Vesicle-mediated Secretion Research Articles

Activation of a mitochondrial ATPase gene induces abnormal seed development in Arabidopsis.

The ATPases associated with various cellular activities (AAA) proteins are widespread in living organisms. Some of the AAA-type ATPases possess metalloprotease activities. Other members constitute the 26S proteasome complexes. In recent years, a few AAA members have been implicated in vesicle-mediated secretion, membrane fusion, cellular organelle biogenesis, and hypersensitive responses (HR) in plants. However, the physiological roles and biochemical activities of plant AAA proteins have not yet been defined at the molecular level, and regulatory mechanisms underlying their functions are largely unknown. In this study, we showed that overexpression of an Arabidopsis gene encoding a mitochondrial AAA protein, ATPase-in-Seed-Development (ASD), induces morphological and anatomical defects in seed maturation. The ASD gene is expressed at a high level during the seed maturation process and in mature seeds but is repressed rapidly in germinating seeds. Transgenic plants overexpressing the ASD gene are morphologically normal. However, seed formation is severely disrupted in the transgenic plants. The ASD gene is induced by abiotic stresses, such as low temperatures and high salinity, in an abscisic acid (ABA)-dependent manner. The ASD protein possesses ATPase activity and is localized into the mitochondria. Our observations suggest that ASD may play a role in seed maturation by influencing mitochondrial function under abiotic stress.

Combining ChIP-chip and Expression Profiling to Model the MoCRZ1 Mediated Circuit for Ca2+/Calcineurin Signaling in the Rice Blast Fungus

Significant progress has been made in defining the central signaling networks in many organisms, but collectively we know little about the downstream targets of these networks and the genes they regulate. To reconstruct the regulatory circuit of calcineurin signal transduction via MoCRZ1, a Magnaporthe oryzae C2H2 transcription factor activated by calcineurin dephosphorylation, we used a combined approach of chromatin immunoprecipitation - chip (ChIP-chip), coupled with microarray expression studies. One hundred forty genes were identified as being both a direct target of MoCRZ1 and having expression concurrently differentially regulated in a calcium/calcineurin/MoCRZ1 dependent manner. Highly represented were genes involved in calcium signaling, small molecule transport, ion homeostasis, cell wall synthesis/maintenance, and fungal virulence. Of particular note, genes involved in vesicle mediated secretion necessary for establishing host associations, were also found. MoCRZ1 itself was a target, suggesting a previously unreported autoregulation control point. The data also implicated a previously unreported feedback regulation mechanism of calcineurin activity. We propose that calcium/calcineurin regulated signal transduction circuits controlling development and pathogenicity manifest through multiple layers of regulation. We present results from the ChIP-chip and expression analysis along with a refined model of calcium/calcineurin signaling in this important plant pathogen.

Progress in mechanism of salt excretion in recretohalopytes

The recretohalophyte with specialized salt-secreting structures including salt glands and salt bladders can secrete salt from their bodies and easily adapt themselves to many kinds of salt habitats. Salt glands and salt bladders, arose from dermatogen cells, are excretory organs specially adapted for dealing with ionic homeostasis in the cells of recretohalophytes. The main function of salt glands or salt bladders is to secrete excess ions that invade the plant. The structures of salt glands or salt bladders differ among plant species. In addition to structural differences, salt glands also differ in their secretion abilities. In this review, we mainly focus on recent progress in the mechanism of salt excretion of salt glands and salt bladders, and in particular, emphasize the vesicle-mediated secretion systems from the vacuole to the plasmalemma and the possibly involved membrane-bound translocating proteins for salt secretion of plant gland secretory cell.

CMTM5-v1, a four-transmembrane protein, presents a secreted form released via a vesicle-mediated secretory pathway

The CKLF-like MARVEL transmembrane domain-containing family (CMTM) is a novel family of proteins linking classical chemokines and the transmembrane 4 superfamily (TM4SF). Our earlier studies indicated several CMTM members (such as CKLF1 and CMTM2) have a secreted form. This is the first report of the secreted form of CMTM5-v1, the major RNA splicing form of CMTM5, which is produced as small vesicles (100 nm diameter) and floats at a peak density of 1.19 g/ml on continuous sucrose gradients. CMTM5-v1 has no obvious co-localization with CD63 or Golgi complex. In addition, brefeldin A but not wortmannin can inhibit the secretion of CMTM5-v1. Our results suggest that CMTM5-v1 might be secreted via a different vesicle-mediated secretory pathway, which will be helpful for the studies of vesicle-mediated secretion and MARVEL domain-containing proteins.

Vesicle-mediated secretion of human eosinophil granule-derived major basic protein

Major basic protein (MBP), the predominant cationic protein of human eosinophil specific granules, is stored within crystalloid cores of these granules. Secretion of MBP contributes to the immunopathogenesis of varied diseases. Prior electron microscopy (EM) of eosinophils in sites of inflammation noted losses of granule cores in the absence of granule exocytosis and suggested that eosinophil granule proteins might be released through piecemeal degranulation (PMD), a secretory process mediated by transport vesicles. Because release of eosinophil granule-derived MBP through PMD has not been studied, we evaluated secretion of this cationic protein by human eosinophils. Intracellular localizations of MBP were studied within nonstimulated and eotaxin-stimulated human eosinophils by both immunofluorescence and a pre-embedding immunonanogold EM method that enables optimal epitope preservation and antigen access to membrane microdomains. In parallel, quantification of transport vesicles was assessed in eosinophils from a patient with hypereosinophilic syndrome (HES). Our data demonstrate vesicular trafficking of MBP within eotaxin-stimulated eosinophils. Vesicular compartments, previously implicated in transport from granules to the plasma membrane, including large vesiculotubular carriers termed eosinophil sombrero vesicles (EoSVs), were found to contain MBP. These secretory compartments were significantly increased in numbers within HES eosinophils. Moreover, in addition to granule-stored MBP, even unstimulated eosinophils contained appreciable amounts of MBP within secretory vesicles, as evidenced by immunonanogold EM and immunofluorescent colocalizations of MBP and CD63. These data suggest that eosinophil MBP, with its multiple extracellular activities, can be mobilized from granules by PMD into secretory vesicles and both granule- and secretory vesicle-stored pools of MBP are available for agonist-elicited secretion of MBP from human eosinophils. The recognition of PMD as a secretory process to release MBP is important to understand the pathological basis of allergic and other eosinophil-associated inflammatory diseases.

Proteomic Characterization of the Whole Secretome of Legionella pneumophila and Functional Analysis of Outer Membrane Vesicles

Secretion of effector molecules is one of the major mechanisms by which the intracellular human pathogen Legionella pneumophila interacts with host cells during infection. Specific secretion machineries which are responsible for the subfraction of secreted proteins (soluble supernatant proteins [SSPs]) and the production of bacterial outer membrane vesicles (OMVs) both contribute to the protein composition of the extracellular milieu of this lung pathogen. Here we present comprehensive proteome reference maps for both SSPs and OMVs. Protein identification and assignment analyses revealed a total of 181 supernatant proteins, 107 of which were specific to the SSP fraction and 33 of which were specific to OMVs. A functional classification showed that a large proportion of the identified OMV proteins are involved in the pathogenesis of Legionnaires' disease. Zymography and enzyme assays demonstrated that the SSP and OMV fractions possess proteolytic and lipolytic enzyme activities which may contribute to the destruction of the alveolar lining during infection. Furthermore, it was shown that OMVs do not kill host cells but specifically modulate their cytokine response. Binding of immunofluorescently stained OMVs to alveolar epithelial cells, as visualized by confocal laser scanning microscopy, suggested that there is delivery of a large and complex group of proteins and lipids in the infected tissue in association with OMVs. On the basis of these new findings, we discuss the relevance of protein sorting and compartmentalization of virulence factors, as well as environmental aspects of the vesicle-mediated secretion.

Prions and retroviruses: an endosomal rendezvous?

The past two decades have seen the emergence of endocytic pathways as highly regulated systems for the sorting, selective degradation and recycling of nearly all cell‐surface membrane proteins. At the centre of these pathways are complex endosomal structures known as multivesicular bodies (MVBs) that house intraluminal vesicles (ILVs) formed by invagination and budding from the limiting membrane (Fig 1A). The ILVs contain membrane proteins from the Golgi complex and cell surface that are destined typically for degradation after the fusion of MVBs with lysosomes. This constitutive fate of ILVs is universal to eukaryotes and represents one of the main pathways for membrane protein degradation (Katzmann et al , 2002). Figure 1. Protein transport in the endo‐lysosomal pathways. ( A ) Topological relationships of the multivesicular body (MVB). Different types of cell‐surface proteins (indicated by blue and red molecules) can be internalized and transported by various routes (arrows) to endosomal compartments. The endosomal compartment is known as an MVB when its lumen contains intraluminal vesicles (ILVs) that are formed by budding from the limiting membrane. If the MVB fuses with the plasma membrane, ILVs are released into the extracellular space and become known as exosomes. ( B ) Transport into and out of the multivesicular body (MVB). Pathways linking the MVB with other compartments, such as the early endosome (EE), Golgi, lysosome and plasma membrane, are all regulated in a substrate‐specific and cell‐type‐specific manner to control the localization and fate of various membrane proteins (blue, red and green molecules). Alterations of these transport pathways is emerging as a key facet of viral pathogenesis and might also influence the intracellular accumulation and cell‐to‐cell transmission of prions. For example, adjacent cells, through interactions among cell surface signalling or adhesion molecules, might stimulate localized MVB fusion with the plasma membrane to facilitate exosome release and intercellular transfer of their cargo. …

Auxin/gibberellin interactions in pea leaf morphogenesis

Pea (Pisum sativum) has been an important model plant for several generations of plant physiologists, geneticists and developmental biologists. There exists an extensive body of knowledge on the genetics of many developmental processes including gibberellic acid (GA) biosynthesis and GA and auxin interactions during stem elongation. Auxin is a morphogen and is transported from cell to cell via vesicle-mediated secretion from sites of synthesis. This creates auxin concentration gradients, which can regulate genes controlling morphogenesis. Among the genes regulated are those of GA biosynthesis and this subsequently sets up secondary, parallel gradients of GA concentration. Both these hormones have been proposed to play roles during leaf development in other plant species. The question posed is whether auxin/GA interactions control leaf morphogenesis in pea. It was addressed by (1) looking at effects of auxin and GA inhibitors on leaf development, (2) attempting to rescue leaf form mutants by hormone application and (3) looking for genes expressed during leaf development that might be regulated by auxin and GA. Auxin and GA inhibitors produce common abnormalities during pea leaf development of wildtype (WT) cultured plantlets, which include: inhibition of leaf initiation, reductions in the number of pinna pairs produced and conversion of terminal tendrils into leaflets. Both GA and auxin rescued the tendrilled acacia (uni-tac) mutant by (1) increasing the number of pinna pairs produced and (2) converting terminal leaflets into tendrils. The Uni gene was transcriptionally up-regulated by auxin in shoot tips of cultured WT plantlets. Stable mRNA levels of three genes (PsPIN1, LE and IAA 4/5) known to be auxin-regulated also increased. These results suggest that both auxin and GA are involved in leaf initiation, promote leaf tip growth, and function in gradients and at multiple levels during pea leaf morphogenesis. © 2006 The Linnean Society of London, Botanical Journal of the Linnean Society, 2006, 150, 45–59.

Interactions of (−)-ilimaquinone with methylation enzymes: implications for vesicular-mediated secretion

The marine sponge metabolite (-)-ilimaquinone has antimicrobial, anti-HIV, anti-inflammatory and antimitotic activities, inhibits the cytotoxicity of ricin and diptheria toxin, and selectively fragments the Golgi apparatus. The range of activities demonstrated by this natural product provides a unique opportunity for studying these cellular processes. Affinity chromatography experiments show that (-)-ilimaquinone interacts with enzymes of the activated methyl cycle: S-adenosylmethionine synthetase, S-adenosylhomocysteinase and methyl transferases. Known inhibitors of these enzymes were found to block vesicle-mediated secretion in a manner similar to (-)-ilimaquinone. Moreover, the antisecretory effects of (-)-ilimaquinone and inhibitors of methylation chemistry, but not brefeldin A, could be reversed in the presence of the cellular methylating agent S-adenosylmethionine. Of the enzymes examined in the activated methyl cycle, S-adenosylhomocysteinase was specifically inhibited by (-)-ilimaquinone. Consistent with these observations, (-)-ilimaquinone was shown to obstruct new methylation events in adrenocorticotrophic hormone (ACTH)-secreting pituitary cells. (-)-ilimaquinone inhibits cellular methylations through its interactions with S-adenosylhomocysteinase. Furthermore, these studies indicate that the inhibition of secretion by ilimaquinone is the result of the natural product's antimethylation activity. It is likely that the ability to fragment the Golgi apparatus, as well as other activities, are also related to ilimaquinone's influence on methylation chemistry.

Enrichment of presenilin 1 peptides in neuronal large dense-core and somatodendritic clathrin-coated vesicles.

Presenilin 1 is an integral membrane protein specifically cleaved to yield an N-terminal and a C-terminal fragment, both membrane-associated. More than 40 presenilin 1 mutations have been linked to early-onset familial Alzheimer disease, although the mechanism by which these mutations induce the Alzheimer disease neuropathology is not clear. Presenilin 1 is expressed predominantly in neurons, suggesting that the familial Alzheimer disease mutants may compromise or change the neuronal function (s) of the wild-type protein. To elucidate the function of this protein, we studied its expression in neuronal vesicular systems using as models the chromaffin granules of the neuroendocrine chromaffin cells and the major categories of brain neuronal vesicles, including the small clear-core synaptic vesicles, the large dense-core vesicles, and the somatodendritic and nerve terminal clathrin-coated vesicles. Both the N- and C-terminal presenilin 1 proteolytic fragments were greatly enriched in chromaffin granule and neuronal large dense-core vesicle membranes, indicating that these fragments are targeted to these vesicles and may regulate the large dense-core vesicle-mediated secretion of neuropeptides and neurotransmitters at synaptic sites. The presenilin 1 fragments were also enriched in the somatodendritic clathrin-coated vesicle membranes, suggesting that they are targeted to the somatodendritic membrane, where they may regulate constitutive secretion and endocytosis. In contrast, these fragments were not enriched in the small clear-core synaptic vesicle or in the nerve terminal clathrin-coated vesicle membranes. Taken together, our data indicate that presenilin 1 proteolytic fragments are targeted to specific populations of neuronal vesicles where they may regulate vesicular function. Although full-length presenilin 1 was present in crude homogenates, it was not detected in any of the vesicles studied, indicating that, unlike the presenilin fragments, full-length protein may not have a vesicular function.

Aggregation reroutes molecules from a recycling to a vesicle-mediated secretion pathway during reticulocyte maturation.

Endocytosis of the Tf/TfR complex is essentially the only pathway active in maturing reticulocytes, while exosomes, formed by invagination of the endosomal membrane, provide a mechanism to eliminate seemingly obsolescent proteins, including the TfR, when their function is completed. In this study, we examined molecular trafficking in the recycling and exosome-directed pathways during endocytosis in maturing reticulocytes. To this end, the flow of two exogenously inserted fluorescent lipid analogs, N-(N-[6-[(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]caproyl]) sphingomyelin (C6-NBD-SM) and N-(lissamine rhodamine B sulfonyl) phosphatidyl ethanolamine (N-Rh-PE) was monitored and compared to that of the transferrin (Tf)/Tf receptor (TfR) complex. Prior to elimination via exosomes, the TfR actively recycles with a half-time of approx. 2 minutes. The recycling kinetics of C6-NBD-SM, as bulk plasma membrane marker, are identical to those of the apoTf/TfR complex, as shown by fluorescence microscopy and biochemical analysis. By contrast, although efficiently internalized along the same pathway, N-Rh-PE does not return to the cell surface. More specifically, sucrose gradient analysis and immunoisolation experiments demonstrated that N-Rh-PE accumulates in exosomes, which are eventually released into the extracellular medium. Fluorometric measurements showed that exogenously inserted N-Rh-PE is present in the reticulocyte plasma membrane as small molecular clusters. Moreover, a close correlation was observed between the fate of crosslinked proteins, including the TfR and acetylcholinesterase (AChE), and the fate of the clustered lipid N-Rh-PE. Thus antibody-induced aggregation of specific proteins like the TfR and AChE, which are normally sorted into exosomes during reticulocyte maturation, enhances their shedding by the exosomal pathway. Taken together, the results support the hypothesis that aggregation of either proteins or lipids act as a general sorting signal for exosomal processing, thereby inhibiting reentry in a recycling pathway and providing an effective means for clearing molecules from the cell surface and their eventual elimination from the cells.

Calcium-dependent phospholipid-binding proteins in plants

There is evidence that Ca(2+) can regulate vesicle-mediated secretion in plant cells, but the mechanism for this is not known. One possibility is that Ca(2+) -dependent phospholipid-binding proteins (annexins) couple the Ca(2+) stimulus to the exocytotic response. Using a protocol developed for the isolation of animal annexins we have identified proteins in maize (Zea mays L.) coleoptiles that have similar characteristics to annexins. The predominant polypeptide species run as a doublet of relative molecular mass (Mr) 33000-35000 on sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE); another less-abundant protein of Mr 23000 is also present. In the presence of Ca(2+) these proteins bind to liposomes composed of acidic phospholipids. Calcium-sensitivity of binding differs for each protein and is also influenced by the pH of the buffer used for the liposome-binding assay. Antiserum raised to the 33 to 35-kDa doublet purified on SDS-PAGE recognises the doublet in crude extracts from maize and proteins of similar Mr in Tradescantia virginiana and tobacco Nicotiana tabacum L. The antiserum also recognises p68 (Annexin VI) from chicken gizzard extracts, indicating homology between animal annexins and the maize proteins. For the maize proteins to be involved in the regulation of exocytosis, binding to phospholipids would be expected to occur at physiological levels of Ca(2+). The characteristics of the maize annexin-like proteins are described and attention drawn to the marked effect of pH in lowering the requirement for Ca(2+) for phospholipid binding.

Sorting and Secretory Pathways in Exocrine Cells

Exocrine secretory cells contain multiple post-Golgi pathways from protein secretion. The major pathway in pancreatic and parotid acinar cells involves protein sorting into storage granules that undergo exocytosis with or without stimulation by secretagogues. This route of release is paralleled by a minor nongranular (but vesicular) pathway that originates by budding from maturing secretory granules. The nongranular pathway carries the same polypeptides that undergo storage in the granules but in different relative amounts. These features indicate that sorting into the stimulus-regulated pathway reflects not only the deposition of secretory proteins into immature granules but may also involve selective aggregation of proteins along with exclusion and vesicle-mediated secretion of other polypeptides that are inefficiently retained. Storage granules represent a distinct compartment of the secretory pathway, as indicated by the specific composition of their limiting membranes. Little is known about processes that maintain the low content and limited diversity of integral proteins of the granule membrane as compared to the membranes with which it fuses during exocytosis and formation. Future studies will examine the role of the nongranular secretory pathway in acinar cells, the branchpoint of pathways that are directed to the apical or basolateral cell surfaces, the structural determinants of secretory sorting, and the distribution and function of specific granule membrane polypeptides.

Secretion and membrane recycling in plant cells: novel intermediary structures visualized in ultrarapidly frozen sycamore and carrot suspension-culture cells

Freeze-fracture electron microscopy of propane-jet-frozen samples has been employed to investigate vesicle-mediated secretion and membrane recycling events in carrot (Daucus carota L.) and sycamore maple (Acer pseudoplatanus L.) suspension-culture cells. Stabilization of the cells by means of ultrarapid freezing has enabled us to preserve the cells in a turgid state and to visualize new intermediate membrane configurations related to these events. Indeed, many of the observed membrane configurations, such as flattened membrane vesicles with slit-shaped membrane fusion sites and horseshoe-shaped membrane infoldings, appear to result from the action of turgor forces on the plasma membrane. Individual cells exhibited great variations in numbers and types of membrane configurations postulated to be related to secretion and membrane-recycling events. In the majority of cells, the different membrane profiles displayed a patchy distribution, and within each patch the membrane configurations tended to be of the same stage. This result indicates that secretory events are triggered in domains measuring from 0.1 to about 10 μm in diameter. Based on an extensive analysis of the different membrane configurations seen in our samples, we have formulated the following model of vesicle-mediated secretion in plant cells: Fusion of a secretory vesicle with the plasma membrane leads to the formation of a single, narrow-necked pore that increases in diameter up to about 60 nm. During discharge, the vesicle is flattened, forming a disc-shaped structure perpendicular to the plane of the plasma membrane. As the vesicle is flattened, the pore is converted to a slit, the maximum length of which coincides with the diameter of the flattened vesicle. The flattened vesicle then tips over and concomitantly the plasma-membrane slit becomes curved into a horseshoe-shaped configuration as it extends along the outer margins of the tipped-over vesicle. Some coated pits are present interspersed between the above-mentioned structures, but their numbers appear insufficient to account for an exclusively endocytotic mechanism of membrane recycling. Instead, our micrographs are more consistent with a mixed mode of recycling of membrane components to the cortical endoplamic reticulum and to Golgi cisternae that involves both internalization of membrane by endocytosis and of individual lippid molecules by unknown mechanisms (lipid exchange proteins?). To this end, overall flattening out of the horseshoe-shaped membrane infoldings is accompanied by a retraction and reduction in size of their central, tongue-like structure.