Colicin E2 release: lysis,leakage or secretion? Possible role of a phospholipase.

Results presented here and by others indicate that the release of colicins from producing cells can be uncoupled from the decline in culture turbidity which usually occurs within 2-3 h after the induction of colicin synthesis. This excludes lysis as a necessary event in colicin release. Conversely, the failure to dissociate colicin release from the normally simultaneous release of a specific subset of soluble proteins argues against the idea of a specific colicin secretion system sensu-stricto. Rather, colicin release appears to be a consequence of semi-specific leakage resulting from an alteration of the permeability properties of the cell envelope. This alteration is caused by the 'lysis protein' known to be encoded by most multiple copy number Col plasmids. The finding that the expression of the lysis gene of plasmid ColE2 renders the cells exquisitely sensitive to lysozyme demonstrates that the permeability of the outer membrane must indeed be altered. Evidence is presented that this alteration could be due at least in part to the activation of the detergent-resistant phospholipase A (pldA product). Lysophosphatidylethanolamine, a product of the action of phospholipase on phosphatidylethanolamine, is a membrane perturbant which could alter the permeability properties of the envelope and allow some proteins such as colicin to leak out of the cell.     

Nucleocytoplasmic trafficking of proteins: With or without Ran?

Proteins and RNAs move between the nucleus and cytoplasm by translocation through nuclear pore complexes in the nuclear envelope. To do this, they require specific targeting signals, energy, and a cellular apparatus that catalyzes their transport. Several of the factors involved in nucleocytoplasmic trafficking of proteins have been identified and characterized in some detail. The emerging picture for nuclear transport proposes a central role for the small GTPase Ran and proteins with which it interacts. In particular, asymmetric distribution of these proteins between nucleus and cytoplasm appears to be responsible for the vectorial nature of nucleocytoplasmic transport. Here, we summarize the role of Ran and Ran-binding proteins in nuclear trafficking of proteins with classical nuclear localisation signals. We also discuss examples of the growing number of alternative pathways that are involved in transport of proteins across the nuclear envelope. BioEssays 21:579–589, 1999. © 1999 John Wiley & Sons, Inc.     

Type V protein secretion: simplicity gone awry?

Since its discovery in the late 1980's, the family of secreted proteins termed the autotransporters has been expanding continuously to become the largest group of secreted proteins in Gram-negative bacteria. The type V secretion pathway, which includes the autotransporters (type Va) together with the two-partner secretion system (type Vb) and the Oca family (type Vc), can be defined by secreted proteins that are (i) translocated across the outer membrane via a transmembrane pore formed by a beta-barrel and (ii) contain all the information required for translocation through the cell envelope. In the light of new discoveries and controversies in this research field, the secretion process of autotransporters, or the type Va secretion system, will be discussed here and placed in the context of the more general field of bacterial protein translocation.     

Apoplastic exosome-like vesicles: A new way of protein secretion in plants?

The presence of apoplastic proteins without predicted signal peptide in the gene sequence suggests the existence of protein secretion independent of the ER/Golgi classical route. In animals, one of the pathways proposed for alternative protein secretion involves the release of exosomes to the extracellular space. Although this pathway has not been dissected in plants some indirect evidence is emerging. We have reported that apoplastic fractions of sunflower seeds contain exosome-like vesicles. Besides, these vesicles are enriched in the lectin Helja, which is immunolocalized in the extracellular space even if it the protein has no predicted signal peptide. Here we show that Helja is not glycosylated and its secretion is insensitive to brefeldin A, two of the major characteristics to discard ER/Golgi-mediated protein transport. Moreover, the levels of Helja in sunflower extracellular vesicles are not affected by brefeldin A treatment. Our results suggest that Helja could be exported through an exosome-mediated pathway and point out that this mechanism may be responsible for the secretion of at least part of the leaderless proteins detected in the extracellular compartment of plants.     

The ordered secretion of bioactive peptides: oldest or newest first?

The order of secretion of newly synthesized and older bioactive peptides was investigated using primary rat intermediate pituitary melanotropes, which synthesize, store, and secrete peptides derived from pro-ACTH/endorphin (PAE; also POMC). PAE-derived peptides produced by the cells were biosynthetically labeled by incubating the cells with radioactive amino acids at various times preceding the period during which secretion was examined; secreted and cellular peptides were characterized and quantitated by immunoprecipitation, using affinity-purified antibodies to selected regions of PAE, followed by polyacrylamide gel electrophoretic analysis. Release in the absence of secretagogues (basal or constitutive release) was compared to release in the presence of maximally effective levels of 8-bromo-cAMP and BaCl2 (stimulated or regulated release). Both cell types showed short-lived preferential basal release of newly synthesized and not fully mature peptides (less than 2-3 h old). Conversely, the cells showed preferential stimulated secretion of older peptides. A process of maturation occurred, taking 2-4 h, after which the secretion of newly synthesized and older peptides in response to secretagogues was nearly indistinguishable for the smallest product peptides. The data support a model of gradual processing of peptides from precursors into smaller products and maturation from molecules only available for basal release into peptides available for stimulated secretion as well as for basal release. Basal secretion was found to include mature peptides as well as intermediates and precursor molecules. The data do not support the existence of any preferential regulated secretion of newly synthesized peptides.     

Autophagy: a broad role in unconventional protein secretion?

Autophagy, a cellular 'self-eating' process in eukaryotic cells, exists in both a basal and in an activated state that is induced in response to starvation. Basal and induced autophagy are associated with the packaging of cellular components, including damaged and/or redundant organelles, into double-membrane vesicles called autophagosomes, followed by autophagosome fusion with lysosomes, in which their contents are degraded and recycled. Recent results highlight a novel role for autophagy that does not involve lysosomal degradation of autophagosomal contents, but instead involves their redirection towards the extracellular delivery of an unconventionally secreted protein. Here, we discuss these findings, evaluate the strength of evidence, consider their implications for the field of protein trafficking, and suggest the next steps required to probe this interesting pathway.     

Daxx: death or survival protein?

The death domain-associated protein (Daxx) was originally cloned as a CD95 (FAS)-interacting protein and modulator of FAS-induced cell death. Daxx accumulates in both the nucleus and the cytoplasm; in the nucleus, Daxx is found associated with the promyelocytic leukaemia (PML) nuclear body and with alpha-thalassemia/mental retardation syndrome protein (ATRX)-positive heterochromatic regions. In the cytoplasm, Daxx has been reported to interact with various proteins involved in cell death regulation. Despite a significant number of studies attempting to determine Daxx function in apoptotic and non-apoptotic cell death, its precise role in this process is only partially understood. Here, we critically review the current understanding of Daxx function and shed new light on this interesting field.     

Contribution of Do?ana Wetlands to Carbon Sequestration

Inland and transitional aquatic systems play an important role in global carbon (C) cycling. Yet, the C dynamics of wetlands and floodplains are poorly defined and field data is scarce. Air-water fluxes in the wetlands of Doñana Natural Area (SW Spain) were examined by measuring alkalinity, pH and other physiochemical parameters in a range of water bodies during 2010–2011. Areal fluxes were calculated and, using remote sensing, an estimate of the contribution of aquatic habitats to gaseous transport was derived. Semi-permanent ponds adjacent to the large Guadalquivir estuary acted as mild sinks, whilst temporal wetlands were strong sources of (−0.8 and 36.3 ). Fluxes in semi-permanent streams and ponds changed seasonally; acting as sources in spring-winter and mild sinks in autumn (16.7 and −1.2 ). Overall, Doñana''s water bodies were a net annual source of (5.2 ). Up–scaling clarified the overwhelming contribution of seasonal flooding and allochthonous organic matter inputs in determining regional air-water gaseous transport (13.1 ). Nevertheless, this estimate is about 6 times < local marsh net primary production, suggesting the system acts as an annual net sink. Initial indications suggest longer hydroperiods may favour autochthonous C capture by phytoplankton. Direct anthropogenic impacts have reduced the hydroperiod in Doñana and this maybe exacerbated by climate change (less rainfall and more evaporation), suggesting potential for the modification of C sequestration.     

Regulation of mucin secretion and inflammation in asthma: a role for MARCKS protein?

BACKGROUND: A major characteristic of asthmatic airways is an increase in mucin (the glycoprotein component of mucus) producing and secreting cells, which leads to increased mucin release that further clogs constricted airways and contributes markedly to airway obstruction and, in the most severe cases, to status asthmaticus. Asthmatic airways show both a hyperplasia and metaplasia of goblet cells, mucin-producing cells in the epithelium; hyperplasia refers to enhanced numbers of goblet cells in larger airways, while metaplasia refers to the appearance of these cells in smaller airways where they normally are not seen. With the number of mucin-producing and secreting cells increased, there is a coincident hypersecretion of mucin which characterizes asthma. On a cellular level, a major regulator of airway mucin secretion in both in vitro and in vivo studies has been shown to be MARCKS (myristoylated alanine-rich C kinase substrate) protein, a ubiquitous substrate of protein kinase C (PKC). GENERAL SIGNIFICANCE: In this review, properties of MARCKS and how the protein may regulate mucin secretion at a cellular level will be discussed. In addition, the roles of MARCKS in airway inflammation related to both influx of inflammatory cells into the lung and release of granules containing inflammatory mediators by these cells will be explored. This article is part of a Special Issue entitled: Biochemistry of Asthma.     

Contribution of the γ-secretase subunits to the formation of catalytic pore of presenilin 1 protein

γ-Secretase is an intramembrane-cleaving protease related to the etiology of Alzheimer disease. γ-Secretase is a membrane protein complex composed of presenilin (PS) and three indispensable subunits: nicastrin, Aph-1, and Pen-2. PS functions as a protease subunit forming a hydrophilic catalytic pore structure within the lipid bilayer. However, it remains unclear how other subunits are involved in the pore formation. Here, we show that the hydrophilic pore adopted with an open conformation has already been formed by PS within the immature γ-secretase complex. The binding of the subunits induces the close proximity between transmembrane domains facing the catalytic pore. We propose a model in which the γ-secretase subunits restrict the arrangement of the transmembrane domains of PS during the formation of the functional structure of the catalytic pore.     

Is there leaderless protein secretion in plants?

The sugar alcohol mannitol and it’s catabolic enzyme mannitol dehydrogenase (MTD), in addition to welldocumented roles in metabolism and osmoprotection, may play roles in hostpathogen interactions. Research suggests that in response to the mannitol that pathogenic fungi secrete to suppress reactive oxygen-mediated host defenses, plants make MTD to catabolize fungal mannitol. Yet previous work suggested that pathogen-secreted mannitol is extracellular, while in healthy plants MTD is cytoplasmic. We have presented results showing that the normally cytoplasmic MTD is exported into the cell wall or extracellular space in response to the endogenous inducer of plant defense responses salicylic acid (SA). This SA-induced secretion is insensitive to brefeldin A, an inhibitor of Golgimediated protein transport. Together with the absence of MTD in Golgi stacks and the lack of a documented extracellular targeting sequence in the MTD protein, this suggests MTD is secreted by a non-Golgi, pathogen-activated secretion mechanism in plants. Here we discuss the potential significance of non-Golgi secretion in response to stress.Key words: protein secretion, mannitol metabolism, plant-pathogen interaction, extracellular space, apoplast     

Response of thirsty rats to absence of water: Frustration,disinhibition or compensation?

Water-deprived rats were allowed to drink in an experimental chamber during a series of training trials, and were then presented with an empty water spout during a single test trial. Their initial response to absence of water was to bite, sniff and paw at the empty spout (spout-directed behaviour), after which they engaged in activities such as eating, grooming and exploration of the chamber. By comparison with control animals that had no expectation of water, experimental rats performed these latter activities in a bizarre ‘speeded-up’ manner, which was quantifiable as an increase in local rate. Both the occurrence of spout-directed behaviour and the increase in local rate of other activities were enhanced by increasing the prior level of water deprivation and by offering a more palatable fluid during training. The results are discussed in relation to current models of behavioural switching.     

Coarse-grained models of protein folding: toy models or predictive tools?

Coarse-grained models are emerging as a practical alternative to all-atom simulations for the characterization of protein folding mechanisms over long time scales. While a decade ago minimalist toy models were mainly designed to test general hypotheses on the principles regulating protein folding, the latest coarse-grained models are increasingly realistic and can be used to characterize quantitatively the detailed folding mechanism of specific proteins. The ability of such models to reproduce the essential features of folding dynamics suggests that each single atomic degree of freedom is not by itself particularly relevant to folding and supports a statistical mechanical approach to characterize folding transitions. When combined with more refined models and with experimental studies, the systematic investigation of protein systems and complexes using coarse-grained models can advance our theoretical understanding of the actual organizing principles that emerge from the complex network of interactions among protein atomic constituents.