Nitric oxide production within rat urothelial cells.

Purpose: Recent studies have suggested that nitric oxide (NO) synthase (NOS) may be localized in the urothelium of the proximal part of the mammalian ureter. We investigated endogenous NO production in the proximal half of the rat ureter, localized its cellular source, characterized the NOS isoforms involved and assessed the impact of NO on ureteral motility.

Materials And Methods: Direct detection of NO production was performed on primary cultures of living rat ureteral cells with the fluorescent indicator diaminofluorescein.

A resident Golgi protein is excluded from peri-Golgi vesicles in NRK cells.

To understand the structure and the function of the Golgi apparatus, it is essential to establish how resident Golgi enzymes are localized in only a few Golgi cisternae. In particular it is crucial to establish whether Golgi enzymes are retained specifically in cisternae, or if they are continuously transported from cisterna to cisterna. Here we report that a resident Golgi enzyme is largely excluded from peri-Golgi transport vesicles in normal rat kidney cells, a cell type in which conflicting results have been reported.

Lst1p and Sec24p cooperate in sorting of the plasma membrane ATPase into COPII vesicles in Saccharomyces cerevisiae.

Formation of ER-derived protein transport vesicles requires three cytosolic components, a small GTPase, Sar1p, and two heterodimeric complexes, Sec23/24p and Sec13/31p, which comprise the COPII coat. We investigated the role of Lst1p, a Sec24p homologue, in cargo recruitment into COPII vesicles in Saccharomyces cerevisiae. A tagged version of Lst1p was purified and eluted as a heterodimer complexed with Sec23p comparable to the Sec23/24p heterodimer.

Exclusion of golgi residents from transport vesicles budding from Golgi cisternae in intact cells.

A central feature of cisternal progression/maturation models for anterograde transport across the Golgi stack is the requirement that the entire population of steady-state residents of this organelle be continuously transported backward to earlier cisternae to avoid loss of these residents as the membrane of the oldest (trans-most) cisterna departs the stack. For this to occur, resident proteins must be packaged into retrograde-directed transport vesicles, and to occur at the rate of anterograde transport, resident proteins must be present in vesicles at a higher concentration than in cisternal membranes. We have tested this prediction by localizing two steady-state residents of medial Golgi cisternae (mannosidase II and N-acetylglucosaminyl transferase I) at the electron microscopic level in intact cells.

Megavesicles implicated in the rapid transport of intracisternal aggregates across the Golgi stack.

Engineered protein aggregates ranging up to 400 nm in diameter were selectively deposited within the cis-most cisternae of the Golgi stack following a 15 degrees C block. These aggregates are much larger than the standard volume of Golgi vesicles, yet they are transported across the stack within 10 min after warming the cells to 20 degrees C. Serial sectioning reveals that during the peak of anterograde transport, about 20% of the aggregates were enclosed in topologically free "megavesicles" which appear to pinch off from the rims of the cisternae.

Anterograde flow of cargo across the golgi stack potentially mediated via bidirectional "percolating" COPI vesicles.

How do secretory proteins and other cargo targeted to post-Golgi locations traverse the Golgi stack? We report immunoelectron microscopy experiments establishing that a Golgi-restricted SNARE, GOS 28, is present in the same population of COPI vesicles as anterograde cargo marked by vesicular stomatitis virus glycoprotein, but is excluded from the COPI vesicles containing retrograde-targeted cargo (marked by KDEL receptor). We also report that GOS 28 and its partnering t-SNARE heavy chain, syntaxin 5, reside together in every cisterna of the stack. Taken together, these data raise the possibility that the anterograde cargo-laden COPI vesicles, retained locally by means of tethers, are inherently capable of fusing with neighboring cisternae on either side.

Coupling of coat assembly and vesicle budding to packaging of putative cargo receptors.

COPI-coated vesicle budding from lipid bilayers whose composition resembles mammalian Golgi membranes requires coatomer, ARF, GTP, and cytoplasmic tails of putative cargo receptors (p24 family proteins) or membrane cargo proteins (containing the KKXX retrieval signal) emanating from the bilayer surface. Liposome-derived COPI-coated vesicles are similar to their native counterparts with respect to diameter, buoyant density, morphology, and the requirement for an elevated temperature for budding. These results suggest that a bivalent interaction of coatomer with membrane-bound ARF[GTP] and with the cytoplasmic tails of cargo or putative cargo receptors is the molecular basis of COPI coat assembly and provide a simple mechanism to couple uptake of cargo to transport vesicle formation.

COPII-coated vesicle formation reconstituted with purified coat proteins and chemically defined liposomes.

COPII vesicle formation requires only three coat assembly subunits: Sar1p, Sec13/31p, and Sec23/24p. PI 4-phosphate or PI 4,5-bisphosphate is required for the binding of these proteins to liposomes. The GTP-bound form of Sar1p recruits Sec23/24p to the liposomes as well as to the ER membranes, and this Sar1p-Sec23/24p complex is required for the binding of Sec13/31p.

Somatostatin-14, somatostatin-28, and prosomatostatin[1-10] are independently and efficiently processed from prosomatostatin in the constitutive secretory pathway in islet somatostatin tumor cells (1027B2).

We have characterized the biosynthetic origin of somatostatin-14 (SS-14), SS-28, and pro-SS[1-10] from pro-SS (PSS) in 1027B2 rat islet tumor cells. Because these cells lack regulated secretion and show unresponsiveness of the SS gene to cAMP, we have additionally carried out morphological and functional studies to elucidate the molecular defect in cAMP signalling and to localize the sites of PSS maturation along the secretory pathway. Cell extracts and secretion media were analysed by high performance liquid chromatography and specific C- and N-terminal radioimmunoassays.

Bidirectional transport by distinct populations of COPI-coated vesicles.

Electron microscope immunocytochemistry reveals that both anterograde-directed (proinsulin and VSV G protein) and retrograde-directed (the KDEL receptor) cargo are present in COPI-coated vesicles budding from every level of the Golgi stack in whole cells; however, they comprise two distinct populations that together can account for at least 80% of the vesicles budding from Golgi cisternae. Segregation of anterograde- from retrograde-directed cargo into distinct sets of COPI-coated vesicles is faithfully reproduced in the cell-free Golgi transport system, in which VSV G protein and KDEL receptor are packaged into separable vesicles, even when budding is driven by highly purified coatomer and a recombinant ARF protein.

ERS-24, a mammalian v-SNARE implicated in vesicle traffic between the ER and the Golgi.

We report the identification and characterization of ERS-24 (Endoplasmic Reticulum SNARE of 24 kD), a new mammalian v-SNARE implicated in vesicular transport between the ER and the Golgi. ERS24 is incorporated into 20S docking and fusion particles and disassembles from this complex in an ATP-dependent manner. ERS-24 has significant sequence homology to Sec22p, a v-SNARE in Saccharomyces cerevisiae required for transport between the ER and the Golgi.

A major transmembrane protein of Golgi-derived COPI-coated vesicles involved in coatomer binding.

Formation of non-clathrin-coated vesicles requires the recruitment of several cytosolic factors to the Golgi membrane. To identify membrane proteins involved in this budding process, a highly abundant type I transmembrane protein (p23) was isolated from mammalian Golgi-derived COPI-coated vesicles, and its cDNA was cloned and sequenced. It belongs to the p24 family of proteins involved in the budding of transport vesicles (Stamnes, M.

Cloning and functional characterization of mammalian homologues of the COPII component Sec23.

We screened a human cDNA library with a probe derived from a partial SEC23 mouse homologue and isolated two different cDNA clones (hSec23A and hSec23B) encoding proteins of a predicted molecular mass of 85 kDa. hSec23Ap and hSec23Bp were 85% identical and shared 48% identity with the yeast Sec23p. Affinity-purified anti-hSec23A recognized a protein of approximately 85 kDa on immunoblots of human, mouse, and rat cell extracts but did not recognize yeast Sec23p.

A v-SNARE implicated in intra-Golgi transport.

We report the identification of a putative v-SNARE (GOS-28), localized primarily to transport vesicles at the terminal rims of Golgi stacks. In vitro, GOS-28, A Golgi SNARE of 28 kD, is efficiently packaged into Golgi-derived vesicles, which are most likely COPI coated. Antibodies directed against GOS-28 block its ability to bind alpha-SNAP, partially inhibit transport from the cis to the medial cisternae, and do not inhibit budding of COP-coated vesicles, but do accumulate docked uncoated vesicles.

COPI- and COPII-coated vesicles bud directly from the endoplasmic reticulum in yeast.

The cytosolic yeast proteins Sec13p-Sec31p, Sec23p-Sec24p, and the small GTP-binding protein Sar1p generate protein transport vesicles by forming the membrane coat termed COPII. We demonstrate by thin section and immunoelectron microscopy that purified COPII components form transport vesicles directly from the outer membrane of isolated yeast nuclei. Another set of yeast cytosolic proteins, coatomer and Arf1p (COPI), also form coated buds and vesicles from the nuclear envelope.

Non-clathrin-coat protein alpha is a conserved subunit of coatomer and in Saccharomyces cerevisiae is essential for growth.

To complete the molecular characterization of coatomer, the preformed cytosolic complex that is involved in the formation of biosynthetic transport vesicles, we have cloned and characterized the gene for non-clathrin-coat protein alpha (alpha-COP) from Saccharomyces cerevisiae. The derived protein, molecular weight of 135,500, contains four WD-40 repeated motifs (Trp/Asp-containing motifs of approximately 40 amino acids). Disruption of the yeast alpha-COP gene is lethal.

Coatomer-rich endoplasmic reticulum.

We identify in normal cells the existence of two distinct sites of the transitional endoplasmic reticulum (ER), one housing the Sec23p protein complex (the classical transitional element), the other the coatomer protein complex (the coatomer-rich ER). Experimental conditions that reduce transport from the ER to the Golgi complex lead to the overexpression of this newly defined coatomer-rich ER.

pH-independent and -dependent cleavage of proinsulin in the same secretory vesicle.

By quantitative immunoelectron microscopy and HPLC, we have studied the effect of disrupting pH gradients, by ammonium chloride, on proinsulin conversion in the insulin-producing B-cells of the islets of langerhans. Proinsulin content and pH in single secretory vesicles were measured on consecutive serial sections immunostained alternately with anti-proinsulin or anti-dinitrophenol (to reveal the pH-sensitive probe DAMP) antibodies. Radioactivity labeled proinsulin, proinsulin cleavage intermediates, and insulin were quantitated by HPLC analysis of extracts of islets treated in the same conditions.

COPII: a membrane coat formed by Sec proteins that drive vesicle budding from the endoplasmic reticulum.

In vitro synthesis of endoplasmic reticulum-derived transport vesicles has been reconstituted with washed membranes and three soluble proteins (Sar1p, Sec13p complex, and Sec23p complex). Vesicle formation requires GTP but can be driven by nonhydrolyzable analogs such as GMP-PNP. However, GMP-PNP vesicles fail to target and fuse with the Golgi complex whereas GTP vesicles are functional.

Sar1 promotes vesicle budding from the endoplasmic reticulum but not Golgi compartments.

Two new members (Sar1a and Sar1b) of the SAR1 gene family have been identified in mammalian cells. Using immunoelectron microscopy, Sar1 was found to be restricted to the transitional region where the protein was enriched 20-40-fold in vesicular carriers mediating ER to Golgi traffic. Biochemical analysis revealed that Sar1 was essential for an early step in vesicle budding.

En bloc incorporation of coatomer subunits during the assembly of COP-coated vesicles.

The cDNA encoding epsilon-COP, the 36-kD subunit of coatomer, was cloned from a bovine liver cDNA library and sequenced. Immunoblotting with an anti-epsilon-COP antibody showed that epsilon-COP exists in COP-coated vesicles as well as in the cytosolic coatomer. Using the cloned cDNA, recombinant His6- tagged epsilon-COP was overexpressed in cultured Chinese hamster ovary (CHO) cells, from which metabolically radiolabeled coatomer was purified by taking advantage of the His6 tag.

ADP-ribosylation factor and coatomer couple fusion to vesicle budding.

The coat proteins required for budding COP-coated vesicles from Golgi membranes, coatomer and ADP-ribosylation factor (ARF) protein, are shown to be required to reconstitute the orderly process of transport between Golgi cisternae in which fusion of transport vesicles begins only after budding ends. When either coat protein is omitted, fusion is uncoupled from budding-donor and acceptor compartments pair directly without an intervening vesicle. Coupling may therefore results from the sequestration of fusogenic membrane proteins into assembling coated vesicles that are only exposed when the coat is removed after budding is complete.

Stepwise assembly of functionally active transport vesicles.

Budding of COP-coated vesicles (the likely carriers of newly synthesized proteins from the endoplasmic reticulum through the Golgi stack) from Golgi cisternae requires ADP-ribosylation factor (ARF), coatomer proteins from the cytosol, GTP, and fatty acyl-coenzyme A (CoA). The assembly of coated buds on the membranes requires coatomer, ARF, and GTP. When palmitoyl-CoA is added, membrane fission occurs at the coated bud, releasing coated vesicles.

zeta-COP, a subunit of coatomer, is required for COP-coated vesicle assembly.

cDNA encoding the 20-kD subunit of coatomer, zeta-COP, predicts a protein of 177-amino acid residues, similar in sequence to AP17 and AP19, subunits of the clathrin adaptor complexes. Polyclonal antibody directed to zeta-COP blocks the binding of coatomer to Golgi membranes and prevents the assembly of COP-coated vesicles on Golgi cisternae. Unlike other coatomer subunits (beta-, beta'-, gamma-, and epsilon-COP), zeta-COP exists in both coatomer bound and free pools.

Hydrolysis of bound GTP by ARF protein triggers uncoating of Golgi-derived COP-coated vesicles.

The cycle of nucleotide exchange and hydrolysis by a small GTP-binding protein, ADP-ribosylation factor (ARF), helps to provide vectoriality to vesicle transport. Coat assembly is triggered when ARF binds GTP, initiating transport vesicle budding, and coat disassembly is triggered when ARF hydrolyzes GTP, allowing the uncoated vesicle to fuse.