Synaptic-like microvesicles of neuroendocrine cells originate from a novel compartment that is continuous with the plasma membrane and devoid of transferrin receptor.

We have characterized the compartment from which synaptic-like microvesicles (SLMVs), the neuroendocrine counterpart of neuronal synaptic vesicles, originate. For this purpose we have exploited the previous observation that newly synthesized synaptophysin, a membrane marker of synaptic vesicles and SLMVs, is delivered to the latter organelles via the plasma membrane and an internal compartment. Specifically, synaptophysin was labeled by cell surface biotinylation of unstimulated PC12 cells at 18 degrees C, a condition which blocked the appearance of biotinylated synaptophysin in SLMVs and in which there appeared to be no significant exocytosis of SLMVs.

Asymmetric division and polarity of neuroepithelial cells.

Neuroepithelial cells, the progenitors to the CNS neurons and glia, undergo both symmetric and asymmetric divisions. Symmetric divisions underlie the proliferation of neuroepithelial cells that predominates early in CNS development. Asymmetric divisions are thought to generate the cell types derived from neuroepithelial cells, such as neurons.

Loss of occludin and functional tight junctions, but not ZO-1, during neural tube closure--remodeling of the neuroepithelium prior to neurogenesis.

Neuroepithelial cells can generate nonepithelial cells, the neurons. Here we have investigated, for chick and mouse embryos, the epithelial character of neuroepithelial cells in the context of neurogenesis by examining the presence of molecular components of tight junctions during the transition from the neural plate to the neural tube. Immunoreactivity for occludin, a transmembrane protein specific to tight junctions, was detected at the apical end of the lateral membrane of neuroepithelial cells throughout the chick neural plate.

Overall lack of regulated secretion in a PC12 variant cell clone.

A stable clone of PC12 neuroendocrine cells, named 27, known from previous studies to exhibit a defect of regulated secretion (lack of regulated secretory proteins, of synaptophysin, of dense granules and of catecholamine uptake and release; Clementi, E., Racchetti, G., Zacchetti, D.

Expression of tyrosine-sulfated secretory proteins in Xenopus laevis oocytes. Differential export of constitutive and regulated proteins.

Xenopus laevis oocytes were used to study the tyrosine sulfation and secretion of exogenous proteins. Secretogranin II (SgII), a tyrosine-sulfated protein found in secretory granules of a wide variety of endocrine cells and neurons, became tyrosine-sulfated by the oocytes when expressed by injection of poly(A)-rich RNA isolated from the neuroendocrine cell line PC12. The same result was observed when SgII was expressed from cloned SgII cRNA, showing that its tyrosine sulfation did not require the coexpression of exogenous tyrosylprotein sulfotransferase (TPST) but occurred by means of the endogenous oocyte TPST.

Chromogranin B (secretogranin I) promotes sorting to the regulated secretory pathway of processing intermediates derived from a peptide hormone precursor.

Chromogranin B (CgB, secretogranin I) is a widespread constituent of neuroendocrine secretory granules whose function is unknown. To determine whether CgB affects the sorting of peptide hormone and neuropeptide precursors to secretory granules, we overexpressed CgB in AtT-20 cells, which exhibit an only moderate capacity to sort proopiomelanocortin and proteolytic fragments derived therefrom. In mock-transfected AtT-20 cells, a substantial proportion of newly synthesized proopiomelanocortin and its two primary proteolytic products generated in the trans-Golgi network, the N-terminal 23-kDa fragment containing adrenocorticotropin and the C-terminal beta-lipotropin fragment, was secreted via the constitutive pathway.

A role for ADP-ribosylation factor 1, but not COP I, in secretory vesicle biogenesis from the trans-Golgi network.

A synthetic N-myristoylated peptide corresponding to the amino-terminal domain of ADP-ribosylation factor 1 (ARF1) markedly increases, in a cell-free system using post-nuclear supernatant from PC12 cells, the biogenesis of constitutive secretory vesicles and immature secretory granules from the trans-Golgi network (TGN). The related N-myristoylated ARF4 peptide only weakly stimulates, and the non-myristoylated ARF1 and ARF4 peptides inhibit, the biogenesis of these secretory vesicles. In a modified cell-free system using TGN membranes, coatomer-depleted cytosol supports the biogenesis of TGN-derived secretory vesicles to the same extent as control cytosol.

Dispersion of chromogranin/secretogranin secretory protein family loci in mammalian genomes.

Chromogranin A, chromogranin B, and secretogranin II, members of the chromogranin/secretogranin secretory protein family, are overexpressed in some human hereditary maladies and may have arisen, in part, from common ancestor genes. To understand better the mammalian chromosomal dispersion of this gene family and to facilitate studies of these genes in human illnesses and their animal models, we positioned the locus of each member in the rat, mouse, and human genomes. Our results indicate that each locus lies in a region of locally syntenic chromosomal homology across the three species.

A role for phosphatidylinositol transfer protein in secretory vesicle formation.

Vesicular traffic in eukaryotic cells is characterized by two steps of membrane rearrangement: the formation of vesicles from donor membranes and their fusion with acceptor membranes. With respect to vesicle formation, several of the cytosolic proteins implicated in budding and fission have been identified. A feature common to all these proteins is that their targets, when known, are other proteins rather than lipids.

Neurosecretory vesicles can be hybrids of synaptic vesicles and secretory granules.

We have investigated the relationship of the so-called small dense core vesicle (SDCV), the major catecholamine-containing neurosecretory vesicle of sympathetic neurons, to synaptic vesicles containing classic neurotransmitters and secretory granules containing neuropeptides. SDCVs contain membrane proteins characteristic of synaptic vesicles such as synaptophysin and synaptoporin. However, SDCVs also contain membrane proteins characteristic of certain secretory granules like the vesicular monoamine transporter and the membrane-bound form of dopamine beta-hydroxylase.

Synaptotagmin I- and II-deficient PC12 cells exhibit calcium-independent, depolarization-induced neurotransmitter release from synaptic-like microvesicles.

Synaptotagmin I- and II-deficient PC12 cells (Shoji-Kasai et al. [1]) were used to compare the role of this protein in the calcium-dependent exocytosis of secretory granules and synaptic-like microvesicles (SLMVs). While neither catecholamine nor protein secretion from secretory granules were altered, the depolarization-induced acetylcholine release from SLMVs was no longer calcium-dependent.

Regulated secretion. Helper proteins for neuroendocrine secretion.

The granins, known to be general constituents of neuroendocrine secretory granules, are regulators of the proteolytic processing of peptide precursors and promote their aggregation-mediated sorting into secretory granules.

XL alpha s is a new type of G protein.

The GTP-binding proteins are well-known regulators of cellular functions, including vesicular transport. Cholera toxin, which is known to catalyse ADP-ribosylation of the alpha s subunit of heterotrimeric G proteins, stimulates secretory vesicle formation from the trans-Golgi network. Here we describe a new cholera toxin target, an 'extra large' G protein (XL alpha s; M(r) 92K) which consists of a new 51K XL-portion linked to a G alpha s truncated at the amino terminus.

An elevation of cytosolic protein phosphorylation modulates trimeric G-protein regulation of secretory vesicle formation from the trans-Golgi network.

The role of protein phosphorylation in the formation of secretory vesicles from the trans-Golgi network (TGN) and in the regulation of this process by TGN-associated trimeric G-proteins was investigated, using a previously established and a novel cell-free system derived from the neuroendocrine cell line PC12. In the absence of exogenous activators of trimeric G-proteins, okadaic acid, an inhibitor of protein serine/threonine phosphatase types 1, 2A, and PPX, had no significant effect on secretory vesicle formation as reconstituted in a postnuclear supernatant. However, okadaic acid antagonized the inhibition of secretory vesicle formation which occurred upon activation of trimeric G-proteins by either aluminum fluoride or guanosine 5'-3-O-(thio)-triphosphate (GTP gamma S).

The disulfide bond in chromogranin B, which is essential for its sorting to secretory granules, is not required for its aggregation in the trans-Golgi network.

Chromogranin B (secretogranin I), a protein sorted to secretory granules in many endocrine cells and neurons, undergoes selective aggregation during the sorting process in the trans-Golgi network. Reduction of the single, highly conserved intramolecular disulfide bond of chromogranin B by exposure of intact PC12 cells to the thiol reducing agent dithiothreitol has previously been shown to cause its missorting to the constitutive pathway of secretion. Using saponin perforation of membrane vesicles in aggregative buffer mimicking the milieu in the lumen of the trans-Golgi network (pH 6.

Protein tyrosine sulfation, 1993--an update.

Sulfation is the most abundant post-translational modification of tyrosine residues and occurs in many soluble and membrane proteins passing through the secretory pathway of metazoan cells. The sulfation reaction is catalysed by tyrosylprotein sulfotransferase, a membrane-bound enzyme of the trans-Golgi-network. Tyrosylprotein sulfotransferase has been purified and its substrate specificity characterized.

Peptides derived from the granins (chromogranins/secretogranins).

This review summarizes the role of granins (chromogranins/secretogranins) as precursors to biologically active peptides. We discuss the structural basis of the proteolytic processing of the granins, the consequences of their processing and the biological effects of granin-derived peptides.

Biogenesis of constitutive secretory vesicles, secretory granules and synaptic vesicles.

The formation of constitutive secretory vesicles and secretory granules from the trans-Golgi network is inhibited by brefeldin A, suggesting a role for ADP ribosylation factor and other coat proteins in the budding process, and is regulated by multiple heterotrimeric G proteins. Membrane proteins are sorted to secretory granules through signals in their cytoplasmic domains and, like secretory proteins, by aggregation of their lumenal domains. Synaptic vesicle membrane proteins undergo exocytotic-endocytotic recycling before synapse formation, consistent with the proposed biogenesis of synaptic vesicles from early endosomes.

Selective storage of acetylcholine, but not catecholamines, in neuroendocrine synaptic-like microvesicles of early endosomal origin.

We have defined, in the neuroendocrine cell line PC12, the catecholamine- and acetylcholine-storing organelles in the context of the biogenesis of secretory granules and synaptic-like microvesicles (SLMVs). SLMVs were found to originate directly from early endosomes. Both early endosomes and SLMVs exhibited uptake and storage of biosynthetic acetylcholine.

Reduction of the disulfide bond of chromogranin B (secretogranin I) in the trans-Golgi network causes its missorting to the constitutive secretory pathways.

The role of the single, highly conserved disulfide bond in chromogranin B (secretogranin I) on the sorting of this regulated secretory protein to secretory granules was investigated in the neuroendocrine cell line PC12. Treatment of PC12 cells with dithiothreitol (DTT), a membrane permeable thiol reducing agent known to prevent disulfide bond formation in intact cells, resulted in the secretion of newly synthesized chromogranin B, but only slightly decreased the intracellular storage of newly synthesized secretogranin II, a regulated secretory protein devoid of cysteines. The secretion of newly synthesized chromogranin B in the presence of DTT occurred with similar kinetics to those of a heparan sulfate proteoglycan, a known marker of the constitutive secretory pathway in PC12 cells.