donors in the form of nucleotide sugars to drive processes such as posttranslational modifications and polysaccharide biosynthesis. Most plant cell wall polysaccharides are biosynthesized in the Golgi apparatus from cytosolic-derived nucleotide sugars, which are actively transferred into the Golgi lumen by nucleotide sugar transporters (NSTs). An exception is UDP-xylose, which is biosynthesized in both the cytosol and the Golgi lumen by a family of UDP-xylose synthases. The NST-based transport of UDP-xylose into the Golgi lumen would appear to be redundant. However, employing a recently developed approach, we identified three UDP-xylose transporters in the Arabidopsis thaliana NST family and designated them UDP-XYLOSE TRANSPORTER1 (UXT1) to UXT3. All three transporters localize to the Golgi apparatus, and UXT1 also localizes to the endoplasmic reticulum. Mutants in UXT1 exhibit ∼30% reduction in xylose in stem cell walls. These findings support the importance of the cytosolic UDP-xylose pool and UDP-xylose transporters in cell wall biosynthesis.     

The Structure and Functions of Xyloglucan

Xyloglucan is a polysaccharide found in the primary cell wallsof all higher plants examined. Its cellulose-like backbone,which is about 0.15 to 1.5 µm long, consists of 300 to3 000 ß-(14)-linked D-glucopyranose residues. About60–75% (or, in grasses, about 30–40%) of the glucoseresidues have side-chains attached to position 6. The majorside-chains are: D-xylopyranosyl--1 -, D-galactopyranosyl-ß-(12)-D-xylopyranosyl--I , L-arabinofuranosyl-(1 -2)-D-xylopyranosyl--1-, and (except in grasses) L.-fucopyranosyl--(1 -2)-D-galactopyranosyl-ß-(1-2)-D-xylopyranosyl--1-. There is some regularity in the distribution of these side-chainsalong the backbone. Xyloglucan plays two very different r?les in the control ofcell growth: (a) as a major building material of the wall [concentrationof xyloglucan in the wall in vivo 10% (w/v)] it probably directlydictates wall extensibility and, therefore, the rate of cellexpansion and (b) it can be broken down to a fucose-containingoligosaccharide which [at a concentration of 0.0000001% (w/v)]exerts a hormone-like anti-auxin effect on growth. In addition,xyloglucan lacking fucose is used by certain dicotyledonousseeds as a food reserve which is mobilized after germination.Xyloglucan is, therefore, the subject of considerable currentinterest in several apparently disparate areas of botany. Key words: Xyloglucan, ‘oligosaccharin’, hemicellulose, auxin, anti-auxin, growth, cell walls, reserve carbohydrate     

The Pathway of Golgi Cluster Formation in Okadaic Acid-Treated Cells

Using stereology and immunoelectron microscopy we examined the pathway of Golgi duster formation during treatment with the phosphatase inhibitor okadaic acid. During the first hour the Golgi stack of suspension HeLa cells lost 90% of its membrane without appreciable reduction in the number of cisternae. During this time clusters of tubules and vesicles (Golgi clusters) appeared and these contained only a fraction of the Golgi membrane present in untreated cells. Despite the overall reduction in membrane the total amount of immunolabeling for galactosyltransferase over the Golgi clusters of a typical cell was maintained, indicating that galactosyltransferase had been retained in Golgi membranes. The observation that, after 40 min okadaic acid treatment, labeling density for galactosyltransferase within trans Golgi cisternae increased 1.6-fold (n = 3, CE 10%) suggests that membrane loss from trans cisternae was selective. Careful evaluation of immunolabeled clusters showed that most of the galactosyltransferase labeling was located over complex tubular profiles and not vesicular profiles. Tubular structures were also observed during disassembly and these were found both connected to disassembling cisternae and within forming Golgi clusters, indicating that they were intermediates in cluster formation. We also investigated the role of vesicular transport in cluster formation. During disassembly we found no accumulation of COP-coated buds and vesicles over Golgi membrane. However, aluminium fluoride, previously found to arrest transport in the Golgi stack, completely inhibited membrane depletion and stack disassembly. Taken together, our results indicate that during Golgi cluster formation, membrane leaves the Golgi but galactosyltransferase is retained within a tubular reticulum which is a direct descendant of trans-Golgi cisternae. Membrane depletion may require ongoing vesicular transport and we postulate that it arises because of an imbalance in membrane traffic into and out of the Golgi apparatus.     

Spatial Organization and Stoichiometry of N-Terminal Domain-Mediated Glycosyltransferase Complexes in Golgi Membranes Determined by Fret Microscopy

The functional link between glycolipid glycosyltransferases (GT) relies on the ability of these proteins to form organized molecular complexes. The organization, stoichiometry and composition of these complexes may impact their sorting properties, sub-Golgi localization, and may determine relative efficiency of GT in different glycolipid biosynthetic pathways. In this work, by using Förster resonance energy transfer microscopy in live CHO-K1 cells, we investigated homo- and hetero-complex formation by different GT as well as their spatial organization and molecular stoichiometry on Golgi membranes. We find that GalNAcT and GalT2 Ntd are able to form hetero-complexes in a 1:2 molar ratio at the trans-Golgi network and that GalT2 but not GalNAcT forms homo-complexes. Also, GalNAcT/GalT2 complexes exhibit a stable behavior reflected by its clustered lateral organization. These results reveals that particular topological organization of GTs may have functional implications in determining the composition of glycolipids in cellular membranes.     

Degradation of Xyloglucan by Wall-Bound Enzymes from Soybean Tissue II: Degradation of the Fragment Heptasaccharide from Xyloglucan and the Characteristic Action Pattern of the {alpha}-D-Xylosidase in the Enzyme System

Hydrolysis of the fragment heptasaccharide (glucose : xylose= 4 : 3) from xyloglucan with an enzyme preparation from soybeancell wall produced a penta- and a trisaccharide. The resultsof fragmentation analysis of these oligosaccharides with Aspergillusoryzae ß-D-glucosidase indicate the following structuresfor the penta- and trisaccharide. The detection of these intermediate products suggested thatdegradation of the heptasaccharide took place by sequentialsplitting of the -D-xylosidic and ß-D-glucosidic linkages.A characteristic action pattern of the a-D-xylosidase in theenzyme preparation was found. ¹Present address: Department of Biology, McGill University,Montreal, Canada. ²Present address: Department of Botany, Iowa State University,Ames, Iowa 50010, U.S.A. (Received August 20, 1982; Accepted December 7, 1982)     

Early Stages of Golgi Vesicle and Tubule Formation Require Diacylglycerol

We have investigated the role for diacylglycerol (DAG) in membrane bud formation in the Golgi apparatus. Addition of propranolol to specifically inhibit phosphatidate phosphohydrolase (PAP), an enzyme responsible for converting phosphatidic acid into DAG, effectively prevents formation of membrane buds. The effect of PAP inhibition on Golgi membranes is rapid and occurs within 3 min. Removal of the PAP inhibitor then results in a rapid burst of buds, vesicles, and tubules that peaks within 2 min. The inability to form buds in the presence of propranolol does not appear to be correlated with a loss of ARFGAP1 from Golgi membranes, as knockdown of ARFGAP1 by RNA interference has little or no effect on actual bud formation. Rather, knockdown of ARFGAP1 results in an increase in membrane buds and a decrease of vesicles and tubules suggesting it functions in the late stages of scission. How DAG promotes bud formation is discussed.     

Protein-dependent Reduction of the Pyrene Excimer Formation in Membranes

The presence of proteins in lipid bilayers always decreases the excimer formation rate of pyrene and pyrene lipid analogues in a way that is related to the protein-to-lipid ratio. Energy transfer measurements from intrinsic tryptophans to pyrene have shown (Engelke et al., 1994), that in microsomal membranes, the excimer formation rate of pyrene and pyrene fatty acids is heterogeneous within the membrane plane, because a lipid layer of reduced fluidity surrounds the microsomal proteins. This study investigates whether of not liposomes prepared from egg yolk phosphatidylcholine with incorporated gramicidin A give results comparable to those from microsomal membranes. The results indicate that the influence of proteins on the lipid bilayer cannot be described by one unique mechanism: Small proteins such as gramicidin A obviously reduce the excimer formation rate by occupying neighboring positions of the fluorescent probe and thus decrease the pyrene collision frequency homogeneously in the whole membrane plane, while larger proteins are surrounded by a lipid boundary layer of lower fluidity than the bulk lipid. The analysis of the time-resolved tryptophan fluorescence of gramicidin A incorporated liposomes reveals, that the tryptophan quenching by pyrene is stronger for tryptophans located closely below the phospholipid headgroup region because of the pyrene enrichment in this area of the lipid bilayer. Received: 29 December 1996/Revised: 15 May 1996     

Calcium Transduces Plasma Membrane Receptor Signals to Produce Diacylglycerol at Golgi Membranes

Protein kinase C and protein kinase D are potently activated by agonist-evoked increases in diacylglycerol. Using live cell-imaging probes for kinase activity, we have shown that both kinases are robustly activated at the Golgi following stimulation of G_q-coupled receptor signaling pathways, displaying activation signatures at the Golgi that are distinct from those at the plasma membrane. Here we report that Ca²⁺ is the mediator that allows signals received at the plasma membrane to activate these two protein kinases at the Golgi. Specifically, using fluorescence resonance energy transfer-based reporters to image diacylglycerol production, we show that Ca²⁺ is necessary and sufficient to elevate diacylglycerol levels at the Golgi. First, raising intracellular Ca²⁺ by treating cells with thapsigargin induces diacylglycerol production at the Golgi. Second, chelation of intracellular Ca²⁺ prevents UTP-stimulated increases in diacylglycerol at the Golgi. Thus, agonist-evoked increases in intracellular Ca²⁺ cause increases in Golgi diacylglycerol, allowing this intracellular membrane to serve as a platform for signaling by protein kinases C and D.     

Characterization of Kiwifruit Xyloglucan

Structural characteristics of xyloglucan are constant in the pericarp cell walls of kiwifruit (Actinidia deliciosa) throughout fruit enlargement and maturation. Most of the xyloglucan (XG) persists in the cell walls of ripe kiwifruit. XG from the pericarp tissues of 36-h ethylene-treated kiwifruit was extracted as hemicellulose Ⅱ (HC-Ⅱ) with 4.28 M KOH containing 0.02% NaBH4, and purified using iodine precipitation and subsequent anion-exchange chromatography. This purifying protocol increased XG purity from 50 mol% in HC-Ⅱ fraction to 62 mol% in the purified XG powder. The molar ratio of glucose: xylose: galactose: fucose in the purified XG was 10: 6.9: 2.1: 0.3. Gel permeation chromatography indicated that purified XG had an average molecular-mass of 161 KDa, a value that exceeds the 95 KDa M_r determined for total polymeric sugars. Sugar linkage analysis confirmed the lack of fucose in the kiwifruit XG, but a small amount of arabinoxylan and low M_r glucomannan remained associated with this fraction.     

Characterization of Kiwifruit Xyloglucan

Structural characteristics of xyloglucan are constant in the pericarp cell walls of kiwifruit ( Actinidia deliciosa ) throughout fruit enlargement and maturation. Most of the xyloglucan (XG) persists in the cell walls of ripe kiwifruit. XG from the pericarp tissues of 36-h ethylene-treated kiwifruit was extracted as hemicellulose II (HC-II) with 4.28 M KOH containing 0.02% NaBH₄, and purified using iodine precipitation and subsequent anion-exchange chromatography. This purifying protocol increased XG purity from 50 mol% in HC-II fraction to 62 mol% in the purified XG powder. The molar ratio of glucose: xylose: galactose: fucose in the purified XG was 10: 6.9: 2.1: 0.3. Gel permeation chromatography indicated that purified XG had an average molecular-mass of 161 KDa, a value that exceeds the 95 KDa M_r determined for total polymeric sugars. Sugar linkage analysis confirmed the lack of fucose in the kiwifruit XG, but a small amount of arabinoxylan and low M_r glucomannan remained associated with this fraction.