Processing and assembly of the integrin, glycoprotein IIb-IIIa, in HEL cells

We examined the biosynthetic processing and assembly of the platelet glycoprotein (GP) IIb-IIIa complex in [35S]methionine-labeled HEL cells, a human cell line with features of megakaryocytes. Both GPIIb and GPIIIa were synthesized as single-chain precursors to which high mannose N-linked oligosaccharides were added in the endoplasmic reticulum (ER). A 5-fold excess of the major IIb precursor, preIIb, was synthesized relative to GPIIIa. Two smaller proteins immunologically related to GPIIb were synthesized in smaller amounts. Assembly of the GPIIb and GPIIIa precursors required 4-6 h for completion. All GPIIIa molecules were eventually assembled; the excess GPIIb precursors were degraded without reaching the cell surface. Following assembly, preIIb-IIIa complexes were rapidly transported to the Golgi apparatus where preIIb underwent modification of high mannose chains into complex oligosaccharides and proteolytic cleavage to yield disulfide-linked heavy and light chains. Pretreating cells with the ionophore monensin blocked cleavage of preIIb but not its carbohydrate modification or its assembly with GPIIIa. These studies suggest that 1) assembly of the precursors of GPIIb and GPIIIa in the ER is a slow process requiring conformational maturation of one or both subunits, and 2) only heterodimers assembled in the ER are transported to the Golgi apparatus for additional processing and, ultimately, expression on the cell surface.     

Inhibition of disulfide bonding of von Willebrand protein by monensin results in small, functionally defective multimers

The biosynthesis of von Willebrand protein by human endothelial cells was impaired by the presence of the carboxylic ionophore monensin. Several processing steps that have been localized to the Golgi apparatus were affected in a dose-dependent manner, including carbohydrate processing, dimer multimerization, and precursor cleavage. Since multimerization was more susceptible to the ionophore than was precursor cleavage, it appears that these processing steps are separate events. As expected, dimer formation, which occurs in the rough endoplasmic reticulum, was unaffected by monensin. Thus, at high concentrations of monensin, only dimer molecules were produced and secreted. The observed inhibition of multimer formation and precursor cleavage were not likely the result of incomplete carbohydrate processing, since inhibition of complex carbohydrate formation by swainsonine did not interfere with the other processing steps. Monensin also affected the capacity of endothelial cells to store von Willebrand protein, as the ratio of secreted to cell-associated protein increased dramatically in the presence of monensin, and the processed forms could not be found in the treated cells. The low molecular weight multimers produced in the presence of monensin did not incorporate in the endothelial cells' extracellular matrix nor did they bind to the matrix of human foreskin fibroblasts. In summary, the presence of monensin in human endothelial cell culture produced experimental conditions that mimic Type IIA von Willebrand disease, in that the cells synthesized and secreted only low molecular weight von Willebrand protein multimers, which were functionally defective.     

Neuropeptide processing activity in Aplysia bag cell homogenates

The neurosecretory bag cells of the mollusk, Aplysia, generate a peptide egg-laying hormone (ELH) from a 29,000 Dalton precursor protein by proteolytic cleavage to a 6-9,000 Dalton intermediate, followed by cleavage of the intermediate. We report here the initial characterization of these cleavage activities. Homogenates of bag cells in low ionic strength buffer process endogenous precursor to a peptide which is indistinguishable from ELH in molecular weight and isoelectric point. Non-specific proteolysis in the homogenates is not detectable. The pH optimum for cleavage of the precursor and the intermediate is 5.5-6.5. The cleavage activities exhibit a substantial degree of membrane association, and the inhibitor profile of each is characteristic of a thiol protease without a metal cofactor requirement. Precursor cleavage activity differs from that of the intermediate cleaving activity in inhibitor profile, solubility, and slightly, in pH optimum.     

A furin-like convertase mediates propeptide cleavage of BACE, the Alzheimer's beta -secretase

The novel transmembrane aspartic protease BACE (for Beta-site APP Cleaving Enzyme) is the beta-secretase that cleaves amyloid precursor protein to initiate beta-amyloid formation. As such, BACE is a prime therapeutic target for the treatment of Alzheimer's disease. BACE, like other aspartic proteases, has a propeptide domain that is removed to form the mature enzyme. BACE propeptide cleavage occurs at the sequence RLPR downward arrowE, a potential furin recognition motif. Here, we explore the role of furin in BACE propeptide domain processing. BACE propeptide cleavage in cells does not appear to be autocatalytic, since an inactive D93A mutant of BACE is still cleaved appropriately. BACE and furin co-localize within the Golgi apparatus, and propeptide cleavage is inhibited by brefeldin A and monensin, drugs that disrupt trafficking through the Golgi. Treatment of cells with the calcium ionophore, leading to inhibition of calcium-dependent proteases including furin, or transfection with the alpha(1)-antitrypsin variant alpha(1)-PDX, a potent furin inhibitor, dramatically reduces cleavage of the BACE propeptide. Moreover, the BACE propeptide is not processed in the furin-deficient LoVo cell line; however, processing is restored upon furin transfection. Finally, in vitro digestion of recombinant soluble BACE with recombinant furin results in complete cleavage only at the established E46 site. Taken together, our results strongly suggest that furin, or a furin-like proprotein convertase, is responsible for cleaving the BACE propeptide domain to form the mature enzyme.     

Sorting within the regulated secretory pathway occurs in the trans-Golgi network

Bioactive peptides cleaved from the egg-laying hormone precursor in the bag cell neurons of Aplysia are sorted into distinct dense core vesicle classes (DCVs). Bag cell prohormone processing can be divided into two stages, an initial cleavage occurring in a late Golgi compartment, which is not blocked by monensin, and later cleavages that occur within DCVs and are blocked by monensin. Prohormone intermediates are sorted in the trans-Golgi network. The large soma-specific DCVs turn over, while the small DCVs are transported to processes for regulated release. Thus, protein trafficking differentially regulates the levels and localization of multiple biologically active peptides derived from a common prohormone.     

Expression of mutant ELH prohormones in AtT-20 cells: the relationship between prohormone processing and sorting

Posttranslational processing of many proteins is essential to the synthesis of fully functional molecules. The ELH (egg-laying hormone) prohormone is cleaved by endoproteases in a specific order at a variety of basic residue processing sites to produce mature peptides. The prohormone is first cleaved at a unique tetrabasic site liberating two intermediates (amino and carboxy) which are sorted to different classes of dense core vesicles in the bag cell neurons of Aplysia. When expressed in AtT-20 cells, the ELH prohormone is also first cleaved at the tetrabasic site. The amino-terminal intermediate is then sorted to the constitutive pathway, and a portion of the carboxy-terminal intermediate is sorted to the regulated pathway. Here, we use mutant constructs of the ELH prohormone expressed in AtT-20 cells to examine the relationship between prohormone processing and consequent sorting. Prohormone which has a dibasic site in place of the tetrabasic site is processed and sorted similarly to wild type. Furthermore, mutant prohormone which lacks the tetrabasic site is processed at an alternative site comprising three basic residues. In these mutant prohormones, mature ELH is still produced and stored in dense core vesicles while amino-terminal products are constitutively secreted. However, deletion of the tetrabasic and tribasic sites results in the rerouting of the amino-terminal intermediate products from the constitutive pathway to the regulated secretory pathway. Thus, in the ELH prohormone, the location of the proteolytic processing events within the secretory pathway and the order of cleavages regulate the sorting of peptide products.     

Multiple neuropeptides derived from a common precursor are differentially packaged and transported

The ELH prohormone is proteolytically processed into at least nine peptides which govern egg-laying behavior in Aplysia. Quantitative immunocytochemistry demonstrates that peptides derived from the prohormone are packaged into distinct vesicle classes. Further experiments suggest the segregation occurs via a rapid initial proteolytic cleavage of the prohormone followed by sorting at the trans Golgi. Egg-laying hormone (ELH) immunoreactivity is localized to the cell body and processes, while bag cell peptide (BCP) immunoreactivity is greater in the cell body. Steady state levels of the amino-terminal set of peptides including the BCPs are 3- to 8-fold lower than the carboxy-terminal cleavage products, such as ELH. Thus, intracellular packaging and routing of the peptides cleaved from a single prohormone regulate their localization and levels in these neurons.     

Biosynthesis of von Willebrand protein by human endothelial cells: processing steps and their intracellular localization

Biosynthesis of von Willebrand protein by human umbilical vein endothelial cells involved distinct processing steps marked by the presence of several intermediate molecular species. Examination of endoglycosidase H sensitivity of these intracellular intermediates indicated that the processing steps occurred in at least two separate cellular compartments. In the pre-Golgi apparatus (most probably the endoplasmic reticulum), the high mannose carbohydrates were added onto the precursor monomer chains and the 260,000-mol-wt monomers dimerized by interchain disulfide bond formation. The other processing steps have been localized to the Golgi apparatus and later compartments (e.g., Weibel-Palade bodies). High mannose carbohydrate was converted to the complex type, leading to the appearance of a larger precursor subunit of 275,000 mol wt. The 275,000-mol-wt species was not formed if carbohydrate processing was inhibited by the ionophore monensin. From the large pool of dimers of precursor subunits, the high molecular weight multimers were built. These dimer molecules appeared to have free sulfhydryls which might have been involved in the interdimer disulfide bond formation. Simultaneously with multimerization, the precursor subunits were cleaved to the 220,000-mol-wt form. The cleavage of the pro-sequence was not likely to be an absolute requirement for von Willebrand protein multimerization or secretion, as the 275,000-mol-wt precursor subunit was present in secreted high molecular weight multimers of the protein.     

Alteration of intracellular traffic by monensin; mechanism,specificity and relationship to toxicity

Monensin, a monovalent ion-selective ionophore, facilitates the transmembrane exchange of principally sodium ions for protons. The outer surface of the ionophore-ion comples is composed largely of nonpolar hydrocarbon, which imparts a high solubility to the complexes in nonpolar solvents. In biological systems, these complexes are freely soluble in the lipid components of membranes and, presumably, diffuse or shuttle through the membranes from one aqueous membrane interface to the other. The net effect for monensin is a trans-membrane exchange of sodium ions for protons. However, the interaction of an ionophore with biological membranes, and its ionophoric expression, is highly dependent on the biochemical configuration of the membrane itself.One apparent consequence of this exchange is the neutralization of acidic intracellular compartments such as the trans Golgi apparatus cisternae and associated elements, lysosomes, and certain endosomes. This is accompanied by a disruption of trans Golgi apparatus cisternae and of lysosome and acidic endosome function. At the same time, Golgi apparatus cisternae appear to swell, presumably due to osmotic uptake of water resulting from the inward movement of ions.Monensin effects on Golgi apparatus are observed in cells from a wide range of plant and animal species. The action of monensin is most often exerted on the trans half of the stacked cisternae, often near the point of exit of secretory vesicles at the trans face of the stacked cisternae, or, especially at low monensin concentrations or short exposure times, near the middle of the stacked cisternae. The effects of monensin are quite rapid in both animal and plant cells; i.e., changes in Golgi apparatus may be observed after only 2–5 min of exposure. It is implicit in these observations that the uptake of osmotically active cations is accompanied by a concomitant efflux of H⁺ and that a net influx of protons would be required to sustain the ionic exchange long enough to account for the swelling of cisternae observed in electron micrographs.In the Golgi apparatus, late processing events such as terminal glycosylation and proteolytic cleavages are most susceptible to inhibition by monensin. Yet, many incompletely processed molecules may still be secreted via yet poorly understood mechanisms that appear to bypass the Golgi apparatus.In endocytosis, monensin does not prevent internalization. However, intracellular degradation of internalized ligands may be prevented. It is becoming clear that endocytosis involves both acidic and non-acidic compartments and that monensin inhibits those processes that normally occur in acidic compartments.Thus, monensin, which is capable of collapsing Na⁺ and H⁺ gradients, has gained wide-spread acceptance as a tool for studying Golgi apparatus function and for localizing and identifying the molecular pathways of subcellular vesicular traffic involving acid compartments. Among its advantages are the low concentrations at which inhibitions are produced (0.01–1.0 μM), a minimum of troublesome side effects (e.g., little or no change of protein synthesis or ATP levels) and a reversible action. Because the affinity of monensin for Na⁺ is ten times that for K⁺, its nearest competitor, monensin mediates primarily a Na⁺-H⁺ exchange. Monensin has little tendency to bind calcium.Not only is monensin of importance as an experimental tool, it is of great commercial value as a coccidiostat for poultry and to promote more efficient utilization of feed in cattle. The mechanisms by which monensin interact with coccidia and rumen microflora to achieved these benefits are reasonably well documented. However, the interactions between monensin and the tissues of the host animal are not well understood although the severe toxicological manifestations of monensin poisoning are well known. Equine species are particularly susceptible to monensin poisoning, and a common effect of monensin poisoning is vacuolization and/or swelling of mitochondria in striated muscle. Other pathological injuries to striated muscle, spleen, lung, liver and kidney also have been noted. A consistent observation is cardiac myocyte degeneration as well as vacuolization. Differences in cellular response resulting from exposure to monensin (i.e., Golgi apparatus swelling in cultured cells, isolated tissues, and plants vs.mitochondrial swelling in animals fed monensin) suggest that myocardial damage is due either to a monensin metabolite or is a secondary response to some other derivation. However, as pointed out by Bergen and Bates [26], the underlying mode of action of ionophores is on transmembrane ion fluxes which dissipate cation and proton gradients. Consequently, some or all of the observed monensin effects in vivo in animals could be secondary phenomena caused by disruption of normal membrane physiology resulting from altered ion fluxes.     

Golgi/granule processing of peptide hormone and neuropeptide precursors: a minireview

Proteolytic processing of precursor proteins is a phylogenetically ancient and widely used mechanism for producing biologically active peptides. Proteolytic cleavage of proproteins begins only after transport to the Golgi apparatus has been completed and in most systems may continue for many hours within newly formed secretory vesicles as these are stored in the cytosol or transported along axons to more peripheral sites of release. Paired basic residues are required for efficient proteolysis in most precursors, suggesting that a small number of specialized tryptic proteases exist that have great site selectivity but can process many sites within the same precursor or in different precursors within the same cell, or in different cells or tissues. Cleavage-site choice may be strongly influenced by other factors, such as secondary and tertiary structure, but definitive structural information on precursor proteins is lacking. Modifications such as glycosylation, phosphorylation, and sulfation also are Golgi associated but are not known to influence proteolytic processing patterns. Golgi/granule processing also rarely occurs at sites other than pairs of basic amino acids, including single basic residues ( trypsinlike ), Leu-Ala, Leu-Ser, or Tyr-Ala bonds ( chymotrysinlike ) as well as other specialized nontryptic cleavages, suggesting that mixtures of proteases coexist in the Golgi/granule system. Cathepsin B-like thiol proteases, or their precursors, have been implicated as the major processing endopeptidases in several systems. Carboxypeptidase B-like enzymes also have been identified in secretion granules in several tissues and appear to be metalloenzymes similar in mechanism to the pancreatic carboxypeptidases, but with a lower pH optimum. The role of the Golgi apparatus in sorting newly formed secreted products from lysosomal hydrolases may have permitted the development in evolution of an intimate relationship between certain of the lysosomal degradative enzymes, such as cathepsin B or its precursors, and the Golgi/granule processing systems. The sequestration of the proteolytic products of precursors within secretion granules leads to the coordinate discharge of highly complex mixtures of peptides having related or overlapping biological activities. The cosecretion of nonfunctional peptide " leftovers ," such as the proinsulin C-peptide, can serve as useful markers of secretion or cellular localization, as well as of evolutionary relation ships. Errors in cleavage due to point mutations in precursors have been identified in several systems, leading to the accumulation of incorrectly processed materials in the circulation. These and/or defects (ABSTRACT TRUNCATED AT 400 WORDS)     

Biologically active APRIL is secreted following intracellular processing in the Golgi apparatus by furin convertase

Tumor necrosis factor (TNF) ligand family members are synthesized as transmembrane proteins, and cleavage of the membrane-anchored proteins from the cell surface is frequently observed. The TNF-related ligands APRIL and BLyS and their cognate receptors BCMA/TACI form a two ligand/two receptor system that has been shown to participate in B- and T-cell stimulation. In contrast to BLyS, which is known to be cleaved from the cell surface, we found that APRIL is processed intracellularly by furin convertase. Blockage of protein transport from the endoplasmic reticulum to the Golgi apparatus by Brefeldin A treatment abrogated APRIL processing, whereas monensin, an inhibitor of post-Golgi transport, did not interfere with cleavage of APRIL, but blocked secretion of processed APRIL. Thus, APRIL shows a unique maturation pathway among the TNF ligand family members, as it not detectable as a membrane-anchored protein at the cell surface, but is processed in the Golgi apparatus prior to its secretion.     

Effect of monensin on intracellular transport and receptor-mediated endocytosis of lysosomal enzymes.

In cultured human fibroblasts we observed that monensin, a Na+/H+-exchanging ionophore, (i) inhibits mannose 6-phosphate-sensitive endocytosis of a lysosomal enzyme, (ii) enhances secretion of the precursor of cathepsin D, while inhibiting secretion of the precursors of beta-hexosaminidase, (iii) induces secretion of mature beta-hexosaminidase and mature cathepsin D, and (iv) inhibits carbohydrate processing in and proteolytic maturation of the precursors remaining within the cells; this last effect appears to be secondary to an inhibition of the transport of the precursors. If the treated cells are transferred to a monensin-free medium, about half of the accumulated precursors are secreted, and the intracellular enzyme is converted into the mature form. Monensin blocks formation of complex oligosaccharides in lysosomal enzymes. In the presence of monensin, total phosphorylation of glycoproteins is partially inhibited, whereas the secreted glycoproteins are enriched in the phosphorylated species. The suggested inhibition by monensin of the transport within the Golgi apparatus [Tartakoff (1980) Int. Rev. Exp. Pathol. 22, 227-250] may be the cause of some of the effects observed in the present study (iv). Other effects (i, ii) are rather explained by interference by monensin with the acidification in the lysosomal and prelysosomal compartments, which appears to be necessary for the transport of endocytosed and of newly synthesized lysosomal enzymes.     

Delta-bag cell peptide from the egg-laying hormone precursor of Aplysia. Processing, primary structure, and biological activity

The bag cells of the marine mollusk Aplysia express a gene encoding a 271-residue egg-laying hormone (ELH) precursor that is processed into at least nine peptide products. Four of the peptides have been identified in bag cell releasates and are known to act as nonsynaptic neurotransmitters in the abdominal ganglion. The isolation, primary structure, and proposed biological activity of a fifth peptide product (delta-bag cell peptide (delta-BCP)) from the ELH precursor are described. delta-BCP was established to be a 39-residue peptide: NH2-Asp-Gln-Asp-Glu-Gly-Asn-Phe-Arg-Arg-Phe-Pro-Thr-Asn-Ala-Val-Ser-Met- Ser-Ala-Asp- Glu-Asn-Ser-Pro-Phe-Asp-Leu-Ser-Asn-Glu-Asp-Gly-Ala-Val-Tyr-Gln-Arg- Asp-Leu-COOH. This sequence corresponds to residues 81-119 of the ELH prohormone and shares sequence identity with atrial gland peptides A and B. Significantly, synthetic delta-BCP stimulated Ca2+ uptake into mitochondria of secretory cells in the albumin gland in vitro, suggesting that the peptide regulates the cellular release of perivitelline fluid by the gland. Similar results were obtained with purified peptide A and a shorter version of delta-BCP (delta-BCP-(14-33)). These results indicate that delta-BCP belongs to a family of structurally related peptides with similar pharmacological activities that center at a conserved region of sequence corresponding to delta-BCP-(14-33).     

Effects of the monovalent ionophore monensin on the intracellular transport and processing of pro-opiomelanocortin in cultured intermediate lobe cells of the rat pituitary

Detailed studies on the effects of the ionophore monensin upon synthesis, maturation, and intracellular transport of pro-opiomelanocortin in cultures of rat pituitary intermediate lobe cells have been carried out. When added at concentrations larger than 5 X 10(-8) M monensin significantly inhibited protein synthesis by cultured intermediate lobe cells. Pro-opiomelanocortin synthesis was also reduced proportionally to the overall rate of protein synthesis. During pulse-chase experiments, monensin when added at a concentration of 10(-5) M at the beginning of the chase incubation completely inhibited the proteolytic processing of pro-opiomelanocortin. Using a subcellular fractionation procedure of intermediate lobe cell extracts on Percoll gradients, we were able to show that after the addition of monensin (10(-5) M), labeled pro-opiomelanocortin molecules synthesized during a 15-min pulse-incubation were recovered intact after a 2-h chase, in the fractions of the density gradient corresponding to the rough endoplasmic reticulum and Golgi elements. No maturation products or precursor molecules entered the granule fractions as observed in nontreated cells. Taken together these results strongly suggest that monensin blocks the intracellular transport of newly synthesized pro-opiomelanocortin molecules at the Golgi level and that inhibition of proteolytic processing is due to the failure of the prohormone to enter the cell compartment (probably the secretion granules) where maturation proteases are located.     

Prosomatostatin is processed in the Golgi apparatus of rat neural cells.

Proteolytic processing of somatostatin precursor produces several peptides including somatostatin-14 (S-14), somatostatin-28 (S-28), and somatostatin-28 (1-12) (S-28(1-12)). The subcellular sites at which these cleavages occur were identified by quantitative evaluation of these products in enriched fractions of the biosynthetic secretory apparatus of rat cortical or hypothalamic cells. Each of the major cellular compartments was obtained by discontinuous gradient centrifugation and was characterized both by specific enzyme markers and electron microscopy. The prosomatostatin-derived fragments were measured by radioimmunoassay after chromatographic separation. Two specific antibodies were used, allowing the identification of either S-28(1-12) or S-14 which results from peptide bond hydrolysis at a monobasic (arginine) and a dibasic (Arg-Lys) cleavage site, respectively. These antibodies also revealed prosomatostatin-derived forms containing at their COOH terminus the corresponding dodeca- and tetradecapeptide sequences. Whereas the reticulum-enriched fractions contained the highest levels of prosomatostatin, the proportion of precursor was significantly lower in the Golgi apparatus. In the latter fraction, other processed forms were also present, i.e. S-14 and S-28(1-12) together with the NH2-terminal domain (1-76) of prosomatostatin (pro-S(1-76). Inhibition of the intracellular transport either by monensin or by preincubation at reduced temperature resulted in an increase of prosomatostatin-derived peptides in the Golgi-enriched fractions. Finally, immunogold labeling using antibodies raised against S-28(1-12) and S-14 epitopes revealed the presence of these forms almost exclusively in the Golgi-enriched fraction mainly at the surface of saccules and vesicles. Together these data demonstrate that in rat neural cells, prosomatostatin proteolytic processing at both monobasic and dibasic sites is initiated at the level of the Golgi apparatus.     

Proteolytic processing of egg-laying hormone-related precursors in Aplysia. Identification of peptide regions critical for biological activity

The atrial gland of the marine mollusk Aplysia californica is an exocrine organ that expresses at least three genes belonging to the egg-laying hormone (ELH) family. In order to study the post-translational processing of the ELH-related gene products in the atrial gland and how it compares to the bag cells, peptides were isolated from the atrial gland and chemically characterized. The A- and B-related precursors were each cleaved in vivo to yield several major and minor peptides including peptides A and B and the ELH-related peptide complexes that caused egg laying. About 13% of the peptide complexes were further enzymically processed by the atrial gland to yield smaller fragments, which included A-AP.A-ELH-(15-36), A-AP.[Ala27]A-ELH-(15-36), and A-AP.[Gln23,Ala27]A-ELH-(16-36), where A-AP is an acidic peptide encoded by the A- and B-related genes and A-ELH is an ELH-related peptide encoded by the A gene. These processed peptide fragments were not active in an egg-laying bioassay, indicating that retention of the 14-residue NH2-terminal segment of the A-ELH-related sequence, or some portion thereof, was critical for the induction of egg laying. Other characterized peptides included two novel 13-residue NH2-terminal peptides, A-NTP and B-NTP, representing residues 22-34 of the A and B precursors, respectively. These two peptides occurred adjacent to the signal peptide region in each precursor, and their characterization established the site of signal peptide cleavage to be the Ser21-Gln22 peptide bond of each precursor. Intermediate peptide fragments (A-NTP-peptide A and B-NTP-peptide B) were also identified indicating that there was a specific ordering in the cleavage of peptide bonds during posttranslational processing. Finally, a new 55-residue atrial gland peptide was also isolated that was not a part of any ELH-related precursor characterized to date.     

Monensin inhibits the processing of herpes simplex virus glycoproteins, their transport to the cell surface, and the egress of virions from infected cells.

HEp-2 cells or Vero cells infected with herpes simplex virus type 1 were exposed to the ionophore monensin, which is thought to block the transit of membrane vesicles from the Golgi apparatus to the cell surface. We found that yields of extracellular virus were reduced to less than 0.5% of control values by 0.2 microM monensin under conditions that permitted accumulation of cell-associated infectious virus at about 20% of control values. Viral protein synthesis was not inhibited by monensin, whereas late stages in the post-translational processing of the viral glycoproteins were blocked. The transport of viral glycoproteins to the cell surface was also blocked by monensin. Although the assembly of nucleocapsids appeared to be somewhat inhibited in monensin-treated cells, electron microscopy revealed that nucleocapsids were enveloped to yield virions, and electrophoretic analyses showed that the isolated virions contained immature forms of the envelope glycoproteins. Most of the virions which were assembled in monensin-treated cells accumulated in large intracytoplasmic vacuoles, whereas most of the virions produced by and associated with untreated cells were found attached to the cell surface. Our results implicate the Golgi apparatus in the egress of herpes simplex virus from infected cells and also suggest that complete processing of the viral envelope glycoproteins is not essential for nucleocapsid envelopment or for virion infectivity.     

A single mutation prevents the normal intracellular transport of multiple lysosomal proteins from the rough endoplasmic reticulum

Dictyostelium discoideum strain HMW-426 has been previously shown to be defective in the proteolytic processing of the lysosomal enzyme precursor to alpha-mannosidase. We have now shown that the mutant is defective in the proteolytic processing of a second lysosomal enzyme, beta-glucosidase. Digestion of the HMW-426 alpha-mannosidase and beta-glucosidase precursors with endoglycosidase H revealed that the majority of oligosaccharide side chains on both precursors were sensitive to cleavage by this enzyme, indicating that both precursors fail to reach the Golgi apparatus. Subcellular fractionation experiments demonstrated that these two mutant precursors accumulated inside the lumen of the rough endoplasmic reticulum. The alpha-mannosidase precursor is conformationally altered, as evidenced by its abnormal protease susceptibility, suggesting that altered conformation is responsible for a generalized defect in transport of lysosomal protein precursors from the rough endoplasmic reticulum in the mutant.     

Evidence that all newly synthesized proteins destined for fast axonal transport pass through the Golgi apparatus

Effects of the sodium ionophore, monensin, were examined on the passage from neuronal cell body to axon of materials undergoing fast intracellular transport. In vitro exposure of bullfrog dorsal root ganglia to concentrations of drug less than 1.0 micron led to a dose-dependent depression in the amount of fast-transported [3H]leucine- or [3H]glycerol-labeled material appearing in the nerve trunk. Incorporation of either precursor was unaffected. Exposure of a desheathed nerve trunk to similar concentrations of monensin, while ganglia were incubated in drug-free medium, had no effect on transport. With [3H]fucose as precursor, fast transport of labeled glycoproteins was depressed to the same extent as with [3H]leucine; synthesis, again, was unaffected. By contrast, with [3H]galactose as precursor, an apparent reduction in transport of labeled glycoproteins was accounted for by a marked depression in incorporation. The inference from these findings, that monensin acts to block fast transport at the level of the Golgi apparatus, was supported by ultrastructural examination of the drug-treated neurons. An extensive and selective disruption of Golgi saccules was observed, accompanied by an accumulation of clumped smooth membranous cisternae. Quantitative analyses of 48 individual fast-transported protein species, after separation by two-dimensional gel electrophoresis, revealed that monensin depresses all proteins to a similar extent. These results indicate that passage through the Golgi apparatus is an obligatory step in the intracellular routing of materials destined for fast axonal transport.