Isolation of a vesicular intermediate in the cell-free transfer of membrane from transitional elements of the endoplasmic reticulum to Golgi apparatus cisternae of rat liver

Preparations enriched in part-smooth (lacking ribosomes), part-rough (with ribosomes) transitional elements of the endoplasmic reticulum when incubated with ATP plus a cytosol fraction responded by the formation of blebbing profiles and approximately 60-nm vesicles. The 60-nm vesicles formed resembled closely transition vesicles in situ considered to function in the transfer of membrane materials between the endoplasmic reticulum and the Golgi apparatus. The transition elements following incubation with ATP and cytosol were resolved by preparative free-flow electrophoresis into fractions of differing electronegativity. The main fraction contained the larger vesicles of the transitional membrane elements, while a less electronegative minor shoulder fraction was enriched in the 60-nm vesicles. If the vesicles concentrated by preparative free-flow electrophoresis were from material previously radiolabeled with [3H]leucine and then added to Golgi apparatus immobilized to nitrocellulose, radioactivity was transferred to the Golgi apparatus membranes. The transfer was rapid (T1/2 of about 5 min), efficient (10-30% of the total radioactivity of the transition vesicle preparations was transferred to Golgi apparatus), and independent of added ATP but facilitated by cytosol. Transfer was specific and apparently unidirectional in that Golgi apparatus membranes were ineffective as donor membranes and endoplasmic reticulum vesicles were ineffective as recipient membranes. Using a heterologous system with transition vesicles from rat liver and Golgi apparatus isolated from guinea pig liver, coalescence of the small endoplasmic reticulum-derived vesicles with Golgi apparatus membranes was demonstrated using immunocytochemistry. Employed were polyclonal antibodies directed against the isolated rat transition vesicle preparations. When localized by immunogold procedures at the electron microscope level, regions of rat-derived vesicles were found fused with cisternae of guinea pig Golgi apparatus immobilized to nitrocellulose strips. Membrane transfer was demonstrated from experiments where transition vesicle membrane proteins were radioiodinated by the Bolton-Hunter procedure. Additionally, radiolabeled peptide bands not present initially in endoplasmic reticulum appeared following coalescence of the derived vesicles with Golgi apparatus. These bands, indicative of processing, required that both Golgi apparatus and transition vesicles be present and did not occur in incubated endoplasmic reticulum preparations or on nitrocellulose strips to which no Golgi apparatus were added.(ABSTRACT TRUNCATED AT 400 WORDS)     

SB401, a pollen-specific protein from Solanum berthaultii, binds to and bundles microtubules and F-actin

We characterize a novel, pollen-specific, microtubule-associated protein, SB401, found in Solanum berthaultii. This protein binds to and bundles taxol-stabilized microtubules and enhances tubulin polymerization in a concentration-dependent manner, particularly at lower temperatures. Electron microscopy revealed that the protein decorates the entire length of microtubules. Cross-linking and electrophoresis studies showed that SB401 protein forms dimers, and suggest that dimerization could account for bundling. Double immunofluorescent staining of pollen tubes of S. berthaultii showed that SB401 protein co-localized with cortical microtubule bundles. SB401 protein also binds to and bundles actin filaments, and could connect actin filaments to microtubules. SB401 protein had a much higher affinity for microtubules than for actin filaments. In the presence of both cytoskeletal elements, the protein preferentially bound microtubules to form bundles. These results demonstrate that SB401 protein may have important roles in organizing the cytoskeleton in pollen tubes.     

Spatial and temporal variability in benthic processes along a mangrove-seagrass transect near the Bangrong Mangrove,Thailand

Benthic primary production and nutrient dynamics were examined along a transect in the Bangrong mangrove forest in Thailand. Six stations were established extending from a high-intertidal site within the mangrove forest to low-intertidal flats and seagrass beds in front of the mangrove forest. Benthic processes (O₂ and CO₂ fluxes) and nutrient dynamics (mineralization, sediment-water fluxes, pore water and sediment pools) were measured under light and dark conditions during wet and dry seasons over a 2-yr period. The sediments were mostly autotrophic, only the mangrove forest sites were net heterotrophic during the wet season. Maximum daily net primary production was found at the non-vegetated tidal flats (40–75 mmol O₂ m^-2d^-1), where light and nutrient availability were highest. The variation in benthic mineralization along the transect was minor (1.6–4.3 mmol CO₂ m^-2h^-1) and did not reflect the large changes inorganic matter content (organic carbon: 0.7–4.2% DW) and quality (C:N ratio varied from 25 to 100), suggesting that the mineralizable pool of organic matter was of similar magnitude at all sites. There was only minor seasonal variation in rates of mineralization. The net primary production showed more variation with lower rates in the mangrove forest (reduced with 74%) and higher rates at the tidal flats (increased with 172%) and in the seagrass beds (increased with 228%) during the wet season. The nutrient pools and fluxes across the sediment-water interface were generally low along the transect, and the sediments were efficient in retaining nitrogen in the nutrient limited mangrove/seagrass environment. Pools and fluxes of phosphorus were generally very low suggesting that benthic primary production was phosphorus limited along the transect. This revised version was published online in July 2006 with corrections to the Cover Date.     

Excessive expression of the plant kinesin TBK5 converts cortical and perinuclear microtubules into a radial array emanating from a single focus

TBK5 is a plant-specific kinesin constantly expressed in tobacco BY-2 cells. An analysis of the distribution of green fluorescent protein-tagged TBK5 (GFP-TBK5) transiently expressed in BY-2 protoplasts revealed that TBK5 could associate with microtubules in vivo. GFP-TBK5 often assembled to form a single particle when accumulated in cells. The particle was located in close proximity to the nucleus, and its formation was accompanied by the development of a radial array of microtubules emanating from it and the loss of cortical microtubules. Microtubule depolymerization by treatment with propyzamide inhibited particle formation and stimulated the formation of dispersed aggregates of GFP-TBK5. Through expression of different TBK5 mutants as GFP fusions, the motor domain, two separated coiled-coil domains and the C-terminal domain of TBK5 were identified as the domains playing essential roles in particle formation. Mutants with putatively non-motile motor domains or lacking the C-terminal domain were localized to cortical and perinuclear microtubules, whereas those lacking either of the coiled-coil domains were preferentially distributed around the nucleus and along perinuclar microtubules. Further, the deletion of one of the coiled-coil domains or the C-terminal domain was sufficient to inhibit the propyzamide-induced formation of dispersed aggregates, whereas the mutation in the motor domain was not. These results led us to propose a model in which the particle is formed through the microtubule-based movement of GFP-TBK5 toward the nucleus and subsequent microtubule-independent aggregation based on coiled-coil interactions. The dramatic microtubule rearrangement would be explained if GFP-TBK5 relocated and gathered newly formed microtubules and/or microtubule-nucleating units.     

Mechanism of inhibition of calcium uptake into sarcoplasmic reticulum by notexin, a neurotoxic and myotoxic polypeptide

Notexin belongs to a class of snake venom neurotoxins and myotoxins that have phospholipase A2 activity. Previous studies have shown that these toxins affect target cells differently from phospholipases that are not neurotoxic or myotoxic. Notexin inhibited the Ca2+ uptake into fragmented sarcoplasmic reticulum from rabbit skeletal muscle, but it did not cause an efflux of previously accumulated Ca2+ or inhibit the Ca2+--ATPase activity. It is suggested that notexin specifically binds to and decreases the conductance for Ca2+ of the Ca2+ pump and/or the conductance of a channel for an ion that facilitates Ca2+ transport. The K+ ionophore valinomycin reversed the notexin-induced inhibition of Ca2+ uptake into sarcoplasmic reticulum, suggesting that the molecular target of notexin could be a K+ channel. Two types of reconstitution experiments make it unlikely that notexin acts by degrading a minor lipid that is resistant to hydrolysis by nontoxic phospholipases A2. Notexin-inactivated sarcoplasmic reticulum vesicles were reactivated (with respect to Ca2+ uptake) by simple solubilization with detergent and subsequent reconstitution by detergent removal. Second, notexin was still active on sarcoplasmic reticulum vesicles after greater than 94% of the lipids were replaced by soybean phosphoglycerides during the reconstitution procedure.     

Ionic selectivity, saturation, and block in a K+-selective channel from sarcoplasmic reticulum

The open-channel conductance properties of a voltage-gated channel from sarcoplasmic reticulum were studied in planar phospholipid membranes. The channel is ideally selective for K+ over Cl- and for K+ over Ca++. In symmetrical 1 M solutions, the single-channel conductance (in pmho) falls in the order: K+ (214) > NH4+ (157) > Rb+ (125) > Na+ (72) > La+ (8.1) > Cs+ (< 3). In neutral bilayers, the channel conductance saturates with ion activity according to a rectangular hyperbolic relation, with half-saturation activities of 54 mM for K+ and 34 mM for Na+. Under symmetrical salt conditions, the K+:Na+ channel conductance ratio increases with salt activity, but the permeability ratio, measured by single-channel bi-ionic potentials, is constant between 20 mM and 2.5 M salt; the permeability ratio is equal to the conductance ratio in the limit of low-salt concentration. The channel conductance varies < 5% in the voltage range -100 to +70 mV. The maximum conductance varies K+ and Na+ is only weakly temperature dependent (delta H++ = 4.6 and 5.3 kcal/mol, respectively), but that of Li+ varies strongly with temperature (delta H++ = 13 kcal/mol). The channel's K+ conductance is blocked asymmetrically by Cs+, and this block is competitive with K+. The results are consistent with an Eyring-type barriers as it permeates the channel. The data conform to Lüger's (1973. Biochem. Biophys. Acta. 311:423-441) predictions for a "pure" single-ion channel.     

Conduction and block by organic cations in a K+-selective channel from sarcoplasmic reticulum incorporated into planar phospholipid bilayers

A collection of organic cations has been used to probe the gross structural features of the ionic diffusion pathway in a K+-selective channel from sarcoplasmic reticulum (SR). Channels were incorporated into planar phospholipid bilayer membranes, and single-channel currents were measured in the presence of ammonium-derived cations in the aqueous phases. Small monovalent organic cations are able to permeate the channel: the channel conductance drops sharply for cations having molecular cross sections larger than 18-20 A2. Impermeant or poorly permeant cations such as tetraethylammonium, choline, and glucosamine, among others, block K+ conduction through the channel. This block is voltage dependent and can be described by a one-site, one-ion blocking scheme. 19 monovalent organic cations blocks primarily from the trans side of the membrane (the side defined as zero voltage), and much more weakly, if at all, from the cis side (to which SR vesicles are added). These blockers all appear to interact with a site located at 63% (average value) of the electric potential drop measured from the trans side. Furthermore, block by 1,3-bis[tris(hydroxymethyl)-methylamino] propane (BTP) shows that the presence of a blocking ion increases the duration of the apparent open state, as expected for a scheme in which the blocking site can be reached only when the channel is open. The results lead to a picture of the channel containing a wide (at least 50 A2) nonselective trans entry in series with a narrow (20 A2) constriction.     

Stanmarkia,a new genus of Melastomataceae from the volcanic highlands of western Guatemala and adjacent Mexico

A new genus,Stanmarkia, is described from the volcanic highlands of western Guatemala and adjacent Chiapas, Mexico to includeS. medialis, comb. nov. andS. spectabilis, sp. nov. Among the Melastomataceae,Stanmarkia is distinguished by its 5-merous flowers, subequal stamens that lack prominently developed appendages or prolonged connectives, terete hypanthia, and clavate-cuneate seeds that have an enlarged excurrent arilloid raphe and a tuberculate seed coat. A gametic chromosome count ofn=40 is reported here forS. spectabilis. This count, which is clearly of polyploid origin, represents a new definitive number for the family. An emphasis on seed characters suggests placement ofStanmarkia in the Sonerileae which is here interpreted in a broad sense to include all genera previously assigned to the New World Bertolonieae and the Old World Oxysporeae. A consideration of other characters such as configuration of the ovary apex, capsule morphology, and androecial details also provides evidence for placement ofStanmarkia in the Merianieae.     

Transport of free polymannose-type oligosaccharides from the endoplasmic reticulum into the cytosol is inhibited by mannosides and requires a thapsigargin-sensitive calcium store

The transport of free polymannose-type oligosaccharides from the lumen of the endoplasmic reticulum into the cytosol has been recently demonstrated (Moore,S.E.H., et al., 1995, EMBO J., 14, 6034-6042), but at present little is known of the characteristics of this process. Here, it is shown that inhibition of the transport of endogenously synthesized metabolically radiolabeled free oligosaccharides out of the endoplasmic reticulum into the cytosol of permeabilized HepG2 cells occurs when assays are conducted in the presence of mannose (IC50, 4.9 mM), or its derivatives modified at the first carbon (C1) of the sugar ring; alpha-methyl mannoside (IC50, 2.0 mM), mannoheptulose (IC50, 1.6 mM), and alpha-benzyl mannoside (IC50, 0.8 mM), whereas other monosaccharides (50 mM), differing from mannose at position; C2 (glucose), C3 (altrose), C4 (talose), C5 (l-rhamnose), and C6 (mannoheptose), have little effect. N-Acetylglucosamine does not inhibit oligosaccharide transport and, furthermore, although mannobioses and a mannotriose inhibit free oligosaccharide transport, di-N-acetylchitobiose is without effect. It is also shown that if the transport assay buffer is either depleted of calcium ions, or supplemented with the Ca2+/Mg2+ATPase inhibitor, thapsigargin, or with calcium ionophores, free oligosaccharide transport out of the endoplasmic reticulum is inhibited. These results demonstrate that the terminal nonreducing mannosyl residues of free polymannose-type oligosaccharides and not their N-acetylglucosamine-containing reducing termini, play an important role in the interaction of the free oligosaccharide with the transport machinery, and that this transport process requires the presence of calcium sequestered in the lumen of the endoplasmic reticulum.      

Adenosine 3',5'-monophosphate-dependent phosphorylation of a 6000 and a 22,000 dalton protein from cardiac sarcoplasmic reticulum

In canine cardiac sarcoplasmic reticulum, adenosine 3',5'-monophosphate (cyclic AMP)-dependent protein kinase specifically phosphorylates two proteins, as seen by sodium dodecyl sulfate-slab gel electrophoresis and autoradiography. One protein has a molecular weight ranging between 22,000 and 24,000 daltons and has previously been identified and named phospholamban (Tada, M., Kirchberger, M.A. and Katz, A.M. (1975) J. Biol. Chem. 250, 2640-2647). The other protein that the 32P label incorporates into has a molecular weight of approximately 6000. Like the 22,000 dalton protein, the 6000 dalton protein has characteristics of phosphoester bonding. The time-dependent course of phosphorylation shows that initially the 32P label is incorporated more rapidly into the 22,000 dalton protein than the 6000 dalton protein, with both proteins reaching a steady-state level of phosphorylation after 10 min of incubation. When both protein kinase and cyclic AMP are eliminated from the incubation medium, both the 22,000 and the 6000 dalton protein are still phosphorylated, but only to about a quarter of the activity found when cyclic AMP and protein kinases are included in the incubation mixture. The addition of phosphodiesterase completely eliminates the phosphorylation of both proteins. Treating the microsomes with trypsin prevents subsequent phosphorylation of either protein. Phosphorylating the microsomes first, then treating with trypsin, renders both the 22,000 and the 6000 dalton proteins resistant to even prolonged trypsin attack. Unphosphorylated, both proteins are solubilized by a very low concentration of deoxycholate. After phosphorylation the proteins cannot be solubilized by deoxycholate. Phosphorylation appears to alter greatly the physical properties of these proteins. Control experiments exclude the possibility that a lipid is being phosphorylated. After phosphorylation the phosphorylated 22,000 dalton protein is separated from the 6000 dalton protein by proteolipid extraction. After first treating the microsomes with methanol, the 22,000 dalton protein is then soluble in acidified chloroform/methanol, while the 6000 dalton protein remains insoluble. The finding that both proteins have much different biochemical properties when phosphorylated than when not, may be relevant in how they regulate calcium transport in the sarcoplasmic reticulum.     

On the balance between niche and neutral processes as drivers of community structure along a successional gradient: insights from alpine and sub-alpine meadow communities

BACKGROUND AND AIMS: Neutral theory predicts that the diversity and relative abundance of species in ecological communities do not depend on their specific traits. This prediction remains controversial, as many studies suggest that variations in the niches of species determine the structure of communities. The aim of this study was to test empirically the relative importance of niche and neutral processes as drivers of species abundance within plant communities along a successional gradient. METHODS: Information on the abundance (density and frequency) and traits (aboveground individual biomass and seed mass) of > 90 species was collected in alpine and sub-alpine meadows of the Tibet Plateau (China). A successional gradient (1, 3, 15 and 30 years after abandonment) was established in a sub-alpine meadow. The relationships between species traits and their abundance were evaluated using regression models. KEY RESULTS: Seed mass was negatively related to both species density (r = -0.6270, P < 0.001) and frequency (r = -0.5335, P = 0.005) in the 1-year meadow. Such relationships disappeared along the successional gradient evaluated (P > 0.07 in the 3-, 15- and 30-year meadows). Data gathered in all sites showed a significant negative relationship between the average individual biomass of a given species and its density within the community (r < -0.30, P < 0.025 in all cases). CONCLUSIONS: The results show that seed mass was a key driver of species abundance in early successional communities, and that niche forces may become more important as succession progresses. They also indicate that predictions from neutral theory, in its current form, do not hold for the meadow communities studied.     

Inhibition of calcium-dependent ATPase from sarcoplasmic reticulum by a new class of indolizidine alkaloids, pumiliotoxins A, B, and 251D

Pumiliotoxins (PTX) A, B, and 251D, members of a new class of indolizidine alkaloids isolated from the skin of poison frogs of the family Dendrobatidae, inhibit Ca2+-ATPase activity in sarcoplasmic reticulum vesicles from frog and rat hind-limb muscles. PTX-B and PTX-A appear to be relatively specific inhibitors of Ca2+-ATPase; PTX-A is much less potent than PTX-B. PTX-251D is a potent inhibitor of Ca2+-ATPase, and was also found to inhibit Na+, K+, and Mg2+-ATPases in rat brain synaptosomes. Caffeine and verapamil, two drugs known to affect calcium translocation, are very weak inhibitors of the Ca2+-ATPase. The Ki values for inhibition of the Ca2+-ATPase of rat and frog sarcoplasmic reticulum by PTX-B were comparable and ranged between 22 and 36 microM. Inhibition of calcium-dependent ATPase in sarcoplasmic reticulum by pumiliotoxin-B is noncompetitive with calcium and is not readily reversible. Based on structure-activity profiles, it is concluded that inhibition of Ca2+-ATPase by the indolizidine alkaloids is responsible for the alkaloid-elicited prolongation of twitch in intact muscle.     

Yeast pyruvate decarboxylase tetramers can dissociate into dimers along two interfaces. Hybrids of low-activity D28A (or D28N) and E477Q variants, with substitution of adjacent active center acidic groups from different subunits, display restored activity

The tetrameric enzyme yeast pyruvate decarboxylase (YPDC) has been known to dissociate into dimers at elevated pH values. However, the interface along which the dissociation occurs, as well as the fundamental kinetic properties of the resulting dimers, remains unknown. The active sites of YPDC are comprised of amino acid residues from two subunits, a property which we utilize to address the issue as to which dimer interface is cleaved under different conditions of dissociation. Hydroxide-induced dissociation of the active site D28A (or D28N) and E477Q variants, each at least 100 times less reactive than wild-type YPDC, followed by reassociation of D28A (or D28N) and E477Q variants led to a remarkable 35-50-fold increase in activity. This result is possible only if the hydroxide-induced dissociation results in a cleavage along the interface between two subunits so that residues D28 and E477 are now separated. Upon reassociation, one of the two active sites of the hybrid dimer will have both residues substituted, whereas the second one will be of the wild-type phenotype. In contrast to the hydroxide-induced dimers, the urea-induced dissociation recently proposed results in dissociation along dimer-dimer interfaces, without separating the active sites, and therefore, on reassociation, these dimers do not regain activity. The significance of the results is discussed in light of a recently proposed alternating sites mechanism for YPDC. A preparative ion-exchange method is reported for the separation and purification of hybrid enzymes.