豆科根瘤拟菌体周膜来源和扩增方式的多样性

豆科植物根瘤内的共生拟菌体需有周膜包围。田菁［Ｓｅｓｂａｎｉａ ｃａｎｎａｂｉｎａ （Ｒｅｔｚ．） Ｐｅｒｓ．］、大豆［Ｇｌｙｃｉｎｅ ｍ ａｘ （Ｌ．） Ｍｅｒｒ．］和豌豆（Ｐｉｓｕｍ ｓａｔｉｖｕｍ Ｌ．）的根瘤拟菌体周膜内有多个拟菌体。在周膜形成和周膜内的拟菌体由１ 个到多个的根瘤发育过程中,要有大量的膜来源和周膜的扩增才能适应这种生理变化。同一种根瘤内周膜扩增有周膜间的镶嵌融合,周膜与内质网及其小泡、小液泡膜融合等多种方式。细胞化学表明：周膜、质膜和内质网膜具有相同型的ＡＴＰ酶,周膜是一种嵌合膜,与质膜、内质网膜和液泡膜有共性。最后对周膜来源和扩增方式多样性的生理意义进行了讨论     

Iron Uptake by Symbiosomes from Soybean Root Nodules

To identify possible iron sources for bacteroids in planta, soybean (Glycine max L. Merr.) symbiosomes (consisting of the bacteroid-containing peribacteroid space enclosed by the peribacteroid membrane [PBM]) and bacteroids were assayed for the ability to transport iron supplied as various ferric [Fe(III)]-chelates. Iron presented as a number of Fe(III)-chelates was transported at much higher rates across the PBM than across the bacteroid membranes, suggesting the presence of an iron storage pool in the peribacteroid space. Pulse-chase experiments confirmed the presence of such an iron storage pool. Because the PBM is derived from the plant plasma membrane, we reasoned that it may possess a ferric-chelate reductase activity similar to that present in plant plasma membrane. We detected ferric-chelate reductase activity associated with the PBM and suggest that reduction of Fe(III) to ferrous [Fe(II)] plays a role in the movement of iron into soybean symbiosomes.     

Ultrastructural morphology suggesting a new hypothesis for development of microsporidians seen in Loma salmonae infecting the gills of rainbow and brook trout

In two variants of Loma salmonae that have specificity for rainbow trout Oncorhynchus mykiss (OA variant) and specificity to brook trout Salvelinus fontinalis (SV variant), the parasitophorous vacuole forms at the onset of sporogony. In the OA variant the merogonial stage is bound by a single plasma membrane in direct contact with host cytoplasm. The parasitophorous vacuole formation is initiated by the host cell surrounding the merogonial stages with endoplasmic reticulum (ER) as occurs in autophagy. Of the two host ER membranes surrounding the parasite, one remains in close association with the plasma membrane of the meront, while the other forms the limiting membrane of the vacuole. The sporogonial stage is bounded by two closely apposed membranes, giving the appearance of a thick electron dense plasmalemma. The observations from this study support the novel hypothesis that this microsporidian uses the intracellular process of autophagy to aid formation of a parasitophorous vacuole. The morphology of the SV‐variant is consistent with that of the OA‐variant suggesting that it uses the same mechanism for development.     

Legionella pneumophila Promotes Functional Interactions between Plasma Membrane Syntaxins and Sec22b

Biogenesis of a specialized organelle that supports intracellular replication of Legionella pneumophila involves the fusion of secretory vesicles exiting the endoplasmic reticulum (ER) with phagosomes containing this bacterial pathogen. Here, we investigated host plasma membrane SNARE proteins to determine whether they play a role in trafficking of vacuoles containing L. pneumophila. Depletion of plasma membrane syntaxins by RNA interference resulted in delayed acquisition of the resident ER protein calnexin and enhanced retention of Rab1 on phagosomes containing virulent L. pneumophila, suggesting that these SNARE proteins are involved in vacuole biogenesis. Plasma membrane‐localized SNARE proteins syntaxin 2, syntaxin 3, syntaxin 4 and SNAP23 localized to vacuoles containing L. pneumophila. The ER‐localized SNARE protein Sec22b was found to interact with plasma membrane SNAREs on vacuoles containing virulent L. pneumophila, but not on vacuoles containing avirulent mutants of L. pneumophila. The addition of α‐SNAP and N‐ethylmaleimide‐sensitive factor (NSF) to the plasma membrane SNARE complexes formed by virulent L. pneumophila resulted in the dissociation of Sec22b, indicating functional pairing between these SNAREs. Thus, L. pneumophila stimulates the non‐canonical pairing of plasma membrane t‐SNAREs with the v‐SNARE Sec22b to promote fusion of the phagosome with ER‐derived vesicles. The mechanism by which L. pneumophila promotes pairing of plasma membrane syntaxins and Sec22b could provide unique insight into how the secretory vesicles could provide an additional membrane reserve subverted during phagosome maturation.     

The Supramolecular Organization of Red Algal Vacuole Membrane Visualized by Freeze-fracture

The supramolecular organization of the vacuole membrane (orof the membranes of mucilage sacs) in 27 species of red algaeis studied in replicas of rapidly frozen and fractured cells.Intramembranous particle complexes composed of four particles('tetrads' with average diameters between 8·5 and 14·5have been observed in the protoplasmic fracture (PF) face butmost clearly and more frequently in the exoplasmic fracture(EF) face of the vacuole membrane of all red algae investigated.The tetrads lie individually within the vacuole membrane orform clusters in several species and are randomly distributed.In the species Ceramium diaphanum var. strictum and Laurenciaobtusa the intramembranous particle complexes ('tetrads') havebeen observed both in the EF and PF faces of the vacuole membrane;the 'membrane tetrads' at least as regards these two speciesseem to span both the outer and inner leaflets of the vacuolemembrane ('transmembrane particles'). The occurrence of particletetrads in the plasma membrane is probably due to exocytosiseither of the Golgi vesicles or of the mucilage sacs. Tetradfrequency in the EF face of the vacuole membranes of the investigatedred algae varies between 2 and 87 µm^-2, while that ofsingle particles varies between 102 and 695 µm^-2. ThePF face of the vacuole membrane is characterized by a higherparticle density than the EF face. The particle densities ofthe PF and EF faces of the plasma membrane for a given speciesare higher than those of the corresponding fracture faces ofthe vacuole membrane. Some members of Bangiophycidae bear smallerprotein particles (diameter between 8·5 and 10·5nm) in comparison with those of Florideophycidae (diameter between10·5 and 14·5 nm). It is suggested, based uponthe particle tetrads lying in depressions of the vacuole membraneand the origin of vacuoles (mucilage sacs) from ER, that theparticle tetrads originate from the ER or the Golgi complex.Since vacuoles (mucilage sacs) in red algae, along with theGolgi complex, are involved in the synthesis and export of cellsurface polysaccharides, it could be assumed that the 'membrane-tetrads'within the vacuole membrane represent a membrane-bound multienzymecomplex, participating in the synthesis of amorphous extracellularmatrix polysaccharides.Copyright 1993, 1999 Academic Press Red algae, freeze-fracture, vacuole membrane, mucilage sacs, membrane tetrads, supramolecular organization     

Quantitative and dynamic assessment of the contribution of the ER to phagosome formation

Phagosomes were traditionally thought to originate from an invagination and scission of the plasma membrane to form a distinct intracellular vacuole. An alternative model implicating the endoplasmic reticulum (ER) as a major component of nascent and maturing phagosomes was recently proposed (Gagnon et al., 2002). To reconcile these seemingly disparate hypotheses, we used a combination of biochemical, fluorescence imaging, and electron microscopy techniques to quantitatively and dynamically assess the contribution of the plasmalemma and of the ER to phagosome formation and maturation. We could not verify even a transient physical continuity between the ER and the plasma membrane, nor were we able to detect a significant contribution of the ER to forming or maturing phagosomes in either macrophages or dendritic cells. Instead, our data indicate that the plasma membrane is the main constituent of nascent and newly formed phagosomes, which are progressively remodeled by fusion with endosomal and eventually lysosomal compartments as phagosomes mature into acidic, degradative organelles.     

The Legionella pneumophila effector DrrA is sufficient to stimulate SNARE-dependent membrane fusion

The intracellular bacterial pathogen Legionella pneumophila subverts host membrane transport pathways to promote fusion of vesicles exiting the endoplasmic reticulum (ER) with the pathogen-containing vacuole. During infection there is noncanonical pairing of the SNARE protein Sec22b on ER-derived vesicles with plasma membrane (PM)-localized syntaxin proteins on the vacuole. We show that the L.?pneumophila Rab1-targeting effector DrrA is sufficient to stimulate this noncanonical SNARE association and promote membrane fusion. DrrA activation of the Rab1 GTPase on PM-derived organelles stimulated the tethering of ER-derived vesicles with the PM-derived organelle, resulting in vesicle fusion through the pairing of Sec22b with the PM syntaxin proteins. Thus, the effector protein DrrA stimulates a host membrane transport pathway that enables ER-derived vesicles to remodel a PM-derived organelle, suggesting that Rab1 activation at the PM is sufficient to promote the recruitment and fusion of ER-derived vesicles.     

Synthesis of an integral protein of the protein-body membrane in Phaseolus vulgaris cotyledons

Protein-body membranes (PBMs) were isolated from cotyledons of Phaseolus vulgaris L. by a procedure involving osmotic shock of purified protein bodies. The purified PBMs have a characteristic density of 1.16 g cm^-3. Treatment of the membranes with increasing concentrations of detergent (Triton X-100) or with a solution at pH 12.0 showed that the membranes contained a characteristic integral protein (IMP) with a relative molecular mass of 25,000. This IMP is not a glycoprotein. When developing cotyledons were labeled with ³H-amino acids for 2–3 h, a radioactive polypeptide with the same mobility on denaturing polyacrylamide gels as IMP was found to be associated with the rough endoplasmic reticulum (ER). During a 24-h chase, a considerable portion of the radioactivity slowly transferred into the IMP associated with more rapidly sedimenting organelles, which sedimented in the same region of the sucrose gradients as the PBMs. Antibodies prepared against purified IMP crossreacted with an ER-associated protein which had the same mobility on denaturing acrylamide gels as authentic IMP. Synthesis of IMP occurred at all stages of cotyledon development examined, but not during seed germination. The results show that a newly synthesized protein of the PBM is associated with the rough ER, just like the soluble matrix proteins, phaseolin (R. Bollini, W. Van der Wilden and M.J. Chrispeels, 1983, J. Cell Biol. 96,999–1007) and phytohemagglutinin (M.J. Chrispeels and R. Bollini, 1982, Plant Physiol. 70, 1425–1428), but that the chase-out from the ER is much slower for IMP than for the matrix proteins.Abbreviations EDTA ethylenediamino-tetraacetic acid - ER endoplasmic reticulum - IMP integral membrane protein - PB protein body - PBM protein-body membrane - PHA phytohemagglutinin - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis     

Fatty Acid Composition of the Peribacteroid Membrane and the ER in Nodules of Glycine max Varies after Infection by Different Strains of the Microsymbiont Bradyrhizobiumjaponicum

The fatty acid composition of ER, Golgi and peribacteroid membrane (PBM) from root nodules formed on Glycine max after infection with different strains of Bradyrhizobium japonicum has been analysed by gas chromatography. In each plant-microsymbiont combination the fatty acid composition (FAC) of the PBM is distinct from ER and Golgi. The similarity between ER and PBM fatty acid composition is significantly stronger than between Golgi and PBM. In addition the fatty acid composition of all membrane systems in nodules is affected by the microsymbiont strain. A comparison of four strains of Bradyrhizobium japonicum grown in agar surface culture and isolated as the symbiotic bacteroids reveals a decrease in oleic acid during bacteroid differentiation.     

Inositol 1,4,5-trisphosphate-sensitive Ca2+ release across nonvacuolar membranes in cauliflower.

Previous studies have indicated that the vacuole represents the major inositol 1,4,5-trisphosphate (InsP3)-mobilizable Ca2+ pool in higher plants. This findings is in contrast to animal cells, in which the endoplasmic reticulum and plasma membrane constitute the dominant InsP3-sensitive membranes. We used membrane vesicles prepared from cauliflower (Brassica oleracae L.) inflorescences that were separated on continuous sucrose gradients to demonstrate that cauliflower possesses at least two distinct membrane populations that are sensitive to InsP3. One of these membrane populations in nonvacuolar in origin and relies upon a Ca(2+)-ATPase to accumulate Ca2+. In addition, we have shown that two polyclonal antibodies, raised against peptides corresponding to the animal type 1 InsP3 receptor, recognize immunologically related proteins in cauliflower, and that the distribution of immunoreactive proteins on a linear sucrose gradient reinforces the notion that cauliflower contains more than one membrane subtype that is sensitive to InsP3. To our knowledge, this is the first report describing an InsP3-sensitive Ca2+ store other than the vacuole in higher plant cells.     

Legionella blocks autophagy by cleaving STX17 (syntaxin 17)

Pathogens subvert host defense systems including autophagy and apoptosis for their survival and proliferation. Legionella pneumophila is a Gram-negative bacterium that grows in alveolar macrophages and causes severe pneumonia. Early during infection Legionella secretes effector proteins that convert the plasma membrane-derived vacuole containing Legionella into an endoplasmic reticulum (ER)-like replicative vacuole. These vacuoles ultimately fuse with the ER, where the pathogen replicates. Recently, we showed that one of the effectors, Lpg1137, is a serine protease that targets the mitochondria-associated ER membrane (MAM) and degrades STX17 (syntaxin 17), a SNARE implicated in macroautophagy/autophagy as well as mitochondria dynamics and membrane trafficking in fed cells. Degradation of STX17 blocks autophagy and BAX-induced apoptosis.     

Plant-specific insertions in the soybean aspartic proteinases, soyAP1 and soyAP2, perform different functions of vacuolar targeting

Most aspartic proteinases (APs) of plant origin are characterized by the presence of plant-specific insertion (PSI) in their primary structure. PSI has been reported to function as signals for both transport of AP molecules from the endoplasmic reticulum (ER) and for their targeting to the vacuole. To determine the functions of the PSIs in soyAP1 and soyAP2 identified in our previous study, we examined their subcellular localization by transient expression of a green fluorescent protein (GFP) fusion protein in the protoplasts of Arabidopsis suspension-cultured cells. Both soyAP1-GFP and soyAP2-GFP were targeted to the vacuole. To confirm the role of the PSI, we prepared PSI-deleted soyAP1 and soyAP2, and investigated their vacuolar targeting by the same method. While the former deletion mutant was always transported to the vacuole, the latter sometimes remained in the ER and was only sometimes transported to the vacuole. These observations indicated that, in the case of soyAP1, the PSI is not involved in vacuolar targeting, also suggesting that the function of the PSI differs depending on its origin.     

Mobilization of Ca2+ by cyclic ADP-ribose from the endoplasmic reticulum of cauliflower florets

The NAD+ metabolite cADP-Rib (cADPR) elevates cytosolic free Ca2+ in plants and thereby plays a central role in signal transduction pathways evoked by the drought and stress hormone abscisic acid. cADPR is known to mobilize Ca2+ from the large vacuole of mature cells. To determine whether additional sites for cADPR-gated Ca2+ release reside in plant cells, microsomes from cauliflower (Brassica oleracea) inflorescences were subfractionated on sucrose density gradients, and the distribution of cADPR-elicited Ca2+ release was monitored. cADPR-gated Ca2+ release was detected in the heavy-density fractions associated with rough endoplasmic reticulum (ER). cADPR-dependent Ca2+ release co-migrated with two ER markers, calnexin and antimycin A-insensitive NADH-cytochrome c reductase activity. To investigate the possibility that contaminating plasma membrane in the ER-rich fractions was responsible for the observed release, plasma membrane vesicles were purified by aqueous two-phase partitioning, everted with Brij-58, and loaded with Ca2+: These vesicles failed to respond to cADPR. Ca2+ release evoked by cADPR at the ER was fully inhibited by ruthenium red and 8-NH2-cADPR, a specific antagonist of cADPR-gated Ca2+ release in animal cells. The presence of a Ca2+ release pathway activated by cADPR at higher plant ER reinforces the notion that, alongside the vacuole, the ER participates in Ca2+ signaling.     

番木瓜乳管结构及木瓜蛋白酶的免疫电镜研究

采用免疫细胞化学方法, 以透射电镜观察, 对番木瓜（Ｃａｒｉｃａ ｐａｐａｙａ Ｌ．）乳管分化及木瓜蛋白酶生成的超微结构环境进行了研究。实验结果表明：１．正在分化的乳管细胞内质网分泌旺盛, 线粒体和聚核糖体非常丰富; 之后细胞器逐渐解体, 内质网断裂、膨胀, 细胞壁多处穿孔; 经过内膜系统的重新组合, 成熟乳管被小泡充满, 小泡内有无定形物质凝聚, 已经没有任何细胞器残留, 但原生质膜一直存在。２．经过纯化的兔抗ｃｈｙｍ ｏｐａｐａｉｎ ＩｇＧ 为第一抗体, 羊抗兔ＩｇＧ－金复合物（１０ ｎｍ 直径）为间接抗体, 进行适当的免疫标记反应, 发现金颗粒主要在乳管细胞内, 附近的薄壁细胞及导管只是偶尔出现金标, 说明免疫标记的特异性较强, 通过各种对照试验, 证明了非特异性吸附相当微弱。显示木瓜蛋白酶的原初生成部位是正在分化的乳管细胞的内质网, 它暂时贮存于分泌泡, 随乳管的发育和乳汁其它成分一起被“组装”为乳汁小泡而充满成熟乳管     

Glycosylated polypeptides of soybean root endomembranes

Summary Endoplasmic reticulum, Golgi apparatus, plasma membrane and mitochondria vesicles were isolated from the roots of four-day-old dark-grown soybean [Glycine max (L.) Merr. cv. Wells II] seedlings and characterized by marker enzyme analyses. Glycoproteins of enriched membrane fractions were identified by concanavalin A (con A)-peroxidase staining of polypeptides separated by two-dimensional IEF-SDS-PAGE and transferred to nitrocellulose.Con A bound to many polypeptides in each endomembrane-enriched fraction with several glycopolypeptides common to all fractions. The mitochondria-enriched fraction possessed few glycopolypeptides and those appeared to be highly glycosylated contaminants of endomembrane origin. Comparison of the endomembrane con A-binding patterns revealed changes in relative stain intensity, molecular weight and isoelectric point of several membrane glycopolypeptides suggestive of processing reactions of the endomembrane complex.Abbreviations con A concanavalin A - PM plasma membrane - GA Golgi apparatus - ER endoplasmic reticulum     

The function of the aleurone layer during galactomannan mobilisation in germinating seeds of fenugreek (Trigonella foenum-graecum L.), crimson clover (Trifolium incarnatum L.) and lucerne (Medicago sativa L.): A correlative biochemical and ultrastructural study

Summary The reserve endosperm galactomannans of fenugreek (Trigonella foenum-graecum L.), crimson clover (Trifolium incarnatum L.) and lucerne (Medicago sativa L.) are broken down to free galactose and mannose in dry-isolated endosperms (devoid of embryo) incubated under germination conditions. Breakdown is prevented by inhibition of protein synthesis or of oxidative phosphorylation in the aleurone layer. Resting aleurone cells contain inter alia a large number of ribosomes more or less regularly distributed in the ground plasma. At the onset of germination, before galactomannan breakdown begins, polysomes are formed and seem, at least partly, to become associated with vesicles and flat cisternae both probably newly formed and derived from ER. Concurrently with galactomannan breakdown in the reserve cells, wall corrosion occurs in the aleurone layer, the contents of the aleurone grains disappear and the rough vesicles and cisternae proliferate. Later a large central vacuole is formed which incorporates smaller vacuoles emerging from the cytoplasm, and at the same time the rough ER vesicles and cisternae become highly distended.It is concluded that the cells of the aleurone layer are responsible for the synthesis and secretion into the storage cells of the enzymes necessary for galactomannan degradation. The physiology of galactomannan breakdown is compared and contrasted with that of starch mobilisation in the endosperm of germinating cereal grains.This is part three in a series of papers dealing with galactomannan metabolism. Part two: Planta (Berl.) 100, 131–142 (1971).     

Intracellular trafficking pathway of yeast long-chain base kinase Lcb4, from its synthesis to its degradation

Sphingoid long-chain base 1-phosphates act as bioactive lipid molecules in eukaryotic cells. In budding yeast, long-chain base 1-phosphates are synthesized mainly by the long-chain base kinase Lcb4. We recently reported that, soon after yeast cells enter into the stationary phase, Lcb4 is rapidly degraded by being delivered to the vacuole in a palmitoylation- and phosphorylation-dependent manner. In this study, we investigated the complete trafficking pathway of Lcb4, from its synthesis to its degradation. After membrane anchoring by palmitoylation at the Golgi apparatus, Lcb4 is delivered to the plasma membrane (PM) through the late Sec pathway and then to the endoplasmic reticulum (ER). The yeast ER consists of a cortical network juxtaposed to the PM (cortical ER) with tubular connections to the nuclear envelope (nuclear ER). Remarkably, the localization of Lcb4 is restricted to the cortical ER. As the cells reach the stationary phase, G(1) cell cycle arrest initiates Lcb4 degradation and its delivery to the vacuole via the Golgi apparatus. The protein transport pathway from the PM to the ER found in this study has not been previously reported. We speculate that this novel pathway is mediated by the PM-ER contact.     

ENDOCYTOSIS OF LANTHANUM NITRATE IN THE ORGANIC ACID-SECRETING TRICHOMES OF CHICKPEA (CICER ARIETINUM L.)

The organic acid-secreting trichomes of chickpea (Cicer arietinum L.) were exposed to 2.5 mm lanthanum nitrate for 24 hr, and this concentration did not inhibit trichome secretion compared with that of controls. We subsequently used this nontoxic concentration of lanthanum to examine endocytosis. In the stalk cells of these secretory trichomes, exogenously applied lanthanum nitrate was present in cell walls and vacuoles, as well as within both invaginations in the plasma membrane and vesicles in the peripheral cytoplasm between the plasma membrane and the tonoplast. In the head cells, lanthanum nitrate was present in cell walls and in vesicles that form a layer in the cytoplasm around the edge of the head cells, but was not present in vacuoles. We propose that fluid phase endocytosis targeted to the vacuole takes place in the stalk cells and that endocytosis occurs in the head cells to remove excess plasma membrane after the fusion of secretory vesicles with the plasma membrane. This is the first demonstration of endocytosis in secretory trichomes.     

ER remodeling by the large GTPase atlastin promotes vacuolar growth of Legionella pneumophila

The pathogenic bacterium Legionella pneumophila replicates in host cells within a distinct ER‐associated compartment termed the Legionella‐containing vacuole (LCV). How the dynamic ER network contributes to pathogen proliferation within the nascent LCV remains elusive. A proteomic analysis of purified LCVs identified the ER tubule‐resident large GTPase atlastin3 (Atl3, yeast Sey1p) and the reticulon protein Rtn4 as conserved LCV host components. Here, we report that Sey1/Atl3 and Rtn4 localize to early LCVs and are critical for pathogen vacuole formation. Sey1 overproduction promotes intracellular growth of L. pneumophila, whereas a catalytically inactive, dominant‐negative GTPase mutant protein, or Atl3 depletion, restricts pathogen replication and impairs LCV maturation. Sey1 is not required for initial recruitment of ER to PtdIns(4)P‐positive LCVs but for subsequent pathogen vacuole expansion. GTP (but not GDP) catalyzes the Sey1‐dependent aggregation of purified, ER‐positive LCVs in vitro. Thus, Sey1/Atl3‐dependent ER remodeling contributes to LCV maturation and intracellular replication of L. pneumophila.