Glycosylphosphatidylinositol is involved in the membrane attachment of proteins in granules of chromaffin cells.
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ABSTRACT:
SUBMITTER: Fouchier F
PROVIDER: S-EPMC1135374 | biostudies-other | 1988 Nov
REPOSITORIES: biostudies-other
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Project description:1. Soluble lysates and membranes were prepared from chromaffin granules isolated from bovine adrenal medulla. The detergent N-cetylpyridinium chloride was used for solubilizing the membrane proteins, including the membrane-bound dopamine (2,4-dihydroxyphenethylamine) beta-hydroxylase. The solubilized proteins were fractionated by Sephadex chromatography in the presence of N-cetylpyridinium chloride. The major component of the membrane proteins, i.e. chromomembrin A, was identified as the enzyme dopamine beta-hydroxylase. 2. The addition of N-cetylpyridinium chloride to the soluble lysate caused precipitation of up to 96% of the proteins, but only a small proportion of the dopamine beta-hydroxylase activity was precipitated. The only protein demonstrable in the supernatant by polyacrylamide-gel electrophoresis was the protein that has a lower mobility than chromogranin A in disc gel electrophoresis. This component has been identified previously as dopamine beta-hydroxylase. Thus, this method provides an extremely simple isolation procedure for dopamine beta-hydroxylase. 3. A comparison of the membrane-bound and soluble dopamine beta-hydroxylases revealed the identity of these two preparations. Both were activated by N-cetylpyridinium chloride, they migrated identically in polyacrylamide-gel electrophoresis, their amino acid composition was very similar and an immunological cross-reaction could be demonstrated.
| S-EPMC1174055 | biostudies-other
Project description:Lipid remodeling of Glycosylphosphatidylinositol (GPI) anchors is required for their maturation and may influence the localization and function of GPI-anchored proteins (GPI-APs). Maturation of GPI-anchors is well characterized in animals and fungi but very little is known about this process in plants. In yeast, the GPI-lipid remodeling occurs entirely at the ER and is initiated by the remodeling enzyme Bst1p (Post-Glycosylphosphatidylinositol Attachment to Proteins inositol deacylase 1 -PGAP1- in mammals and Arabidopsis). Next, the remodeling enzyme Per1p (Post-Glycosylphosphatidylinositol Attachment to Proteins phospholipase 3 -PGAP3- in mammals) removes a short, unsaturated fatty acid of phosphatidylinositol (PI) that is replaced with a very long-chain saturated fatty acid or ceramide to complete lipid remodeling. In mammals, lipid remodeling starts at the ER and is completed at the Golgi apparatus. Studies of the Arabidopsis PGAP1 gene showed that the lipid remodeling of the GPI anchor is critical for the final localization of GPI-APs. Here we characterized loss-of-function mutants of Arabidopsis Per1/PGAP3 like genes (AtPGAP3A and AtPGAP3B). Our results suggest that PGAP3A function is required for the efficient transport of GPI-anchored proteins from the ER to the plasma membrane/cell wall. In addition, loss of function of PGAP3A increases susceptibility to salt and osmotic stresses that may be due to the altered localization of GPI-APs in this mutant. Furthermore, PGAP3B complements a yeast strain lacking PER1 gene suggesting that PGAP3B and Per1p are functional orthologs. Finally, subcellular localization studies suggest that PGAP3A and PGAP3B cycle between the ER and the Golgi apparatus.
| S-EPMC8874281 | biostudies-literature
Project description:Positioned at the C-terminus of many eukaryotic proteins, the glycosylphosphatidylinositol (GPI) anchor is a posttranslational modification that anchors the modified protein in the outer leaflet of the cell membrane. The GPI anchor is a complex structure comprising a phosphoethanolamine linker, glycan core, and phospholipid tail. GPI-anchored proteins are structurally and functionally diverse and play vital roles in numerous biological processes. While several GPI-anchored proteins have been characterized, the biological functions of the GPI anchor have yet to be elucidated at a molecular level. This review discusses the structural diversity of the GPI anchor and its putative cellular functions, including involvement in lipid raft partitioning, signal transduction, targeting to the apical membrane, and prion disease pathogenesis. We specifically highlight studies in which chemically synthesized GPI anchors and analogues have been employed to study the roles of this unique posttranslational modification.
| S-EPMC2663890 | biostudies-literature
Project description:The roles of nonmuscle myosin II and cortical actin filaments in chromaffin granule exocytosis were studied by confocal fluorescence microscopy, amperometry, and cell-attached capacitance measurements. Fluorescence imaging indicated decreased mobility of granules near the plasma membrane following inhibition of myosin II function with blebbistatin. Slower fusion pore expansion rates and longer fusion pore lifetimes were observed after inhibition of actin polymerization using cytochalasin D. Amperometric recordings revealed increased amperometric spike half-widths without change in quantal size after either myosin II inhibition or actin disruption. These results suggest that actin and myosin II facilitate release from individual chromaffin granules by accelerating dissociation of catecholamines from the intragranular matrix possibly through generation of mechanical forces.
| S-EPMC2768403 | biostudies-literature
Project description:Pore-spanning membranes (PSMs) composed of supported membrane parts as well as freestanding membrane parts are shown to be very versatile to investigate SNARE-mediated fusion on the single-particle level. They provide a planar geometry readily accessible by confocal fluorescence microscopy, which enabled us for the first time, to our knowledge, to investigate the fusion of individual natural secretory granules (i.e., chromaffin granules (CGs)) on the single-particle level by two-color fluorescence microscopy in a time-resolved manner. The t-SNARE acceptor complex ΔN49 was reconstituted into PSMs containing 2 mol % 1,2-dipalmitoyl-sn-glycero-3-phosphatidylinositol-4,5-bisphosphate and Atto488-1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine, and CGs were fluorescently labeled with 2-((1E,3E)-5-((Z)-3,3-dimethyl-1-octadecylindolin-2-ylidene)penta-1,3-dien-1-yl)-3,3-dimethyl-1-octadecyl-3H-indol-1-ium perchlorate. We compared the dynamics of docked and hemifused CGs as well as their fusion efficacy and kinetics with the results obtained for synthetic synaptobrevin 2-doped vesicles fusing with PSMs of the same composition. Whereas the synthetic vesicles were fully immobile on supported PSMs, docked as well as hemifused CGs were mobile on both PSM parts, which suggests that this system resembles more closely the natural situation. The fusion process of CGs proceeded through three-dimensional post-lipid-mixing structures, which were readily resolved on the gold-covered pore rims of the PSMs and which are discussed in the context of intermediate states observed in live cells.
| S-EPMC6350068 | biostudies-literature
Project description:The glycosylphosphatidylinositol (GPI) anchor is a lipid and glycan modification added to the C terminus of certain proteins in the endoplasmic reticulum by the activity of a multiple subunit enzyme complex known as the GPI transamidase (GPIT). Several subunits of GPIT have increased expression levels in breast carcinoma. In an effort to identify GPI-anchored proteins and understand the possible role of these proteins in breast cancer progression, we employed a combination of strategies. First, alpha toxin from Clostridium septicum was used to capture GPI-anchored proteins from human breast cancer tissues, cells, and serum for proteomic analysis. We also expressed short interfering RNAs targeting the expression of the GPAA1 and PIGT subunits of GPIT in breast cancer cell lines to identify proteins in which membrane localization is dependent on GPI anchor addition. Comparative membrane proteomics using nano-ESI-RPLC-MS/MS led to the discovery of several new potential diagnostic and therapeutic targets for breast cancer. Furthermore, we provide evidence that increased levels of GPI anchor addition in malignant breast epithelial cells promotes the dedifferentiation of malignant breast epithelial cells in part by increasing the levels of cell surface markers associated with mesenchymal stem cells.
| S-EPMC3408190 | biostudies-literature
Project description:Arabinogalactan-proteins (AGPs) are a class of proteoglycans found in cell secretions and plasma membranes of plants. Attention is currently focused on their structure and their potential role in growth and development. We present evidence that two members of a major class of AGPs, the classical AGPs, AGPNa1 from styles of Nicotiana alata and AGPPc1 from cell suspension cultures of Pyrus communis, undergo C-terminal processing involving glycosylphosphatidylinositol membrane anchors. The evidence is that (i) the transmembrane helix at the C terminus predicted from the cDNA encoding these proteins is not present-the C-terminal amino acid is Asn87 and Ser97 for AGPNa1 and AGPPc1, respectively; (ii) both AGP protein backbones are substituted with ethanolamine at the C-terminal amino acid; and (iii) inositol, glucosamine, and mannose are present in the native AGPs. An examination of the deduced amino acid sequences of other classical AGP protein backbones shows that glycosylphosphatidylinositol-anchors may be a common feature of this class of AGPs.
| S-EPMC20905 | biostudies-literature
Project description:Adrenomedullary chromaffin cells respond to sympathetic nervous system activation by secreting a cocktail of potent neuropeptides and hormones into the circulation. The distinct phases of the chromaffin cell secretory response have been attributed to the progressive fusion of distinct populations of dense core granules with different activation kinetics. However, it has been difficult to define what distinguishes these populations at the molecular level. Functional segregation of granule pools may depend on selective sorting of synaptotagmin-1 (Syt-1) and synaptotagmin-7 (Syt-7), which our previous work showed are rarely cosorted to the same granule. Here we assess the consequences of selective sorting of Syt isoforms in chromaffin cells, particularly with respect to granule dynamics and activation kinetics. Upon depolarization of cells expressing fluorescent Syt isoforms using elevated K+, we find that Syt-7 granules fuse with faster kinetics than Syt-1 granules, irrespective of stimulation strength. Pharmacological blockade of Ca2+ channels reveals differential dependence of Syt-1 versus Syt-7 granule exocytosis on Ca2+ channel subtypes. Syt-7 granules also show a greater tendency to fuse in clusters than Syt-1 granules, and granules harboring Syt-1 travel a greater distance before fusion than those with Syt-7, suggesting that there is spatial and fusion-site heterogeneity among the two granule populations. However, the greatest functional difference between granule populations is their responsiveness to Ca2+ Upon introduction of Ca2+ into permeabilized cells, Syt-7 granules fuse with fast kinetics and high efficacy, even at low Ca2+ levels (e.g., when cells are weakly stimulated). Conversely, Syt-1 granules require a comparatively larger increase in intracellular Ca2+ for activation. At Ca2+ concentrations above 30 µM, activation kinetics are faster for Syt-1 granules than for Syt-7 granules. Our study provides evidence for functional specialization of chromaffin cell granules via selective expression of Syt isoforms with different Ca2+ sensitivities.
| S-EPMC5560776 | biostudies-literature
Project description:Soluble N-ethylmaleimide-sensitive factor activating protein receptor (SNARE) proteins are the main catalysts for membrane fusion in the secretory pathway of eukaryotic cells. In vitro, SNAREs are sufficient to mediate effective fusion of both native and artificial membranes. Here we have established, to our knowledge, a new platform for monitoring SNARE-mediated docking and fusion between giant unilamellar vesicles (GUVs) and smaller liposomes or purified secretory granules with high temporal and spatial resolution. Analysis of fusion is restricted to the free-standing part of the GUV-membrane exhibiting low curvature and a lack of surface contact, thus avoiding adhesion-mediated interference with the fusion reaction as in fusion with supported bilayers or surface-immobilized small vesicles. Our results show that liposomes and chromaffin granules fuse with GUVs containing activated SNAREs with only few milliseconds delay between docking and fusion. We conclude that after initial contact in trans, SNAREs alone can complete fusion at a rate close to fast neuronal exocytosis.
| S-EPMC5607038 | biostudies-literature
Project description:Upon fusion of the secretory granule with the plasma membrane, small molecules are discharged through the immediately formed narrow fusion pore, but protein discharge awaits pore expansion. Recently, fusion pore expansion was found to be regulated by tissue plasminogen activator (tPA), a protein present within the lumen of chromaffin granules in a subpopulation of chromaffin cells. Here, we further examined the influence of other lumenal proteins on fusion pore expansion, especially chromogranin A (CgA), the major and ubiquitous lumenal protein in chromaffin granules. Polarized TIRF microscopy demonstrated that the fusion pore curvature of granules containing CgA-EGFP was long lived, with curvature lifetimes comparable to those of tPA-EGFP-containing granules. This was surprising because fusion pore curvature durations of granules containing exogenous neuropeptide Y-EGFP (NPY-EGFP) are significantly shorter (80% lasting <1 s) than those containing CgA-EGFP, despite the anticipated expression of endogenous CgA. However, quantitative immunocytochemistry revealed that transiently expressed lumenal proteins, including NPY-EGFP, caused a down-regulation of endogenously expressed proteins, including CgA. Fusion pore curvature durations in nontransfected cells were significantly longer than those of granules containing overexpressed NPY but shorter than those associated with granules containing overexpressed tPA, CgA, or chromogranin B. Introduction of CgA to NPY-EGFP granules by coexpression converted the fusion pore from being transient to being longer lived, comparable to that found in nontransfected cells. These findings demonstrate that several endogenous chromaffin granule lumenal proteins are regulators of fusion pore expansion and that alteration of chromaffin granule contents affects fusion pore lifetimes. Importantly, the results indicate a new role for CgA. In addition to functioning as a prohormone, CgA plays an important role in controlling fusion pore expansion.
| S-EPMC6363410 | biostudies-literature