Signal integration at the level of ion channel and exocytotic function in pancreatic β-cells

Whole body energy balance is ensured by the exquisite control of insulin secretion, the dysregulation of which has serious consequences. Although a great deal has been learned about the control of insulin secretion from pancreatic β-cells in the past 30 years, there remains much to be understood about the molecular mechanisms and interactions that underlie the precise control of this process. Numerous molecular interactions at the plasma membrane mediate the excitatory and amplifying events involved in insulin secretion; this includes interactions between ion channels, signal transduction machinery, and exocytotic proteins. The present Perspectives article considers evidence that key membrane and membrane-associated proteins essential to insulin secretion are regulated in concert as a functional unit, ensuring an integrated excitatory and exocytotic response to the signals that control insulin secretion.     

The story of cell secretion: events leading to the discovery of the 'porosome' - the universal secretory machinery in cells

Cell secretion has come of age, and a century old quest has been elegantly solved. We have come a long way since earlier observations of what appeared to be 'fibrillar regions' at the cell plasma membrane, and electrophysiological studies suggesting the presence of 'fusion pores' at the cell plasma membrane where secretion occurs. Finally, the fusion pore or 'porosome' has been discovered, and its morphology and dynamics determined at nm resolution and in real time in live secretory cells. The porosome has been isolated, its composition determined and it has been both structurally and functionally reconstituted in artificial lipid membrane. The discovery of the porosome as the universal secretory machinery in cells and the discovery of the molecular mechanism of vesicular content expulsion during cell secretion have finally enabled a clear understanding of this important cellular process. This review outlines the fascinating and exciting journey leading to the discovery of the porosome, ultimately solving one of the most difficult, significant, and fundamental cellular process -cell secretion.     

Reciprocity in the Developmental Regulation of Aquaporins 1, 3 and 5 during Pregnancy and Lactation in the Rat

Milk secretion involves significant flux of water, driven largely by synthesis of lactose within the Golgi apparatus. It has not been determined whether this flux is simply a passive consequence of the osmotic potential between cytosol and Golgi, or whether it involves regulated flow. Aquaporins (AQPs) are membrane water channels that regulate water flux. AQP1, AQP3 and AQP5 have previously been detected in mammary tissue, but evidence of developmental regulation (altered expression according to the developmental and physiological state of the mammary gland) is lacking and their cellular/subcellular location is not well understood. In this paper we present evidence of developmental regulation of all three of these AQPs. Further, there was evidence of reciprocity since expression of the rather abundant AQP3 and less abundant AQP1 increased significantly from pregnancy into lactation, whereas expression of the least abundant AQP5 decreased. It would be tempting to suggest that AQP3 and AQP1 are involved in the secretion of water into milk. Paradoxically, however, it was AQP5 that demonstrated most evidence of expression located at the apical (secretory) membrane. The possibility is discussed that AQP5 is synthesized during pregnancy as a stable protein that functions to regulate water secretion during lactation. AQP3 was identified primarily at the basal and lateral membranes of the secretory cells, suggesting a possible involvement in regulated uptake of water and glycerol. AQP1 was identified primarily at the capillary and secretory cell cytoplasmic level and may again be more concerned with uptake and hence milk synthesis, rather than secretion. The fact that expression was developmentally regulated supports, but does not prove, a regulatory involvement of AQPs in water flux through the milk secretory cell.     

Dynamic role of microvilli in peritrophic membrane formation

Although the peritrophic membrane (PM) is a common extracellular construction in many invertebrate groups, evidence of the location of its secretion has never been reported. In this study a specific marker for chitin has been developed, enabling a separate examination of secretion of the chitinous and proteinaceous components of the PM in the millipede, Glomeris marginata. Chitin appears first at the base of the microvilli (MV), synchronized in adjacent cells along the entire length of the midgut. Evidence showing that it originates at the plasma membrane is discussed. Proteinaceous components appear to be added from the MV to the chitinous sheet as it moves along the MV toward the lumen. Precedence for such a dynamic role for MV in formation of extracellular structures is reviewed. The completed PM extends around individual items in the gut contents as well as forming a multilayered envelope; this may enhance both its digestive and protective functions.     

Secretory vesicles transiently dock and fuse at the porosome to discharge contents during cell secretion

In contrast with the observation in electron micrographs of partially empty vesicles in cells following secretion, it has been believed since the 1950s that during cell secretion, secretory vesicles completely merge at the cell plasma membrane, resulting in the diffusion of intravesicular contents to the cell exterior and the compensatory retrieval of the excess membrane by endocytosis. In the interim, a large body of work has been published arguing both for and against the complete merger of secretory vesicle membrane at the cell plasma membrane during secretion. The only definitive determination of the mechanism of cell secretion remained in its direct observation at nanometre resolution in live cells. In the past decade, this finally became a reality through the power and scope of the atomic force microscope, which has made it possible to resolve a major conundrum in cell biology. This paradigm shift in our understanding of cell secretion is briefly outlined here.     

Unconventional secretion of fibroblast growth factor 2 is mediated by direct translocation across the plasma membrane of mammalian cells

Fibroblast growth factor 2 (FGF-2) is a pro-angiogenic mediator that is secreted by both normal and neoplastic cells. Intriguingly, FGF-2 has been shown to be exported by an endoplasmic reticulum/Golgi-independent pathway; however, the molecular machinery mediating this process has remained elusive. Here we introduce a novel in vitro system that functionally reconstitutes FGF-2 secretion. Based on affinity-purified plasma membrane inside-out vesicles, we demonstrate post-translational membrane translocation of FGF-2 as shown by protease protection experiments. This process is blocked at low temperature but apparently does not appear to be driven by ATP hydrolysis. FGF-2 membrane translocation occurs in a unidirectional fashion requiring both integral and peripheral membrane proteins. These findings provide direct evidence that FGF-2 secretion is based on its direct translocation across the plasma membrane of mammalian cells. When galectin-1 and macrophage migration inhibitory factor, other proteins exported by unconventional means, were analyzed for translocation into plasma membrane inside-out vesicles, galectin-1 was found to be transported as efficiently as FGF-2. By contrast, migration inhibitory factor failed to traverse the membrane of inside-out vesicles. These findings establish the existence of multiple distinct secretory routes that are operational in the absence of a functional endoplasmic reticulum/Golgi system.     

Insights into the autotransport process of a trimeric autotransporter,Yersinia Adhesin A (YadA)

Trimeric autotransporter adhesins (TAAs) are a subset of a larger protein family called the type V secretion systems. They are localized on the cell surface of Gram‐negative bacteria, function as mediators of attachment to inorganic surfaces and host cells, and thus include important virulence factors. Yersinia adhesin A (YadA) from Yersinia enterocolitica is a prototypical TAA that is used extensively to study the structure and function of the type Vc secretion system. A solid‐state NMR study of the membrane anchor domain of YadA previously revealed a flexible stretch of small residues, termed the ASSA region, that links the membrane anchor to the stalk domain. In this study, we present evidence that single amino acid proline substitutions produce two different conformers of the membrane anchor domain of YadA; one with the N‐termini facing the extracellular surface, and a second with the N‐termini located in the periplasm. We propose that TAAs adopt a hairpin intermediate during secretion, as has been shown before for other subtypes of the type V secretion system. As the YadA transition state intermediate can be isolated from the outer membrane, future structural studies should be possible to further unravel details of the autotransport process.     

Insights into the Assembly of the Alginate Biosynthesis Machinery in Pseudomonas aeruginosa

Pseudomonas aeruginosa is an opportunistic pathogen of particular significance to cystic fibrosis patients. This bacterium produces the exopolysaccharide alginate, which is an indicator of poor prognosis for these patients. The proteins required for alginate polymerization and secretion are encoded by genes organized in a single operon; however, the existence of internal promoters has been reported. It has been proposed that these proteins form a multiprotein complex which extends from the inner to outer membrane. Here, experimental evidence supporting such a multiprotein complex was obtained via mutual stability analysis, pulldown assays, and coimmunoprecipitation. The impact of the absence of single proteins or subunits on this multiprotein complex, i.e., on the stability of potentially interacting proteins, as well as on alginate production was investigated. Deletion of algK in an alginate-overproducing strain, PDO300, interfered with the polymerization of alginate, suggesting that in the absence of AlgK, the polymerase and copolymerase subunits, Alg8 and Alg44, are destabilized. Based on mutual stability analysis, interactions between AlgE (outer membrane), AlgK (periplasm), AlgX (periplasm), Alg44 (inner membrane), Alg8 (inner membrane), and AlgG (periplasm) were proposed. Coimmunoprecipitation using a FLAG-tagged variant of AlgE further demonstrated its interaction with AlgK. Pulldown assays using histidine-tagged AlgK showed that AlgK interacts with AlgX, which in turn was also copurified with histidine-tagged Alg44. Detection of AlgG and AlgE in PAO1 supported the existence of internal promoters controlling expression of the respective genes. Overall experimental evidence was provided for the existence of a multiprotein complex required for alginate polymerization and secretion.     

Secretory vesicles externalize the major plasma membrane ATPase in yeast

Yeast cell surface growth is accomplished by constitutive secretion and plasma membrane assembly, culminating in the fusion of vesicles with the bud membrane. Coordination of secretion and membrane assembly has been investigated by examining the biogenesis of plasma membrane ATPase (PM ATPase) in secretion-defective (sec) strains of Saccharomyces cerevisiae. PM ATPase is synthesized as a approximately 106-kD polypeptide that is not detectably modified by asparagine-linked glycosylation or proteolysis during transit to the plasma membrane. Export of the PM ATPase requires the secretory pathway. In sec1, a mutant defective in the last step of secretion, large amounts of Golgi-derived vesicles are accumulated. Biochemical characterization of this organelle has demonstrated that PM ATPase and the secretory enzyme, acid phosphatase, are transported in a single vesicle species.     

Acid phosphatase localization in the digestive glands of Dionaea muscipula Ellis flytraps

The intracellular localization of acid phosphatases in stimulated digestive glands of Dionaea flytraps has been studied to provide evidence for the route taken by this enzyme during secretion. Previous studies have either included or excluded a role for the dictyosomes in this pathway. Both p-nitrophenyl phosphate and beta-glycerophosphate were used as substrates, and both gave similar localization patterns. Unstimulated glands contained little phosphatase activity in the endomembrane system, whereas 24 and 48 hr after stimulation, heavy deposits of lead were located in the endoplasmic reticulum cisternae, including the nuclear envelope, the dictyosome cisternae, and secretory vesicles. Since dictyosome activation, as judged by the presence of secretory vesicles in the cytoplasm, also coincides with gland stimulation, we conclude that secretion of the hydrolase enzymes occurs via this route and not, as suggested elsewhere, via direct endoplasmic reticulum to plasma membrane connections.     

Protein-translocating outer membrane porins of Gram-negative bacteria

Five families of outer membrane porins that function in protein secretion in Gram-negative bacteria are currently recognized. In this report, these five porin families are analyzed from structural and phylogenetic standpoints. They are the fimbrial usher protein (FUP), outer membrane factor (OMF), autotransporter (AT), two-partner secretion (TPS) and outer membrane secretin (Secretin) families. All members of these families in the current databases were identified, and all full-length homologues were multiply aligned for structural and phylogenetic analyses. The organismal distribution of homologues in each family proved to be unique with some families being restricted to proteobacteria and others being widespread in other bacterial kingdoms as well as eukaryotes. The compositions of and size differences between subfamilies provide evidence for specific orthologous relationships, which agree with available functional information and intra-subfamily phylogeny. The results reveal that horizontal transfer of genes encoding these proteins between phylogenetically distant organisms has been exceptionally rare although transfer within select bacterial kingdoms may have occurred. The resultant in silico analyses are correlated with available experimental evidence to formulate models relevant to the structures and evolutionary origins of these proteins.     

Insertion of the bacterial type III translocon: not your average needle stick

Bacterial type III secretion systems are thought to translocate virulence proteins directly from the bacterial cytoplasm into host cells through a continuous molecular channel. Little is known about how the apparatus itself interacts with membranes and whether insertion of this structure into the host membrane has consequences for the bacteria apart from its beneficial role in delivering virulence proteins. New evidence suggests that membrane insertion of the bacterial type III apparatus might turn on a calcium-dependent signaling pathway resulting in phagolysosomal fusion.     

Protein import and export across the bacterial outer membrane

The bacterial outer membrane forms an impermeable barrier to the environment, but a wide variety of substances must cross it without compromising the membrane. Perhaps, the most fascinating transport phenomenon is the import and export of very large protein toxins using relatively small β-barrel proteins residing in the outer membrane. Progress has been made on three systems in recent years that shed light on this process. In this review, we summarize bacteriocin (toxin) import using TonB-dependent transporters and protein secretion by autotransporters and two partner secretion systems.     

Regulated secretion of conventional lysosomes

Regulated secretion has been traditionally regarded as a specialized process present in only a few cell types. Similarly, the secretory lysosomes of hematopoietic cells have been viewed as 'modified' organelles that acquired the machinery for regulated exocytosis. However, there is evidence that conventional lysosomes can, in many cell types, respond to rises in the intracellular free Ca2+ concentration by fusing with the plasma membrane. These findings profoundly change the current view of lysosomes as a 'final' station of the endocytic pathway and suggest a previously unsuspected active role for this organelle.     

PilM/N/O/P Proteins Form an Inner Membrane Complex That Affects the Stability of the Pseudomonas aeruginosa Type IV Pilus Secretin

The highly conserved pilM/N/O/P/Q gene cluster is among the core set of genes required for cell surface expression of type IV pili and associated twitching motility. With the exception of the outer membrane secretin, a multimer of PilQ subunits, the specific functions of the products encoded by this gene cluster are poorly characterized. Orthologous proteins in the related bacterial type II secretion system have been shown to interact to form an inner membrane complex required for protein secretion. In this study, we provide evidence that the PilM/N/O/P proteins form a functionally equivalent type IVa pilus complex. Using Pseudomonas aeruginosa as model organism, we found that all four proteins, including the nominally cytoplasmic PilM, colocalized to the inner membrane. Stability studies via Western blot analyses revealed that loss of one component has a negative impact on the levels of other members of the putative complex. Furthermore, complementation studies revealed that the stoichiometry of the components is important for the correct formation of a stable complex in vivo. We provide evidence that an intact inner membrane complex is required for optimal formation of the outer membrane complex of the type IVa pilus system in P. aeruginosa, as PilQ stability is negatively affected in its absence. Finally, we show that, in the absence of the pilin subunit, the levels of membrane-bound components of the inner membrane complex are negatively regulated by the PilR/S two-component system, suggesting a role for PilR/S in sensing the piliation status of the cell.     

Regulation of transmitter release by Unc-13 and its homologues

Neurotransmitters are released by Ca(2+)-triggered exocytotic fusion of synaptic vesicles. Before fusion, vesicles dock at a specialised presynaptic plasma membrane region, the active zone, where they are primed to a fusion competent state. The nature of this priming reaction has long been enigmatic. Recent evidence demonstrates that priming is an essential and rate-limiting step in secretion from neurons and neuroendocrine cells. Members of the Unc-13 protein family, which are highly conserved during evolution and act as novel targets of the diacylglycerol second-messenger pathway, have been identified to play an essential role in this process.     

Role of the pilot protein YscW in the biogenesis of the YscC secretin in Yersinia enterocolitica

The YscC secretin is a major component of the type III protein secretion system of Yersinia enterocolitica and forms an oligomeric structure in the outer membrane. In a mutant lacking the outer membrane lipoprotein YscW, secretion is strongly reduced, and it has been proposed that YscW plays a role in the biogenesis of the secretin. To study the interaction between the secretin and this putative pilot protein, YscC and YscW were produced in trans in a Y. enterocolitica strain lacking all other components of the secretion machinery. YscW expression increased the yield of oligomeric YscC and was required for its outer membrane localization, confirming the function of YscW as a pilot protein. Whereas the pilot-binding site of other members of the secretin family has been identified in the C terminus, a truncated YscC derivative lacking the C-terminal 96 amino acid residues was functional and stabilized by YscW. Pulse-chase experiments revealed that approximately 30 min were required before YscC oligomerization was completed. In the absence of YscW, oligomerization was delayed and the yield of YscC oligomers was strongly reduced. An unlipidated form of the YscW protein was not functional, although it still interacted with the secretin and caused mislocalization of YscC even in the presence of wild-type YscW. Hence, YscW interacts with the unassembled YscC protein and facilitates efficient oligomerization, likely at the outer membrane.     

Secretion machinery at the cell plasma membrane

Secretion is a fundamental cellular process involving the regulated release of intracellular products from cells. Physiological functions such as neurotransmission, or the release of hormones and digestive enzymes, are all governed by cell secretion. Three critical activities occur at the cell plasma membrane to ensure secretion. Membrane-bound secretory vesicles dock, fuse, and expel their contents to the outside via specialized and permanent plasma membrane structures, called porosomes or fusion pores. In recent years, significant progress has been made in our understanding of these three key cellular activities required for cell secretion. The molecular machinery and mechanism involving them is summarized in this article.     

Effect of Membrane Phospholipid Composition and Charge of the Signal Peptide of Escherichia coli Alkaline Phosphatase on Efficiency of Its Secretion

The secretion of the Escherichia coli alkaline phosphatase with a different charge of signal peptide due to replacement of positively charged Lys(–20) has been studied depending on the phospholipid composition of the membranes and the activity of the translocational ATPase—protein SecA. Changing the signal peptide charge, along with a change in phospholipid composition, has been shown to reduce the efficiency of secretion. In the absence of phosphatidylethanolamine the membrane contains anionic phospholipids only, and the dependence of secretion on the signal peptide charge decreases. The dependence of secretion on membrane phospholipid composition and the signal peptide charge is also determined by the activity of SecA protein. If SecA is inactivated by sodium azide, then the dependence of secretion on anionic phospholipids increases; on the contrary, higher content of anionic phospholipids (in the absence of phosphatidylethanolamine) decreases the dependence of secretion on the SecA activity. The results suggest a direct interaction of positively charged signal peptide with negatively charged membrane phospholipids under initiation of secretion and also interdependent contribution of the signal peptide charge, anionic phospholipids, and translocational ATPase to secretion.