Mutant alleles of SEC4, an essential gene required for the final stage of secretion in yeast, have been generated by in vitro mutagenesis. Deletion of the two cysteine residues at the C terminus of the protein results in a soluble non‐functional protein, indicating that those two residues are required for normal localization of Sec4p to secretory vesicles and the plasma membrane. A mutant allele of SEC4 generated to mimic an activated, transforming allele of H‐ras, as predicted, does not bind GTP. The presence of this allele in cells containing wild‐type SEC4 causes a secretory defect and the accumulation of secretory vesicles. The results of genetic studies indicate that this allele behaves as a dominant loss of function mutant and as such prevents wild‐type protein from functioning properly. We propose a model in which Sec4p cycles between an active and an inactive state in order to mediate the fusion of vesicles to the plasma membrane.

Arachidonic acid-derived epoxides, epoxyeicosatrienoic acids, are important regulators of vascular homeostasis and inflammation, and therefore manipulation of their levels is a potentially useful pharmacological strategy. Soluble epoxide hydrolase converts epoxyeicosatrienoic acids to their corresponding diols, dihydroxyeicosatrienoic acids, modifying or eliminating the function of these oxylipins. To better understand the phenotypic impact of Ephx2 disruption, two independently derived colonies of soluble epoxide hydrolase-null mice were compared. We examined this genotype evaluating protein expression, biofluid oxylipin profile, tissue oxylipin production capacity, and blood pressure. Ephx2 gene disruption eliminated soluble epoxide hydrolase protein expression and activity in liver, kidney, and heart from each colony. Plasma levels of epoxy fatty acids were increased, and fatty acid diols levels were decreased, while measured levels of lipoxygenase-and cyclooxygenase-dependent oxylipins were unchanged. Liver and kidney homogenates also show elevated epoxide fatty acids. However, in whole kidney homogenate a 4-fold increase in the formation of 20-hydroxyeicosatetraenoic acid was measured along with a 3-fold increase in lipoxygenase-derived hydroxylation and prostanoid production. Unlike previous reports, however, neither Ephx2-null colony showed alterations in basal blood pressure. Finally, the soluble epoxide hydrolase-null mice show a survival advantage following acute systemic inflammation. The data suggest that blood pressure homeostasis may be achieved by increasing production of the vasoconstrictor, 20-hydroxyeicosatetraenoic acid in the kidney of the Ephx2-null mice. This shift in renal metabolism is likely a metabolic compensation for the loss of the soluble epoxide hydrolase gene. Soluble epoxide hydrolase (sEH)3 is a ubiquitous enzyme found in many tissues such as liver, kidney, heart, and ovary (1). sEH catalyzes the degradation of endogenous epoxy lipids such as epoxyeicosatrienoic acids (EETs) to their less active diols (dihydroxyeicosatrienoic acids, DHETs) and hence plays a critical role in the control of EET levels (2). These epoxy lipids are potent vasodilators, regulating cerebral and renal homodynamic and blood pressure (3-5). In addition, EETs inhibit platelet aggregation (6), promote fibrinolysis (7) and have antiinflammatory properties (8, 9). Whereas deletion of the sEH gene, Ephx2, has been reported to reduce blood pressure in male mice (10), the inhibition of endogenous EET hydrolysis may provide pharmacological benefit in hypertension and acute inflammation (11).The human Ephx2 gene encodes sEH and consists of 19 exons encoding 555 amino acids (12). There is 73% homology between the human and mouse sEH protein sequences (13), with 100% conservation in the catalytic residues (14). Each monomer of the homodimeric mouse sEH has two distinct domains (14,15). The N-terminal domain exhibits phosphatase activity, and the C-terminal domain is responsible for the epoxide hydrolase activities (...

Sec4, a GTP-binding protein of the ras superfamily, is required for exocytosis in the budding yeast Saccharomyces cerevisiae. To test the role of GTP hydrolysis in Sec4 function, we constructed a mutation, Q-79-*L, analogous to the oncogenic mutation of Q-61--->L in Ras, in a region of Sec4 predicted to interact with the phosphoryl group of GTP. The sec4-eu79 mutation lowers the intrinsic hydrolysis rate forms is important for its function in vesicular transport, supporting a mechanism for Sec4 function which is distinct from that of the Ras protein.Numerous GTP-binding proteins have been identified which play a variety of roles in diverse intracellular processes (for a recent review, see reference 8). These proteins bind both GDP and GTP and possess an intrinsic GTPase activity. They can be thought to function in a cycle in which GTP is first bound and then hydrolyzed. Following hydrolysis, the resulting GDP dissociates from the protein, allowing the association of a new GTP. The conformational state of the protein depends upon the nucleotide bound and can thus be regulated by the exchange of GDP for GTP and the hydrolysis of GTP to GDP.Various lines of evidence have implicated a subfamily of Ras-like GTP-binding proteins in the regulation of vesicular traffic in eukaryotic cells. Sec4, identified by its role in protein transport from the Golgi apparatus to the cell surface in yeast cells (33), was found to share significant sequence similarity with the Ras protein in the domains required for interaction with guanine nucleotides (36). More extensive similarity was seen between Sec4 and the Yptl protein of Saccharomyces cerevisiae, which has since been shown to play a role in protein transport at an earlier stage of the secretory pathway (2,3,(37)(38)(39). A large number of mammalian homologs of SEC4 and YPT1, known principally as the rab genes, have now been identified (11,12,20,27,45,52). Many of the proteins encoded by these genes have been found to localize to a unique stage of the exocytotic or endocytic pathways (11-13, 18, 19, 30, 47 (18), Yptl is associated with the Golgi (39) as well as endoplasmic reticulum-Golgi carrier vesicles (38), and Rab5, which has been implicated in endosome-endosome fusion (17), has been localized to early endosomes and the plasma membrane. Together, these findings suggest that the Sec4/Yptl homologs play analogous roles, each regulating a distinct vesicular transport event.Bourne (7) has proposed a model for the role of GTPbinding proteins in secretion in which the cycle of GTP binding and hydrolysis serves to ensure the vectorial flow of the vesicular transport process. In this model, a GTPbinding protein on the surface of a carrier vesicle directs delivery of the vesicle to the appropriate acceptor compartment by virtue of an interaction of the protein in its GTPbound form with an effector protein on the target membrane. Once fusion occurs, release of the GTP-binding protein from the acceptor compartment is coupled to a change in conformation resulting from hydrolysis of...

The synthesis and oligosaccharide processing of the glycoproteins of SA11 rotavirus in infected Mal04 cells was examined. Rotavirus assembles in the rough endoplasmic reticulum (RER) and encodes two glycoproteins: VP7, a component of the outer viral capsid, and NCVP5, a nonstructural protein. A variety of evidence suggests the molecules are limited to the ER, a location consistent with the high mannose N-linked oligosaccharides modifying these proteins.VP7 and NCVP5 were shown to be integral membrane proteins. In an in vitro translation system supplemented with dog pancreas microsomes, they remained membrane associated after high salt treatment and sodium carbonate-mediated release of microsomal contents. In infected cells, the oligosaccharide processing of these molecules proceeded in a timedependent manner. For VP7, ManBGIcNAc2 and Man6GIcNAc2 were the predominant intracellular species after a 5-min pulse with [3H]mannose and a 90 min chase, while in contrast, trimming of NCVP5 halted at Man~GIcNAc2. VP7 on mature virus was processed to MansGIcNAc2. It is suggested that the c~-mannosidase activities responsible for the formation of these structures reside in the ER. In the presence of the energy inhibitor carbonyl cyanide m-chlorophenylhydrazone (CCCP), processing of VP7 and the vesicular stomatitis virus G protein was blocked at Man~GIcNAc2. After a 20-min chase of [3H]mannose-labeled molecules followed by addition of CCCP, trimming of VP7 could continue while processing of G protein remained blocked. Thus, an energy-sensitive translocation step within the ER may mark the divergence of the processing pathways of these glycoproteins.Membrane glycoproteins locate with a high degree of specificity to a number of subcellular compartments. Due to the complex distribution of proteins normally present in membranes, simple model systems have been sought where the behavior of a few or a single molecular species can be readily defined. To this end, studies of the highly abundant glycoproteins of such membrane maturing viruses as vesicular stomatitis (VSV), ~ sindbis, semliki forest, and influenza have greatly expanded our knowledge of the mechanism of transport to the plasma membrane (reviewed in reference 31). The fine structure of the oligosaccharide moiety serves as a useful ~,lhhreviations used in this paper." act D, actinomycin D: CCCP. carbonyl cyanide m-chlorophenylhydrazone; DME, Dulbecco's modified Eagle's medium; endo H, endo-~-N-acetylglucosaminidase H; PMSF, phenylmethylsulfonyl fluoride; RER, rough endoplasmic reticulum: TBS, Tris-buffered saline; VSV, vesicular stomatitis virus. 1270marker to indicate the subcellular compartments a specific glycoprotein has traversed. In N-linked glycosylation reactions, a glucose3-mannose9-N-acetylglucosamine2 (Glc3Man9-GlcNAc2) core is transferred from a dolichol pyrophosphate cartier to an asparagine residue on the nascent polypeptide chain. This carbohydrate core is extensively trimmed before the addition, in some cases, of terminal sugars such as Nacetylgluc...

Abstract. Using either permeabilized cells or microsomes we have reconstituted the early events of the yeast secretory pathway in vitro. In the first stage of the reaction ,'o50-70% of the prepro-ct-factor, synthesized in a yeast translation lysate, is translocated into the endoplasmic reticulum (ER) of permeabilized yeast cells or directly into yeast microsomes. In the second stage of the reaction 48-66 % of the ER form of a-factor (26,000 D) is then converted to the high molecular weight Golgi form in the presence of ATP, soluble factors and an acceptor membrane fraction; GTPyS inhibits this transport reaction. Donor, acceptor, and soluble fractions can be separated in this assay. This has enabled us to determine the defective fraction in sec23, a secretory mutant that blocks ER to Golgi transport in vivo. When fractions were prepared from mutant cells grown at the permissive or restrictive temperature and then assayed in vitro, the acceptor Golgi fraction was found to be defective.