p23, a major COPI-vesicle membrane protein, constitutively cycles through the early secretory pathway

A novel type I transmembrane protein of COPI-coated vesicles, p23, has been demonstrated to be localized mainly to the Golgi complex. This protein and p24, another member of the p24 family, have been shown to bind coatomer via their short cytoplasmic tails. Here we demonstrate that p23 continuously cycles through the early secretory pathway. The cytoplasmic tail of p23 is shown to act as a functional retrieval signal as it confers endoplasmic reticulum (ER) residence to a CD8–p23 fusion protein. This ER localization is, at least in part, a result of retrieval from post-ER compartments because CD8–p23 fusion proteins receive post-ER modifications. In contrast, the cytoplasmic tail of p24 has been shown not to retrieve a CD8–p24 fusion protein. The coatomer binding motifs FF and KK in the cytoplasmic tail of p23 are reported to influence the steady-state localization of the CD8–p23 fusion protein within the ER–Golgi recycling pathway. It appears that the steady-state Golgi localization of endogenous p23 is maintained by its lumenal domain, as a fusion protein with the lumenal domain of CD8, and the membrane span as well as the cytoplasmic tail of p23 is no longer detected in the Golgi.

Gi regulation of secretory vesicle swelling examined by atomic force microscopy

In the last decade, several monomeric and heterotrimeric guanine nucleotide binding proteins have been identified to associate with secretory vesicles and to be implicated in exocytosis. Vesicle volume also has been proposed to play a regulatory role in secretory vesicle fusion at the plasma membrane. However, the molecular mechanism of function of the guanine nucleotide binding proteins and of the regulation of secretory vesicle volume in the exocytotic process remains unclear. In this study, we report association of the secretory vesicle membrane with the α subunit of a heterotrimeric GTP binding protein Gαi3 and implicate its involvement in vesicle swelling. Using an atomic force microscope in combination with confocal microscopy, we were able to study the dynamics of isolated zymogen granules, the secretory vesicles in exocrine pancreas. Exposure of zymogen granules to GTP resulted in a 15–25% increase in vesicle height as measured by the atomic force microscope and a similar increase in vesicle diameter as determined by confocal microscopy. Mas7, an active mastoparan analog known to stimulate Gi proteins, was found to stimulate the GTPase activity of isolated zymogen granules and cause swelling. Increase in vesicle size in the presence of GTP, NaF, and Mas7 were irreversible and KCl-sensitive. Ca2+ had no effect on zymogen granule size. Taken together, the results indicate that Gαi3 protein localized in the secretory vesicle membrane mediates vesicle swelling, a potentially important prerequisite for vesicle fusion at the cell plasma membrane.

Altered peptide ligands act as partial agonists by inhibiting phospholipase C activity induced by myasthenogenic T cell epitopes

Myasthenia gravis (MG) is a T cell-regulated, antibody-mediated autoimmune disease. Two peptides representing sequences of the human acetylcholine receptor α-subunit, p195–212 and p259–271, previously were shown to stimulate the proliferation of peripheral blood lymphocytes of patients with MG and were found to be immunodominant T cell epitopes in SJL and BALB/c mice, respectively. Single amino acid-substituted analogs of p195–212 and p259–271, as well as a dual analog composed of the tandemly arranged two single analogs, were shown to inhibit, in vitro and in vivo, MG-associated autoimmune responses. Stimulation of T cells through the antigen-specific T cell receptor activates tyrosine kinases and phospholipase C (PLC). Therefore, in attempts to understand the mechanism of action of the analogs, we first examined whether the myasthenogenic peptides trigger tyrosine phosphorylation and activation of phospholipase C. For that purpose, we measured generation of inositol phosphates and tyrosine phosphorylation of PLC after stimulation of the p195–212- and p259–271-specific T cell lines with these myasthenogenic peptides. Both myasthenogenic peptides stimulated generation of inositol phosphates as well as tyrosine phosphorylation of PLC. However, the single and dual analogs, although inducing tyrosine phosphorylation of PLC, could not induce PLC activity. Furthermore, the single and dual analogs inhibited the induced PLC activity whereas they could not inhibit tyrosine phosphorylation of PLC that was caused by the myasthenogenic peptides. Thus, the altered peptides and the dual analog act as partial agonists. The down-regulation of PLC activity by the analogs may account for their capacity to inhibit in vitro MG-associated T cell responses.

Neuropeptide release by efficient recruitment of diffusing cytoplasmic secretory vesicles

Neuropeptides are slowly released from a limited pool of secretory vesicles. Despite decades of research, the composition of this pool has remained unknown. Endocrine cell studies support the hypothesis that a population of docked vesicles supports the first minutes of hormone release. However, it has been proposed that mobile cytoplasmic vesicles dominate the releasable neuropeptide pool. Here, to determine the cellular basis of the releasable pool, single green fluorescent protein-labeled secretory vesicles were visualized in neuronal growth cones with the use of an inducible construct or total internal reflection fluorescence microscopy. We report that vesicle movement follows the diffusion equation. Furthermore, rapidly moving secretory vesicles are used more efficiently than stationary vesicles near the plasma membrane to support stimulated release. Thus, randomly moving cytoplasmic vesicles participate in the first minutes of neuropeptide release. Importantly, the preferential recruitment of diffusing cytoplasmic secretory vesicles contributes to the characteristic slow kinetics and limited extent of sustained neuropeptide release.

Phospholipase C-linked receptors regulate the ATP-sensitive potassium channel by means of phosphatidylinositol 4,5-bisphosphate metabolism

In the COS7 cells transfected with cDNAs of the Kir6.2, SUR2A, and M1 muscarinic receptors, we activated the ATP-sensitive potassium (KATP) channel with a K+ channel opener and recorded the whole-cell KATP current. The KATP current was reversibly inhibited by the stimulation of the M1 receptor, which is linked to phospholipase C (PLC) by the Gq protein. The receptor-mediated inhibition was observed even when protein kinase C (PKC) was inhibited by H-7 or by chelating intracellular Ca2+ with 10 mM 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetate (BAPTA) included in the pipette solution. However, the receptor-mediated inhibition was blocked by U-73122, a PLC inhibitor. M1-receptor stimulation failed to inhibit the KATP current activated by the injection of exogenous phosphatidylinositol 4,5-bisphosphate (PIP2) through the whole-cell patch pipette. The receptor-mediated inhibition became irreversible when the replenishment of PIP2 was blocked by wortmannin (an inhibitor of phosphatidylinositol kinases), or by including adenosine 5′-[β,γ–imido]triphosphate (AMPPNP, a nonhydrolyzable ATP analogue) in the pipette solution. In inside-out patch experiments, the ATP sensitivity of the KATP channel was significantly higher when the M1 receptor in the patch membrane was stimulated by acetylcholine. The stimulatory effect of pinacidil was also attenuated under this condition. We postulate that stimulation of PLC-linked receptors inhibited the KATP channel by increasing the ATP sensitivity, not through PKC activation, but most probably through changing PIP2 levels.

Inhibition of phospholipase D by lysophosphatidylethanolamine, a lipid-derived senescence retardant

Phospholipid signaling mediated by lipid-derived second messengers or biologically active lipids is still new and is not well established in plants. We recently have found that lysophosphatidylethanolamine (LPE), a naturally occurring lipid, retards senescence of leaves, flowers, and postharvest fruits. Phospholipase D (PLD) has been suggested as a key enzyme in mediating the degradation of membrane phospholipids during the early stages of plant senescence. Here we report that LPE inhibited the activity of partially purified cabbage PLD in a cell-free system in a highly specific manner. Inhibition of PLD by LPE was dose-dependent and increased with the length and unsaturation of the LPE acyl chain whereas individual molecular components of LPE such as ethanolamine and free fatty acid had no effect on PLD activity. Enzyme-kinetic analysis suggested noncompetitive inhibition of PLD by LPE. In comparison, the related lysophospholipids such as lysophosphatidylcholine, lysophosphatidylglycerol, and lysophosphotidylserine had no significant effect on PLD activity whereas PLD was stimulated by lysophosphatidic acid and inhibited by lysophosphatidylinositol. Membrane-associated and soluble PLD, extracted from cabbage and castor bean leaf tissues, also was inhibited by LPE. Consistent with acyl-specific inhibition of PLD by LPE, senescence of cranberry fruits as measured by ethylene production was more effectively inhibited according to the increasing acyl chain length and unsaturation of LPE. There are no known specific inhibitors of PLD in plants and animals. We demonstrate specific inhibitory regulation of PLD by a lysophospholipid.

The Small Mr Ras-like GTPase Rap1 and the Phospholipase C Pathway Act to Regulate Phagocytosis in Dictyostelium discoideum

The function of the small-Mr Ras-like GTPase Rap1 remains largely unknown, but this protein has been demonstrated to regulate cortical actin-based morphologic changes in Dictyostelium and the oxidative burst in mammalian neutrophils. To test whether Rap1 regulates phagocytosis, we biochemically analyzed cell lines that conditionally and modestly overexpressed wild-type [Rap1 WT(+)], constitutively active [Rap1 G12T(+)], and dominant negative [Rap1 S17N(+)] forms of D. discoideum Rap1. The rates of phagocytosis of bacteria and latex beads were significantly higher in Rap1 WT(+) and Rap1 G12T(+) cells and were reduced in Rap1 S17N(+) cells. The addition of inhibitors of protein kinase A, protein kinase G, protein tyrosine kinase, or phosphatidylinositide 3-kinase did not affect phagocytosis rates in wild-type cells. In contrast, the addition of U73122 (a phospholipase C inhibitor), calphostin C (a protein kinase C inhibitor), and BAPTA-AM (an intracellular Ca2+ chelator) reduced phagocytosis rates by 90, 50, and 65%, respectively, suggesting both arms of the phospholipase C signaling pathways played a role in this process. Other protein kinase C–specific inhibitors, such as chelerythrine and bisindolylmaleimide I, did not reduce phagocytosis rates in control cells, suggesting calphostin C was affecting phagocytosis by interfering with a protein containing a diacylglycerol-binding domain. The addition of calphostin C did not reduce phagocytosis rates in Rap1 G12T(+) cells, suggesting that the putative diacylglycerol-binding protein acted upstream in a signaling pathway with Rap1. Surprisingly, macropinocytosis was significantly reduced in Rap1 WT(+) and Rap1 G12T(+) cells compared with control cells. Together our results suggest that Rap1 and Ca2+ may act together to coordinate important early events regulating phagocytosis.

Enhanced Glycosylation and Sulfation of Secretory Proteoglycans Is Coupled to the Expression of a Basic Secretory Protein

We have used coexpression of a salivary basic proline-rich protein (PRP) along with a proline-rich proteoglycan (PRPg) in pituitary AtT-20 cells to examine the regulation of glycosaminoglycan (GAG) biosynthesis and the storage of these secretory products for regulated secretion. The basic PRP caused a dose-dependent increase in sulfation of PRPg and also increased the extent to which PRPg polypeptide backbones are modified by a GAG chain. The sulfation of an endogenous proteoglycan was similarly increased in the presence of basic PRP; however, other sulfated secretory products of AtT-20 cells were unaffected. These results imply that enzymes functioning in elongation and sulfation of proteoglycans are coordinately regulated and that their activities respond to a change in the milieu of the intracellular transport pathway. Analysis of the regulated secretion of both the basic PRP and PRPg has indicated that while the presence of the GAG chain improves the storage of PRPg, the presence of PRPg does not increase the storage of basic PRP. Therefore, sulfation of GAGs does not appear to be a primary factor in regulated secretory sorting.

SNAP-25 Palmitoylation and Plasma Membrane Targeting Require a Functional Secretory Pathway

Synaptosomal-associated protein of 25 kDa (SNAP-25) is a palmitoylated membrane protein essential for neurotransmitter release from synaptic terminals. We used neuronal cell lines to study the biosynthesis and posttranslational processing of SNAP-25 to investigate how palmitoylation contributes to the subcellular localization of the protein. SNAP-25 was synthesized as a soluble protein that underwent palmitoylation approximately 20 min after synthesis. Palmitoylation of the protein coincided with its stable membrane association. Treatment of cells with brefeldin A or other disrupters of transport inhibited palmitoylation of newly synthesized SNAP-25 and abolished membrane association. These results demonstrate that the processing of SNAP-25 and its targeting to the plasma membrane depend on an intact transport mechanism along the exocytic pathway. The kinetics of SNAP-25 palmitoylation and membrane association and the sensitivity of these parameters to brefeldin A suggest a novel trafficking pathway for targeting proteins to the plasma membrane. In vitro, SNAP-25 stably associated with membranes was not released from the membrane after chemical deacylation. We propose that palmitoylation of SNAP-25 is required for initial membrane targeting of the protein but that other interactions can maintain membrane association in the absence of fatty acylation.

The Secretory Route of the Leaderless Protein Interleukin 1β Involves Exocytosis of Endolysosome-related Vesicles

Interleukin 1β (IL-1β), a secretory protein lacking a signal peptide, does not follow the classical endoplasmic reticulum-to-Golgi pathway of secretion. Here we provide the evidence for a “leaderless” secretory route that uses regulated exocytosis of preterminal endocytic vesicles to transport cytosolic IL-1β out of the cell. Indeed, although most of the IL-1β precursor (proIL-1β) localizes in the cytosol of activated human monocytes, a fraction is contained within vesicles that cofractionate with late endosomes and early lysosomes on Percoll density gradients and display ultrastructural features and markers typical of these organelles. The observation of organelles positive for both IL-1β and the endolysosomal hydrolase cathepsin D or for both IL-1β and the lysosomal marker Lamp-1 further suggests that they belong to the preterminal endocytic compartment. In addition, similarly to lysosomal hydrolases, secretion of IL-1β is induced by acidotropic drugs. Treatment of monocytes with the sulfonylurea glibenclamide inhibits both IL-1β secretion and vesicular accumulation, suggesting that this drug prevents the translocation of proIL-1β from the cytosol into the vesicles. A high concentration of extracellular ATP and hypotonic medium increase secretion of IL-1β but deplete the vesicular proIL-1β content, indicating that exocytosis of proIL-1β–containing vesicles is regulated by ATP and osmotic conditions.

The medial-Golgi Ion Pump Pmr1 Supplies the Yeast Secretory Pathway with Ca2+ and Mn2+ Required for Glycosylation, Sorting, and Endoplasmic Reticulum-Associated Protein Degradation

The yeast Ca2+ adenosine triphosphatase Pmr1, located in medial-Golgi, has been implicated in intracellular transport of Ca2+ and Mn2+ ions. We show here that addition of Mn2+ greatly alleviates defects of pmr1 mutants in N-linked and O-linked protein glycosylation. In contrast, accurate sorting of carboxypeptidase Y (CpY) to the vacuole requires a sufficient supply of intralumenal Ca2+. Most remarkably, pmr1 mutants are also unable to degrade CpY*, a misfolded soluble endoplasmic reticulum protein, and display phenotypes similar to mutants defective in the stress response to malfolded endoplasmic reticulum proteins. Growth inhibition of pmr1 mutants on Ca2+-deficient media is overcome by expression of other Ca2+ pumps, including a SERCA-type Ca2+ adenosine triphosphatase from rabbit, or by Vps10, a sorting receptor guiding non-native luminal proteins to the vacuole. Our analysis corroborates the dual function of Pmr1 in Ca2+ and Mn2+ transport and establishes a novel role of this secretory pathway pump in endoplasmic reticulum-associated processes.

Tyrosine Phosphorylation of Selected Secretory Carrier Membrane Proteins, SCAMP1 and SCAMP3, and Association with the EGF Receptor

Secretory carrier membrane proteins (SCAMPs) are ubiquitously expressed proteins of post-Golgi vesicles. In the presence of the tyrosine phosphatase inhibitor vanadate, or after overexpression in Chinese hamster ovary (CHO) cells, SCAMP1 and SCAMP3 are phosphorylated selectively on tyrosine residue(s). Phosphorylation is reversible after vanadate washout in situ or when isolated SCAMP3 is incubated with the recombinant tyrosine phosphatase PTP1B. Vanadate also causes the partial accumulation of SCAMP3, but not SCAMP1, in “patches” at or near the cell surface. A search for SCAMP kinase activities has shown that SCAMPs 1 and 3, but not SCAMP2, are tyrosine phosphorylated in EGF-stimulated murine fibroblasts overexpressing the EGF receptor (EGFR). EGF catalyzes the progressive phosphorylation of the SCAMPs up to 1 h poststimulation and may enhance colocalization of the EGFR and SCAMP3 within the cell interior. EGF also induces SCAMP–EGFR association, as detected by coimmunoprecipitation, and phosphorylation of SCAMP3 is stimulated by the EGFR in vitro. These results suggest that phosphorylation of SCAMPs, either directly or indirectly, may be functionally linked to the internalization/down-regulation of the EGFR.

ADP-Ribosylation Factors Do Not Activate Yeast Phospholipase Ds but Are Required for Sporulation

ADP-ribosylation factor (ARF) proteins in Saccharomyces cerevisiae are encoded by two genes, ARF1 and ARF2. The addition of the c-myc epitope at the C terminus of Arf1 resulted in a mutant (arf1-myc arf2) that supported vegetative growth and rescued cells from supersensitivity to fluoride, but homozygous diploids failed to sporulate. arf1-myc arf2 mutants completed both meiotic divisions but were unable to form spores. The SPO14 gene encodes a phospholipase D (PLD), whose activity is essential for mediating the formation of the prospore membrane, a prerequisite event for spore formation. Spo14 localized normally to the developing prospore membrane in arf1-myc arf2 mutants; however, the synthesis of the membrane was attenuated. This was not a consequence of reduced PLD catalytic activity, because the enzymatic activity of Spo14 was unaffected in meiotic arf1-myc arf2 mutants. Although potent activators of mammalian PLD1, Arf1 proteins did not influence the catalytic activities of either Spo14 or ScPld2, a second yeast PLD. These results demonstrate that ARF1 is required for sporulation, and the mitotic and meiotic functions of Arf proteins are not mediated by the activation of any known yeast PLD activities. The implications of these results are discussed with respect to current models of Arf signaling.

Secretagogue-triggered Transfer of Membrane Proteins from Neuroendocrine Secretory Granules to Synaptic-like Microvesicles

The membrane proteins of all regulated secretory organelles (RSOs) recycle after exocytosis. However, the recycling of those membrane proteins that are targeted to both dense core granules (DCGs) and synaptic-like microvesicles (SLMVs) has not been addressed. Since neuroendocrine cells contain both RSOs, and the recycling routes that lead to either organelle overlap, transfer between the two pools of membrane proteins could occur during recycling. We have previously demonstrated that a chimeric protein containing the cytosolic and transmembrane domains of P-selectin coupled to horseradish peroxidase is targeted to both the DCG and the SLMV in PC12 cells. Using this chimera, we have characterized secretagogue-induced traffic in PC12 cells. After stimulation, this chimeric protein traffics from DCGs to the cell surface, internalizes into transferrin receptor (TFnR)-positive endosomes and thence to a population of secretagogue-responsive SLMVs. We therefore find a secretagogue-dependent rise in levels of HRP within SLMVs. In addition, the levels within SLMVs of the endogenous membrane protein, synaptotagmin, as well as a green fluorescent protein-tagged version of vesicle-associated membrane protein (VAMP)/synaptobrevin, also show a secretagogue-dependent increase.

Quality Control in the Secretory Pathway: The Role of Calreticulin, Calnexin and BiP in the Retention of Glycoproteins with C-Terminal Truncations

Unlike properly folded and assembled proteins, most misfolded and incompletely assembled proteins are retained in the endoplasmic reticulum of mammalian cells and degraded without transport to the Golgi complex. To analyze the mechanisms underlying this unique sorting process and its fidelity, the fate of C-terminally truncated fragments of influenza hemagglutinin was determined. An assortment of different fragments was generated by adding puromycin at low concentrations to influenza virus-infected tissue culture cells. Of the fragments generated, <2% was secreted, indicating that the system for detecting defects in newly synthesized proteins is quite stringent. The majority of secreted species corresponded to folding domains within the viral spike glycoprotein. The retained fragments acquired a partially folded structure with intrachain disulfide bonds and conformation-dependent antigenic epitopes. They associated with two lectin-like endoplasmic reticulum chaperones (calnexin and calreticulin) but not BiP/GRP78. Inhibition of the association with calnexin and calreticulin by the addition of castanospermine significantly increased fragment secretion. However, it also caused association with BiP/GRP78. These results indicated that the association with calnexin and calreticulin was involved in retaining the fragments. They also suggested that BiP/GRP78 could serve as a backup for calnexin and calreticulin in retaining the fragments. In summary, the results showed that the quality control system in the secretory pathway was efficient and sensitive to folding defects, and that it involved multiple interactions with endoplasmic reticulum chaperones.