Retrograde transport from the Golgi complex to the ER of both Shiga toxin and the nontoxic Shiga B-fragment is regulated by butyric acid and cAMP

Endocytosed Shiga toxin is transported from the Golgi complex to the endoplasmic reticulum in butyric acid-treated A431 cells. We here examine the extent of this retrograde transport and its regulation. The short B fragment of Shiga toxin is sufficient for transport to the ER. The B fragment of cholera toxin, which also binds to glycolipids, is transported to all the Golgi cisterns, but cannot be localized in the ER even after butyric acid treatment. Under all conditions the toxic protein ricin was found predominantly in the trans-Golgi network. There is no transport of endocytosed fluid to the Golgi apparatus or to the ER even after butyric acid treatment and in the presence of Shiga toxin, indicating that transport to the ER, through the trans-Golgi network and the cisterns of the Golgi apparatus, involves several sorting stations. Since Shiga toxin receptors (Gb3) in butyric acid- treated A431 cells seem to have a ceramide moiety with longer fatty acids than in untreated cells, the possibility exists that fatty acid composition of the receptor is important for sorting to the ER. Both retrograde transport and intoxication with Shiga toxin can also be induced by cAMP, supporting the idea that retrograde transport from the Golgi to the ER is required for intoxication. The data suggest that transport to the ER in cells in situ may depend on fatty acid composition and is regulated by physiological signals.     

Marked changes in volume of mesophyll protoplasts of pea (Pisum sativum) on exposure to growth hormones

The present study reports quick and significant changes induced by plant hormones in the volume of mesophyll protoplasts of pea (Pisum sativum). Four plant hormones: gibberellic acid (GA3), indole 3-acetic acid (IAA), abscisic acid (ABA)(+/-) and methyl jasmonate (MJ), caused marked changes in the volume of mesophyll protoplasts. GA3 and IAA increased the volume of the protoplasts (up to 90%) whereas the ABA and MJ decreased (by about 40%) the volume. Aquaporins or water channels appear to play an important role in swelling/shrinkage of the protoplasts as indicated by the suppression of volume changes by HgCl2 and reversal by mercaptoethanol. The possible role of secondary messengers in volume changes induced by GA3 was investigated by using selected pharmacological reagents. The GA3 induced swelling was restricted by GDP-beta-S (G-protein antagonist), U73122 (phospholipase C inhibitor), and TFP (calmodulin antagonist), but was not affected by 1-butanol (phospholipase D inhibitor), GTP-gamma-S (G-protein agonist), or verapamil (calcium channel blocker). The results suggest that the mesophyll protoplasts can be a simple and useful system for further studies on volume changes in plant tissues.     

The fine structural effect of sialectomy on the taste bud cells in the rat.

Taste buds in the rat and other mammals share a secretory activity with their transduction function as taste receptor. The present work shows the effect of bilateral removal of the main salivary glands on taste bud cells' components related to secretion in the vallate papilla of the rat. In the sialectomized rats remarkable changes were evidence in the dark and intermediate types of taste bud cells, which are known to be the secretory components. Such changes involve hypertrophy of either the protein synthetizing machinery, the smooth endoplasmic reticulum or the Golgi complex. Lucent and coated vesicles associated to Golgi cisternae increased in number but the amount of dense-core vesicles (secretory vesicles) at the apical cytoplasm of cells decreased. Images of exocytosis of secretory products were observed. The hypertrophy of Golgi complex components was clearly detected with the OsO4 impregnation method for light and electron microscopy. Alteration in the acid phosphatase activity of taste bud cells was not observed in the sialectomized rats. These findings suggest that sialectomy stimulates the entire secretory cycle of dark and intermediate taste bud cells. The light taste bud cells, which are not engaged in secretion, are hardly affected by the treatment. Although taste buds in mammals are neuro-dependent structures, present evidence indicates that they are also sensitive to non-neural influences.     

Poliovirus infection and expression of the poliovirus protein 2B provoke the disassembly of the Golgi complex, the organelle target for the antipoliovirus drug Ro-090179.

Infection of Vero cells with poliovirus results in complete disassembly of the Golgi complex. Milestones of the process of disassembly are the release to the cytosol of the beta-COP bound to Golgi membranes, the disruption of the cis-Golgi network into fragments scattered throughout the cytoplasm, and the disassembly of the stacked cisternae by a process mediated by long tubular structures. Transient expression of the viral protein 2B in COS-7 cells also causes the disassembly of the Golgi complex by a process preceded by the accumulation of the protein in the Golgi area. Vero cells infected for 3 h show no recognizable Golgi complexes at the ultrastructural level and display an enormously swollen endoplasmic reticulum (ER) with extensive areas of its surface heavily coated. Ro-090179 (Ro), a flavonoid isolated from the herb Agastache rugosa, provokes the specific swelling and disruption of the Golgi complex and strongly inhibits poliovirus infection. Ro provokes the swelling and the disruption of the stacked cisternae and trans-Golgi elements without affecting the cis-most Golgi cisternae much. Moreover, Ro inhibits the fusion of the Golgi complex with the ER in cells treated with brefeldin A and provokes the accumulation of the intermediate compartment membrane protein p58 into ERD2-positive Golgi elements but has no effect on the anterograde transport involved in protein secretion. Our results indicate that the secretory pathway and specifically the Golgi complex are preferential targets of poliovirus.     

Structural organization of ultrarapidly frozen barley aleurone cells actively involved in protein secretion

The ultrastructural organization of actively secreting barley (Hordeum vulgare L. cv. Himalaya) aleurone cells was examined using ultrarapid-freezing (<-10 000°C s^-1) followed by freeze-fracture and freeze-substitution. Our analysis indicates that much of the evidence supporting a direct pathway from the endoplasmic reticulum (ER) to the plasma membrane (i.e. bypassing the Golgi apparatus) for the secretion of -amylase (EC 3.2.1.1) may not be valid. Cryofixed ER cisternae show no sign of vesiculation during active -amylase secretion in gibberellic acid (GA₃)-treated cells. At the same time, Golgi complexes are abundant and numerous small vesicles are associated with the edges of the cisternae. Vesicles appear to be involved in the delivery of secretory products to the plasma membrane since depressions containing excess membrane material appear there. Treatment with GA₃ also induces changes in the composition of Golgi membranes; most notably, the density of intramembrane particles increases from 2700 m^-2 to 3800 m^-2 because of an increase of particles in the 3–8.5-nm size range. A slight decrease in 9–11-nm particles also occurs. These changes in membrane structure appear to occur as the Golgi complex becomes committed to the processing and packaging of secretory proteins. We suggest that secretory proteins in this tissue are synthesized in the abundant rough ER, packaged in the Golgi apparatus, and transported to the plasma membrane via Golgi-derived secretory vesicles. Mobilization of reserves is also accompanied by dynamic membrane events. Our micrographs show that the surface monolayer of the lipid bodies fuses with the outer leaflet of the bilayer of protein-body membranes during the mobilization of lipid reserves. Following the breakdown of the protein reserves, the protein bodies assume a variety of configurations.Abbreviations ER endoplasmic reticulum - GA₃ gibberellic acid - P protoplasmic - E exoplasmic     

MAIGO2 is involved in gibberellic acid, sugar, and heat shock responses during germination and seedling development in Arabidopsis

Our previous study has shown that MAIGO2 (MAG2) is a subunit of the Golgi/endoplasmic reticulum (ER) multi-subunit tethering complex, and is required for tolerance to general osmotic stresses and abscisic acid and response to ER stress during seed germination and early growth. MAG2 is crucial for multi-environmental stress responses. To verify this hypothesis, the response of mag2 mutants to gibberellic acid (GA), sugar, and heat shock was described in this study. The mag2 mutants showed defects during seed germination and early seedling development under treatments with the GA biosynthesis inhibitor paclobutrazol, sucrose, and glucose. MAG2 is also essential for basal thermotolerance. However, the MAG2 homolog (MAG2L) is not necessary for these responses. MAG2 is an important regulator in the response to multi-environmental stimuli, supposedly through its roles in Golgi/ER retrograde trafficking and ER stress response.     

Membrane traffic from the endoplasmic reticulum to the Golgi apparatus is disturbed by an inhibitor of the Ca2+-ATPase in the ER

Summary An electron microscopic study of cress (Lepidium sativum L.) roots treated with cyclopiazonic acid (CPA), an inhibitor of the Ca²⁺-ATPase in the endoplasmic reticulum (ER) has been carried out. Drastic changes in the endomembrane system of the secretory root cap cells were observed. After treatment with CPA dense spherical or elliptoidal aggregates of ER (diameter 2–4 m) were formed in addition to the randomly distributed ER cisternae characteristic for control cells. The formation of ER aggregates indicates that in spite of an inhibition of the Ca²⁺ -ATPase in the ER by CPA, membrane synthesis in the ER continued. The ER aggregates are interpreted as a reservoir of ER membrane material newly synthesized during the 2 h CPA-treatment. Hypertrophied Golgi cisternae and secretory vesicles, which are characteristic for secretory cells under control conditions, were completely absent. Additionally the shape of the Golgi stacks was flat and the diameter of the cisternae was shortened by about one third. These phenomena are indicative of an inactive state of the Golgi apparatus. The cellular organization of both other cell types of the root cap, meristematic cells and statocytes, was not visibly affected by CPA, both having a relatively low secretory activity. The formation of ER aggregates as well as the reduction of Golgi compartments are indications for the existence of a unidirectional transport of membrane material from the ER to the Golgi. It is suggested that the membrane traffic from the ER to the Golgi apparatus is regulated by the cytosolic and/or luminal calcium concentration in secretory cells of the root cap.Abbreviations CPA cyclopiazonic acid - ER endoplasmic reticulum     

Reevaluation of the effects of brefeldin A on plant cells using tobacco Bright Yellow 2 cells expressing Golgi-targeted green fluorescent protein and COPI antisera

Brefeldin A (BFA) causes a block in the secretory system of eukaryotic cells by inhibiting vesicle formation at the Golgi apparatus. Although this toxin has been used in many studies, its effects on plant cells are still shrouded in controversy. We have reinvestigated the early responses of plant cells to BFA with novel tools, namely, tobacco Bright Yellow 2 (BY-2) suspension-cultured cells expressing an in vivo green fluorescent protein-Golgi marker, electron microscopy of high-pressure frozen/freeze-substituted cells, and antisera against Atgamma-COP, a component of COPI coats, and AtArf1, the GTPase necessary for COPI coat assembly. The first effect of 10 microg/mL BFA on BY-2 cells was to induce in <5 min the complete loss of vesicle-forming Atgamma-COP from Golgi cisternae. During the subsequent 15 to 20 min, this block in Golgi-based vesicle formation led to a series of sequential changes in Golgi architecture, the loss of distinct Golgi stacks, and the formation of an endoplasmic reticulum (ER)-Golgi hybrid compartment with stacked domains. These secondary effects appear to depend in part on stabilizing intercisternal filaments and include the continued maturation of cis- and medial cisternae into trans-Golgi cisternae, as predicted by the cisternal progression model, the shedding of trans-Golgi network cisternae, the fusion of individual Golgi cisternae with the ER, and the formation of large ER-Golgi hybrid stacks. Prolonged exposure of the BY-2 cells to BFA led to the transformation of the ER-Golgi hybrid compartment into a sponge-like structure that does not resemble normal ER. Thus, although the initial effects of BFA on plant cells are the same as those described for mammalian cells, the secondary and tertiary effects have drastically different morphological manifestations. These results indicate that, despite a number of similarities in the trafficking machinery with other eukaryotes, there are fundamental differences in the functional architecture and properties of the plant Golgi apparatus that are the cause for the unique responses of the plant secretory pathway to BFA.     

A kinetic analysis of the effects of gibberellic Acid, zeatin, and abscisic Acid on leaf tissue senescence in rumex

Hormones which inhibit senescence in Rumex leaf tissue in the dark include gibberellic acid and the cytokinin zeatin. Abscisic acid accelerates senescence in this tissue. Other workers have proposed that cytokinins, but not gibberellins, interact with abscisic acid in senescing Rumex leaf tissue. The present study reinvestigates the question of interaction using measurements of chlorophyll degradation kinetics as parameters of senescence rate and draws the conclusion that neither zeatin nor gibberellic acid interact with abscisic acid in this system. In support of this conclusion are these results. Zeatin clearly cannot overcome the effects of abscisic acid when hormone solutions are replaced every other day. The kinetics of chlorophyll breakdown for tissue treated with unreplaced saturating zeatin solutions is different from that of tissue exposed to saturating zeatin plus abscisic acid. The observed rates of chlorophyll breakdown for tissue treated with abscisic acid and zeatin agree closely with predicted rates using a multiplicative model for independent action of the two hormones.     

Effects of Ga3 and Ca2+ on barley aleurone protoplasts: a freeze-fracture study

Summary Freeze-fracture electron microscopy was used to study changes in the endomembrane system of barley (Hordeum vulgare L. cv. Himalaya) aleurone protoplasts. Protoplasts were used for this study because their response to calcium and the plant hormone gibberellic acid (Ga₃) can be monitored prior to rapid freezing of cells for electron microscopy. Protoplasts incubated in Ga₃ plus Ca²⁺ secrete elevated levels of a-amylase relative to cells incubated in Ga₃ or Ca²⁺ alone. The endoplasmic reticulum (ER) and Golgi apparatus of protoplasts incubated in Ga₃ plus Ca²⁺ undergo changes that are well correlated with the synthesis and secretion of a-amylase. The ER, which appears as short, single sheets of membrane in Ca²⁺-and Ga₃-treated protoplasts, exists as a series of long fenestrated stacks of membranes following incubation in Ga₃ plus Ca²⁺. The Golgi apparatus is also more highly developed in protoplasts treated with Ga₃ plus Ca²⁺. This organelle is larger and has more vesicles associated with its periphery in protoplasts that actively secrete a-amylase. Evidence that the Golgi apparatus participates in a-amylase secretion is also provided by experiments with the ionophore monensin, which causes pronounced swelling of Golgi cisternae and inhibits the secretion of a-amylase. We interpret these observations as showing that the ER and Golgi apparatus of barley aleurone participate in the intracellular transport and secretion of a-amylase. The plasmalemma (PF face) of barley aleurone protoplasts shows a high density of intramembranous particles (IMPs) which, in general, are evenly distributed. Occasionally, ordered arrays of IMPs are observed, possibly resulting fro m osmotic stress. after 48 hours the plasmalemma of some Ga₃-treated protoplasts show particle-free areas considered to be indications of senescence.abbreviations ER endoplasmic reticulum - Ga₃ gibberellic acid - IEF isoelectric focusing - IMP intramembranous particle - PF protoplasmic fracture - PL plasmalemma     

MAIGO2 is involved in abscisic acid‐mediated response to abiotic stresses and Golgi‐to‐ER retrograde transport

The central role of multisubunit tethering complexes in intracellular trafficking has been established in yeast and mammalian systems. However, little is known about their roles in the stress responses and the early secretory pathway in Arabidopsis. In this study, Maigo2 (MAG2), which is equivalent to the yeast Tip20p and mammalian Rad50‐interacting protein, is found to be required for the responses to salt stress, osmotic stress and abscisic acid in seed germination and vegetative growth, and MAG2‐like (MAG2L) is partially redundant with MAG2 in response to environmental stresses. MAG2 strongly interacts with the central region of ZW10, and both proteins are important as plant endoplasmic reticulum (ER)‐stress regulators. ER morphology and vacuolar protein trafficking are unaffected in the mag2, mag2l and zw10 mutants, and the secretory marker to the apoplast is correctly transported in mag2 plants, which indicate that MAG2 functions as a complex with ZW10, and is potentially involved in Golgi‐to‐ER retrograde trafficking. Therefore, a new role for ER–Golgi membrane trafficking in abiotic‐stress and ER‐stress responses is discovered.     

Glucosylceramide Biosynthesis is Involved in Golgi Morphology and Protein Secretion in Plant Cells

Lipids have an established role as structural components of membranes or as signalling molecules, but their role as molecular actors in protein secretion is less clear. The complex sphingolipid glucosylceramide (GlcCer) is enriched in the plasma membrane and lipid microdomains of plant cells, but compared to animal and yeast cells, little is known about the role of GlcCer in plant physiology. We have investigated the influence of GlcCer biosynthesis by glucosylceramide synthase (GCS) on the efficiency of protein transport through the plant secretory pathway and on the maintenance of normal Golgi structure. We determined that GlcCer is synthesized at the beginning of the plant secretory pathway [mainly endoplasmic reticulum (ER)] and that d ,l ‐threo‐1‐phenyl‐2‐decanoyl amino‐3‐morpholino‐propanol (PDMP) is a potent inhibitor of plant GCS activity in vitro and in vivo. By an in vivo confocal microscopy approach in tobacco leaves infiltrated with PDMP, we showed that the decrease in GlcCer biosynthesis disturbed the transport of soluble and membrane secretory proteins to the cell surface, as these proteins were partly retained intracellularly in the ER and/or Golgi. Electron microscopic observations of Arabidopsis thaliana root cells after high‐pressure freezing and freeze substitution evidenced strong morphological changes in the Golgi bodies, pointing to a link between decreased protein secretion and perturbations of Golgi structure following inhibition of GlcCer biosynthesis in plant cells.     

Selective Accumulation of the Endoplasmic Reticulum–Golgi Intermediate Compartment Induced by the Antitumor Drug KRN5500

KRN5500 is a semisynthetic spicamycin analogue consisting of a seven-carbon amino sugar linked to a C₁₄ unsaturated fatty acid through glycine and to the amino group of adenine. The drug inhibits cell growth potently and has antitumor activity in in vivo models. The mechanism of the antiproliferative effect of KRN5500 remains to be elucidated. We have found that acute exposure of drug-sensitive HT-29 colon adenocarcinoma cells to the drug results initially in swelling of the Golgi apparatus. Continuous exposure to the drug resulted in the emergence of a resistant population of cells characterized by numerous intracellular vacuoles. These KRN5500-resistant tumor cells exhibited increased staining with the Golgi stain NBD C₆–ceramide and the ER–Golgi fluorescent dye BODIPY–brefeldin A, which, unlike the parental drug-sensitive cells, was dispersed throughout the cytoplasm. Marker enzymes associated with the ER (glucose 6-phosphatase) and cis-Golgi (GalNAc transferase) were elevated >2-fold and nearly 4-fold, respectively, in drug-resistant cell lines while the trans-Golgi marker enzyme, galactosyltransferase, was not. The additional findings that the KRN5500-resistant cells have a >2-fold elevation in ERGIC-53, a cis-Golgi marker protein of the ER–Golgi intermediate compartment (ERGIC), as well as increased 58K, a 58-kDa microtubule-binding protein with formiminotransferase cyclodeaminase activity, and tubulin indicate that the cellular secretory pathway is a primary determinant of sensitivity to KRN5500, as resistance to this agent corresponds with accumulation of several components relatable to ER and cis-Golgi function. Further support for this conclusion is provided by studies which demonstrate that KRN5500 alters the distribution of newly synthesized carcinoembryonic antigen within the secretory pathway, including arrest of this N-glycosylated protein in the Golgi of LS-174T colon carcinoma cells.     

Role of plant hormones in plant defence responses

Plant hormones play important roles in regulating developmental processes and signaling networks involved in plant responses to a wide range of biotic and abiotic stresses. Significant progress has been made in identifying the key components and understanding the role of salicylic acid (SA), jasmonates (JA) and ethylene (ET) in plant responses to biotic stresses. Recent studies indicate that other hormones such as abscisic acid (ABA), auxin, gibberellic acid (GA), cytokinin (CK), brassinosteroids (BR) and peptide hormones are also implicated in plant defence signaling pathways but their role in plant defence is less well studied. Here, we review recent advances made in understanding the role of these hormones in modulating plant defence responses against various diseases and pests.     

Phytochrome and hormonal control of expansion and greening of etiolated wheat leaves

Summary Unrolling of etiolated wheat leaf segments is stimulated by short periods of exposure to red light. Both gibberellic acid and kinetin will stimulate unrolling in the dark, whereas abscisic acid (ABA) inhibits the unrolling response to these two hormones and to red light. Exposure to 5 minutes of red light leads to a rapid increase in endogenous gibberellin levels in etiolated wheat leaves, and this increase is followed by a rapid decline. Pre-treatment with ABA inhibits the increase in gibberellin levels in response to red light, but the ihibitory effect of ABA on unrolling cannot be ascribed only to its effect on gibberellin levels. Pre-treatment with red light reduces the lag-phase in chlorophyll development when wheat leaf segments are subsequently exposed to white light; the effect of red light may be replaced by pre-treatment with kinetin, but gibberellic acid is relatively ineffective in this respect.     

Grab a Golgi: Laser Trapping of Golgi Bodies Reveals in vivo Interactions with the Endoplasmic Reticulum

In many vacuolate plant cells, individual Golgi bodies appear to be attached to tubules of the pleiomorphic cortical endoplasmic reticulum (ER) network. Such observations culminated in the controversial mobile secretory unit hypothesis to explain transport of cargo from the ER to Golgi via Golgi attached export sites. This proposes that individual Golgi bodies and an attached‐ER exit machinery move over or with the surface of the ER whilst collecting cargo for secretion. By the application of infrared laser optical traps to individual Golgi bodies within living leaf cells, we show that individual Golgi bodies can be micromanipulated to reveal their association with the ER. Golgi bodies are physically attached to ER tubules and lateral displacement of individual Golgi bodies results in the rapid growth of the attached ER tubule. Remarkably, the ER network can be remodelled in living cells simply by movement of laser trapped Golgi dragging new ER tubules through the cytoplasm and new ER anchor sites can be established. Finally, we show that trapped Golgi ripped off the ER are ‘sticky’ and can be docked on to and attached to ER tubules, which will again show rapid growth whilst pulled by moving Golgi.     

PERK inhibition attenuates the abnormalities of the secretory pathway and the increased apoptotic rate induced by SIL1 knockdown in HeLa cells

Loss-of-function mutations in the SIL1 gene are linked to Marinesco-Sjögren syndrome (MSS), a rare multisystem disease of infancy characterized by cerebellar and skeletal muscle degeneration. SIL1 is a ubiquitous adenine nucleotide exchange factor for the endoplasmic reticulum (ER) chaperone BiP. The complexity of mechanisms by which loss of SIL1 causes MSS is not yet fully understood. We used HeLa cells to test the hypothesis that impaired protein folding in the ER due to loss of SIL1 could affect secretory trafficking, impairing the transport of cargoes essential for the function of MSS vulnerable cells. Immunofluorescence and ultrastructural analysis of SIL1-knocked-down cells detected ER chaperone aggregation, enlargement of the Golgi complex, increased autophagic vacuoles, and mitochondrial swelling. SIL1-interefered cells also had delayed ER-to-plasma membrane transport with retention of Na⁺/K⁺-ATPase and procollagen-I in the ER and Golgi, and increased apoptosis. The PERK pathway of the unfolded protein response was activated in SIL1-interfered cells, and the PERK inhibitor GSK2606414 attenuated the morphological and functional alterations of the secretory pathway, and significantly reduced cell death. These results indicate that loss of SIL1 is associated with alterations of secretory transport, and suggest that inhibiting PERK signalling may alleviate the cellular pathology of SIL1-related MSS.     

Effect of brefeldin A on the structure of the Golgi apparatus and on the synthesis and secretion of proteins and polysaccharides in sycamore maple (Acer pseudoplatanus) suspension-cultured cells.

Brefeldin A (BFA), a specific inhibitor of Golgi-mediated secretion in animal cells, has been used to study the organization of the secretory pathway and the function of the Golgi apparatus in plant cells. To this end, we have employed a combination of electron microscopical, immunocytochemical, and biochemical techniques to investigate the effects of this drug on the architecture of the Golgi apparatus as well as on the secretion of proteins and complex cell wall polysaccharides in sycamore maple (Acer pseudoplatanus) suspension-cultured cells. We have used 2.5 and 7.5 micrograms/mL of BFA, which is comparable to the 1 to 10 micrograms/mL used in experiments with animal cells. Electron micrographs of high-pressure frozen and freeze-substituted cells show that although BFA causes swelling of the endoplasmic reticulum cisternae, unlike in animal cells, it does not induce the disassembly of sycamore maple Golgi stacks. Instead, BFA induces the formation of large clusters of Golgi stacks, an increase in the number of trans-like Golgi cisternae, and the accumulation in the cytoplasm of very dense vesicles that appear to be derived from trans Golgi cisternae. These vesicles contain large amounts of xyloglucan (XG), the major hemicellulosic cell wall polysaccharide, as shown by immunocytochemical labeling with anti-XG antibodies. All of these structural changes disappear within 120 min after removal of the drug. In vivo labeling experiments using [3H]leucine demonstrate that protein secretion into the culture medium, but not protein synthesis, is inhibited by approximately 80% in the presence of BFA. In contrast, the incorporation of [3H]fucose into N-linked glycoproteins, which occurs in trans-Golgi cisternae, appears to be affected to a greater extent than the incorporation of [3H]xylose, which has been localized to medial Golgi cisternae. BFA also affects secretion of complex polysaccharides as evidenced by the approximate 50% drop in incorporation of [3H]xylose and [3H]fucose into cell wall hemicelluloses. Taken together, these findings suggest that at concentrations of 2.5 to 7.5 mu g/mL BFA causes the following major changes in the secretory pathway of sycamore maple cells: (a) it inhibits the transport of secretory proteins to the cell surface by about 80% and of hemicelluloses by about 50%; (b) it changes the patterns of glycosylation of N-linked glycoproteins and hemicelluloses; (c) it reduces traffic between trans Golgi cisternae and secretory vesicles; (d) it produces a major block in the transport of XG-containing, dense secretory vesicles to the cell surface; and (e) it induces the formation of large aggregates of Golgi apparatus of plant and animal cels share many functional and structural characteristics, the plant Golgi apparatus possesses properties that make its response to BFA unique.     

Arginine/lysine residues in the cytoplasmic tail promote ER export of plant glycosylation enzymes

Plant N -glycan processing enzymes are arranged along the early secretory pathway, forming an assembly line to facilitate the step-by-step modification of oligosaccharides on glycoproteins. Thus, these enzymes provide excellent tools to study signals and mechanisms, promoting their localization and retention in the endoplasmic reticulum (ER) and Golgi apparatus. Herein, we focused on a detailed investigation of amino acid sequence motifs present in their short cytoplasmic tails in respect to ER export. Using site-directed mutagenesis, we determined that single arginine/lysine residues within the cytoplasmic tail are sufficient to promote rapid Golgi targeting of Golgi-resident N -acetylglucosaminyltransferase I (GnTI) and α-mannosidase II (GMII). Furthermore, we reveal that an intact ER export motif is essential for proper in vivo function of GnTI. Coexpression studies with Sar1p provided evidence for COPII-dependent transport of GnTI to the Golgi. Our data provide evidence that efficient ER export of Golgi-resident plant N -glycan processing enzymes occurs through a selective mechanism based on recognition of single basic amino acids present in their cytoplasmic tails.     

The hormonal regulation of bud outgrowth in Phaseolus vulgaris L.

Summary Aqueous solutions of indole acetic acid, kinetin, gibberellic acid and abscisic acid were applied singly and in combination to the decapitated stem stump of Phaseolus seedlings. Application of indole acetic acid will not completely replace the intact stem apex with regard to the inhibition of lateral bud extension. The greatest inhibition of bud growth is obtained when indole acetic acid is applied in combination with both kinetin and abscisic acid. Treatment with gibberellic acid causes massive bud growth even in the presence of indole acetic acid, kinetin and abscisic acid. Although both abscisic acid and kinetin have only a slight promoting effect on bud outgrowth when applied singly, these hormones will modify the effects of indole acetic acid and gibberellic acid.