The Golgi-associated protein GRASP is required for unconventional protein secretion during development

During Dictyostelium development, prespore cells secrete acyl-CoA binding protein (AcbA). Upon release, AcbA is processed to generate a peptide called spore differentiation factor-2 (SDF-2), which triggers terminal differentiation of spore cells. We have found that cells lacking Golgi reassembly stacking protein (GRASP), a protein attached peripherally to the cytoplasmic surface of Golgi membranes, fail to secrete AcbA and, thus, produce inviable spores. Surprisingly, AcbA lacks a signal sequence and is not secreted via the conventional secretory pathway (endoplasmic reticulum-Golgi-cell surface). GRASP is not required for conventional protein secretion, growth, and the viability of vegetative cells. Our findings reveal a physiological role of GRASP and provide a means to understand unconventional secretion and its role in development.     

Unconventional Secretion of AcbA in Dictyostelium discoideum through a Vesicular Intermediate

The acyl coenzyme A (CoA) binding protein AcbA is secreted unconventionally and processed into spore differentiation factor 2 (SDF-2), a peptide that coordinates sporulation in Dictyostelium discoideum. We report that AcbA is localized in vesicles that accumulate in the cortex of prespore cells just prior to sporulation. These vesicles are not observed after cells are stimulated to release AcbA but remain visible after stimulation in cells lacking the Golgi reassembly stacking protein (GRASP). Acyl-CoA binding is required for the inclusion of AcbA in these vesicles, and the secretion of AcbA requires N-ethylmaleimide-sensitive factor (NSF). About 1% of the total cellular AcbA can be purified within membrane-bound vesicles. The yield of vesicles decreases dramatically when purified from wild-type cells that were stimulated to release AcbA, whereas the yield from GRASP mutant cells was only modestly altered by stimulation. We suggest that these AcbA-containing vesicles are secretion intermediates and that GRASP functions at a late step leading to the docking/fusion of these vesicles at the cell surface.The acyl coenzyme A (CoA) binding protein (ACBP) has been well characterized for its role in intracellular lipid trafficking, but it also serves as the precursor of peptides that function as intercellular signals. ACBPs are involved in the transport and metabolism of long-chain acyl-CoA esters and steroid biosynthesis (15, 16, 32). In the mammalian brain ACBP is also secreted and processed to generate a diazepam binding inhibitor (DBI) peptide that regulates γ-aminobutyric acid A (GABA_A) ionotropic receptors in neurons (12). Qian and colleagues recently demonstrated the secretion of ACBP from Muller glial cells of the retina (36). In Dictyostelium discoideum the ACBP homolog AcbA is secreted and processed extracellularly into spore differentiation factor 2 (SDF-2), a 34-amino-acid peptide that is highly similar to DBI (2). However, neither ACBP nor AcbA carries a signal sequence that is necessary for entering the endoplasmic reticulum/Golgi pathway. Alternative, unconventional pathways for the secretion of proteins, including ACBPs, have been proposed over the past 20 years, involving the direct membrane transport of proteins, novel membrane trafficking, or autophagy (29, 39).In Dictyostelium, SDF-2 signaling controls the terminal cell differentiation of prespore cells into encapsulated spores during fruiting body formation. Prespore cells within the nascent sorus climb the elongating stalk in a process that requires their active motility (8). The extracellular processing of AcbA into SDF-2 by prestalk cells is thought to coordinate spore encapsulation with fruiting body morphogenesis such that immobile spores are not produced before the stalk begins to form. Approximately halfway through this process of culmination, sporulation occurs as a wave from the top to the bottom of the nascent sorus (38).An understanding of the regulation of SDF-2 signaling is now emerging. During culmination, prespore cells respond to a steroid signal by rapidly releasing GABA, which binds to the GABA_B-like receptor GrlE and stimulates a signal transduction pathway leading to the release of AcbA by prespore cells (3, 5). AcbA is processed into SDF-2 by TagC protease, which is displayed on the surface of prestalk cells in response to GABA. The 34-amino-acid peptide SDF-2 binds to the receptor histidine kinase DhkA, leading to elevated levels of intracellular cyclic AMP (cAMP), which induces spore encapsulation (2, 47). Low levels of SDF-2 also trigger the release of additional AcbA proteins, forming a positive-feedback loop (2, 6). Although only 1 to 3% of the total AcbA is secreted, the levels of SDF-2 in the sorus are far above that required to rapidly induce sporulation (5, 19).The release of AcbA is a critical step in this cascade, but the mechanism of its secretion is largely unknown. The Golgi-associated protein GRASP (Golgi reassembly stacking protein) appears to play an essential role in the process since grpA-null mutants lacking GRASP fail to produce SDF-2 (19). To further explore the role of GRASP and understand the regulation of AcbA secretion, we have determined the subcellular localization of AcbA before and after stimulating its release. Secreted AcbA appears to be localized within membrane-bound vesicles, which accumulate in the cortex of prespore cells during culmination. When AcbA secretion is stimulated by GABA or SDF-2, the cortical vesicles containing AcbA are lost from wild-type cells but remain in cells lacking GRASP. It appears that GRASP is not involved in the production or positioning of AcbA within the cortical vesicles, but it is essential for events leading to their regulated release.     

Genetic evidence that the acyl coenzyme A binding protein AcbA and the serine protease/ABC transporter TagA function together in Dictyostelium discoideum cell differentiation

The acyl coenzyme A (CoA) binding protein AcbA is cleaved to form a peptide (SDF-2) that coordinates spore encapsulation during the morphogenesis of Dictyostelium discoideum fruiting bodies. We present genetic evidence that the misspecification of cell types seen in mutants of the serine protease/ABC transporter TagA results from the loss of normal interactions with AcbA. Developmental phenotypes resulting from aberrant expression of the TagA protease domain, such as the formation of supernumerary tips on aggregates and the production of excess prestalk cells, are suppressed by null mutations in the acbA gene. Phenotypes resulting from the deletion of tagA, such as overexpression of the prestalk gene ecmB and the misexpression of the prespore gene cotB in stalk cells, are also observed in acbA mutants. Moreover, tagA- mutants fail to produce SDF-2 during fruiting body morphogenesis but are able to do so if they are stimulated with exogenous SDF-2. These results indicate that the developmental program depends on TagA and AcbA working in concert with each other during cell type differentiation and suggest that TagA is required for normal SDF-2 signaling during spore encapsulation.     

The polyketide MPBD initiates the SDF-1 signaling cascade that coordinates terminal differentiation in Dictyostelium

Dictyostelium uses a wide array of chemical signals to coordinate differentiation as it switches from a unicellular to a multicellular organism. MPBD, the product of the polyketide synthase encoded by stlA, regulates stalk and spore differentiation by rapidly stimulating the release of the phosphopeptide SDF-1. By analyzing specific mutants affected in MPBD or SDF-1 production, we delineated a signal transduction cascade through the membrane receptor CrlA coupled to Gα1, leading to the inhibition of GskA so that the precursor of SDF-1 is released. It is then processed by the extracellular protease of TagB on prestalk cells. SDF-1 apparently acts through the adenylyl cyclase ACG to activate the cyclic AMP (cAMP)-dependent protein kinase A (PKA) and trigger the production of more SDF-1. This signaling cascade shows similarities to the SDF-2 signaling pathway, which acts later to induce rapid spore encapsulation.     

Cytokinins induce sporulation in Dictyostelium

The social amoeba Dictyostelium discoideum diverged from the line leading to animals shortly after the separation of plants and animals but it retained characteristics of both kingdoms. A GABA(B)-like receptor and a peptide, SDF-2, with homologs found only in animals, control sporulation, while cytokinins, which act as hormones in plants, keep spores dormant. When SDF-2 binds its receptor DhkA, it reduces the activity of the cAMP phosphodiesterase RegA such that cAMP levels can increase. It has been proposed that the cytokinin discadenine also produces in an increase in cAMP but acts through a different histidine kinase, DhkB. We have found that discadenine and its precursor, isopentenyl adenine, not only maintain spore dormancy but also initiate rapid encapsulation independently of the SDF-2 signal transduction pathway. DhkB and the adenylyl cyclase of late development, AcrA, are members of two component signal transduction families and both are required to transduce the cytokinin signal. As expected, strains lacking the isopentenyl-transferase enzyme chiefly responsible for cytokinin synthesis are defective in sporulation. It appears that SDF-2 and cytokinins are secreted during late development to trigger signal transduction pathways that lead to an increase in the activity of the camp-dependent protein kinase, PKA, which triggers rapid encapsulation as well as ensuring spore dormancy.     

Lithium respecifies cyclic AMP-induced cell-type specific gene expression in Dictyostelium

We investigated the effect of LiCl on pattern formation and cAMP-regulated gene expression in Dictyostelium discoideum. In intact slugs, 5 mM LiCl induces an almost complete redifferentiation of prespore into prestalk cells. We found that LiCl acts by interfering with the transduction of extracellular cAMP to cell-type-specific gene expression; LiCl inhibits the induction of prespore-specific gene expression by cAMP, while it promotes the induction of prestalk-associated gene expression by cAMP. Our results indicate that two divergent pathways transduce the extracellular cAMP signal to, respectively, prestalk and prespore gene expression.     

Folic acid stimulation of the Gα4 G protein-mediated signal transduction pathway inhibits anterior prestalk cell development in Dictyostelium

In Dictyostelium discoideum, several G proteins are known to mediate the transduction of signals that direct chemotactic movement and regulate developmental morphogenesis. The G protein alpha subunit encoded by the Galpha4 gene has been previously shown to be required for chemotactic responses to folic acid, proper developmental morphogenesis, and spore production. In this study, cells overexpressing the wild type Galpha4 gene, due to high copy gene dosage (Galpha4HC), were found to be defective in the ability to form the anterior prestalk cell region, express prespore- and prestalk-cell specific genes, and undergo spore formation. In chimeric organisms, Galpha4HC prespore cell-specific gene expression and spore production were rescued by the presence of wild-type cells, indicating that prespore cell development in Galpha4HC cells is limited by the absence of an intercellular signal. Transplanted wild-type tips were sufficient to rescue Galpha4HC prespore cell development, suggesting that the rescuing signal originates from the anterior prestalk cells. However, the deficiencies in prestalk-specific gene expression were not rescued in the chimeric organisms. Furthermore, Galpha4HC cells were localized to the prespore region of these chimeric organisms and completely excluded from the anterior prestalk region, suggesting that the Galpha4 subunit functions cell-autonomously to prevent anterior prestalk cell development. The presence of exogenous folic acid during vegetative growth and development delayed anterior prestalk cell development in wild-type but not galpha4 null mutant aggregates, indicating that folic acid can inhibit cell-type-specific differentiation by stimulation of the Galpha4-mediated signal transduction pathway. The results of this study suggest that Galpha4-mediated signals can regulate cell-type-specific differentiation by promoting prespore cell development and inhibiting anterior prestalk-cell development.     

A two-component histidine kinase gene that functions in Dictyostelium development.

A mutant which failed to complete development was isolated from a population of cells that had been subjected to insertional mutagenesis using restriction enzyme-mediated integration. The disrupted gene, dhkA, encodes the conserved motifs of a histidine kinase as well as the response regulator domain. It is likely that the histidine in DhkA is autophosphorylated and the phosphate passed to one or more response regulators. Such two-component systems function in a variety of bacterial signal transduction pathways and have been characterized recently in yeast and Arabidopsis. In Dictyostelium, we found that DhkA functions both in the regulation of prestalk gene expression and in the control of the terminal differentiation of prespore cells.     

Hypertonic signal promotes stability of Dictyostelium spores via a PKA-independent pathway

Differentiation of Dictyostelium spores initiates with rapid encapsulation of prespore cells under the control of cAMP-dependent protein kinase (PKA), followed by further maturation processes involving cytoskeletal reorganization. Constitutive activation of PKA induces precocious formation of viable spores in development and confers the ability to encapsulate under specific submerged conditions. In this study, we show that the stability of these spores depends upon conditions of high osmotic strength during spore differentiation, indicating that a hypertonic signal is required in addition to PKA to induce maturation to stable spores. The formation of stable spores under hypertonic conditions requires high cell density, suggesting the involvement of additional cellular signaling.     

Production and turnover of cAMP signals by prestalk and prespore cells in Dictyostelium discoideum cell aggregates

Dictyostelium discoideum prestalk cells and prespore cells from migrating slugs and culminating cell aggregates were isolated by Percoll density centrifugation. Several activities relevant to the generation, detection, and turnover of extracellular cyclic AMP (cAMP) signals were determined. It was found that: the two cell types have the same basal adenylate cyclase activity; prespore cells and prestalk cells are able to relay the extracellular cAMP signal equally well; intact prestalk cells show a threefold higher cAMP phosphodiesterase activity on the cell surface than prespore cells, whereas their cytosolic activity is the same; intact prestalk cells bind three to four times more cAMP than prespore cells; no large differences in cAMP metabolism and detection were observed between cells derived from migrating slugs and culminating aggregates. The results are discussed in relation to the possible morphogenetic role of extracellular cAMP in Dictyostelium cell aggregates. On the basis of the properties of the isolated cells we assume that a gradient of extracellular cAMP exists in Dictyostelium aggregates. This gradient appears to be involved in the formation and stabilization of the prestalk-prespore cell pattern.     

Local Differences in GABA Release Induced by Excitatory Amino Acids During Retina Development: Selective Activation of NMDA Receptors by Aspartate in the Inner Retina

Glutamate and GABA are the major excitatory and inhibitory neurotransmitters in the CNS. In the retina, it has been shown that glutamate and aspartate and their agonists kainate and NMDA promote the release of GABA. In the chick retina, at embryonic day 14 (E14), glutamate and kainate were able to induce the release of GABA from amacrine and horizontal cells as detected by GABA-immunoreactivity. NMDA also induced GABA release restricted to amacrine cell population and its projections to the inner plexiform layer (E14 and E18). Although aspartate reduced GABA immunoreactivity, specifically in amacrine cells of E18 retinas, it was not efficient to promote GABA release from retinas at E14. As observed in differentiated retinas, dopamine inhibited the GABA release promoted by NMDA and aspartate but not by kainate. Our data show that different retinal sites respond to distinct EAAs via different receptor systems.     

胰岛中谷氨酸和γ-氨基丁酸受体的研究进展

谷氨酸和γ-氨基丁酸受体主要分布在中枢神经系统,在神经信号转导中发挥着重要作用.近年来在胰岛中也发现了这类受体,且胰岛中几乎含有这些受体的所有亚型.本文详细描述了胰岛中这些受体的各亚型,并着重讨论了它们的生理功能和相互作用关系,以及研究它们在胰岛中的生理功能的新技术方法.这些问题的探讨不但为研究胰岛功能机制提供了新思路,也将有助于从新的视角理解神经科学问题.     

Mechanism of glutamate stimulation of CO production in cerebral microvessels

Dilation of piglet pial arterioles to glutamate involves carbon monoxide (CO) produced from heme by heme oxygenase-2 (HO-2). Piglet cerebral microvessels and endothelial and smooth muscle cells grown on microcarrier beads were used to address the hypothesis that glutamate increases endothelial CO production by increasing HO-2 catalytic activity. CO was measured by gas chromatography/mass spectrometry. Glutamate increased CO production from endogenous heme by cerebral microvessels, endothelial cells, and smooth muscle cells. Glutamate increased the conversion of exogenous heme to CO. Protein tyrosine kinase inhibition blocked glutamate stimulation of CO production. Inhibition of protein tyrosine phosphatases stimulated CO production. Conversely, neither phorbol myristate acetate nor H-7 changed glutamate stimulation of CO production. The mechanism of HO-2 stimulation by glutamate appears to be independent of cytosolic Ca, because stimulation of CO production by glutamate was the same in Careplete medium, Ca-free medium with ionomycin, and Careplete medium with ionomycin. Therefore, glutamate appears to increase HO-2 catalytic activity in cerebral microvessels via a tyrosine kinase mediated pathway.     

SDF-2 Induction of Terminal Differentiation in Dictyostelium discoideum Is Mediated by the Membrane-Spanning Sensor Kinase DhkA

SDF-2 is a peptide released by prestalk cells during culmination that stimulates prespore cells to encapsulate. Genetic evidence indicates that the response is dependent on the dhkA gene. This gene encodes a member of the histidine kinase family of genes that functions in two-component signal transduction pathways. The sequence of the N-terminal half of DhkA predicts two hydrophobic domains separated by a 310-amino-acid loop that could bind a ligand. By inserting MYC6 epitopes into DhkA, we were able to show that the loop is extracellular while the catalytic domain is cytoplasmic. Cells expressing the MYC epitope in the extracellular domain of DhkA were found to respond only if induced with 100-fold-higher levels of SDF-2 than required to induce dhkA+ cells; however, they could be induced to sporulate by addition of antibodies specific to the MYC epitope. To examine the enzymatic activity of DhkA, we purified the catalytic domain following expression in bacteria and observed incorporation of labelled phosphate from ATP consistent with histidine autophosphorylation. Site-directed mutagenesis of histidine1395 to glutamine in the catalytic domain blocked autophosphorylation. Furthermore, genetic analyses showed that histidine1395 and the relay aspartate2075 of DhkA are both critical to its function but that another histidine kinase, DhkB, can partially compensate for the lack of DhkA activity. Sporulation is drastically reduced in double mutants lacking both DhkA and DhkB. Suppressor studies indicate that the cyclic AMP (cAMP) phosphodiesterase RegA and the cAMP-dependent protein kinase PKA act downstream of DhkA.     

Distribution of cAMP-dependent protein kinase during development in Dictyostelium discoideum

During the developmental cycle of Dictyostelium discoideum cyclic AMP functions as both a chemotactic signal for aggregation and a regulatory molecule during later events of differentiation. Morphological and biochemical data suggest that cAMP may direct cells during morphogenesis and differentiation. We utilized microtechniques to determine the stage- and cell-specific levels of the cAMP-dependent protein kinase, the probable intracellular cAMP receptor. Kinase activity was low and non-cAMP-dependent in amoebae and early aggregates but increased and became cAMP-dependent in aggregates after the formation of tight cell contacts. Maximum kinase activity and cAMP dependency occurred during the slug and culmination stages. The only differential distribution of the kinase within a single stage occurred during culmination when the activity in the stalks was approximately one-fourth of that in the prespore mass. Preliminary evidence indicates that this difference is not due to an inhibitor. In all other stages tested cAMP-dependent protein kinase activity was equal in prespore and prestalk cells.