Brefeldin A induces endoplasmic reticulum-associated O-glycosylation of galactosyltransferase

Recent data from several laboratories show that Brefeldin A (BFA) induces a microtubule-dependent back-flow of Golgi components to the endoplasmic reticulum (ER) thereby causing disassembly of the Golgi apparatus and its fusion with ER membranes. In order to delineate the effect of BFA on resident Golgi proteins, we investigated its effect on biosynthesis, maturation and intracellular transport of galactosyltransferase (gal-T), an established trans-Golgi enzyme. Using a protocol of metabolic labeling/immunoprecipitation followed by electrophoretic/fluorographic analysis, we show that in the presence of BFA, gal-T matures to a molecular form of 48.5 kD, a size intermediate between the 2 precursor forms of 44 and 47 and the mature form of 54 kD (Strous and Berger: J. Biol. Chem., 257:7623-28, 1982). Little mature form was detectable in the presence of BFA even after prolonged chase times of up to 28 hr. The intermediate form was sensitive to O-glycanase and endoglycosidase H, indicating early O-glycosylation without sialylation and lack of complex N-glycosylation, respectively. In order to define the compartment responsible for O-glycosylation in the presence of BFA, a temperature block of 25 degrees C was applied which inhibited recovery of Golgi elements from BFA-induced fusion with ER. At this temperature and in absence of BFA, biosynthesis of gal-T was not appreciably affected, while maturation was completely inhibited as indicated by the presence of unmodified precursor forms of gal-T. After 60 min preincubation with BFA, a time period sufficient to demonstrate complete fusion of Golgi with ER, subsequent biosynthesis of gal-T at 25 degrees C in absence of BFA led to the intermediate form, while precursor forms were not detectable. These data provide direct evidence for BFA-induced redistribution to the EF of Golgi enzymes involved in O-glycosylation and their early functional involvement in biosynthesis of newly synthesized gal-T.     

Compartmentation of the rough endoplasmic reticulum

It has become evident during recent years that a wide variety of proteins are synthesized on membrane-bound polysomes, very many of which are not ultimately secreted from the cell. The majority of proteins appear to go through some form of post-translational modification before the final appearance of an 'active' product, and in some cases the polypeptide chain may be modified before the completed protein molecule is released from the ribosome. This then raises the question concerning the possibility of the organization of the rough endoplasmic reticulum into individual domains, or compartments, each of which may have the responsibility of performing definite and well defined functions. During recent years the behaviour of two subfractions of the rough endoplasmic reticulum in a variety of cell types and under a variety of conditions has been studied in order to gain insight into a possible compartmentation of this organelle. Throughout the studies disruption of cells has been performed by nitrogen cavitation. This technique was chosen in order to provide conditions of homogenization which were extremely reproducible since shearing forces, mechanical damage and the effects of local heating were eliminated. Endoplasmic reticulum (ER) membranes isolated from the post-mitochondrial supernatant have been separated into subfractions by centrifugation on discontinuous sucrose gradients. By virtue of their high density imparted by the association of ribosomes, rough ER (RER) membranes penetrate 1.4 M sucrose accumulating above either 2.0 M sucrose (light rough -LR membranes) or a cushion of 2.3 M sucrose (heavy rough -HR membranes). Smooth (S) membranes, which are virtually devoid of ribosomes, collect above 1.4 M sucrose. The HR, LR and S subfractions in MPC-11 cells differ in a number of respects: RNA/protein and RNA/phospholipid ratios, polysome profiles and marker enzymes. When cells were homogenized in buffer containing 25 mM KCl then all three ER subfractions were observed, however, when the buffer contained 100 mM KCl then only the LR and S subfractions were observed in gradients, radioactivity equivalent to that in the HR fraction was not recovered in the other two subfractions. Four times as many light chain immunoglobulin polypeptides were found associated with polysomes of HR membranes compared to LR membranes. The nuclear associated ER (NER), though very active in protein synthesis, was only 20% as active in the synthesis of light chain as the combined LR/HR fraction. Studies with MPC-11 cells showed that the relative amounts of the three ER subfractions were related to the phase of the cell cycle.(ABSTRACT TRUNCATED AT 400 WORDS)     

Brefeldin A redistributes resident and itinerant Golgi proteins to the endoplasmic reticulum

Brefeldin A (BFA) has been reported to block protein transport from the ER and cause disassembly of the Golgi complex. We have examined the effects of BFA on the transport and processing of the vesicular stomatitis virus G protein, a model integral membrane protein. Delivery of G protein to the cell surface was reversibly blocked by 6 micrograms/ml BFA. Pulse-label experiments revealed that in the presence of BFA, G protein became completely resistant to endoglycosidase H digestion. Addition of sialic acid, a trans-Golgi event, was not observed. Despite processing by cis- and medial Golgi enzymes, G protein was localized by indirect immunofluorescence to a reticular distribution characteristic of the ER. By preventing transport of G protein from the ER with the metabolic inhibitor carbonyl cyanide m-chlorophenylhydrazone or by use of the temperature-sensitive mutant ts045, which is restricted to the ER at 40 degrees C, we showed that processing of G protein occurred in the ER and was not due to retention of newly synthesized Golgi enzymes. Rather, redistribution of preexisting cis and medial Golgi enzymes to the ER occurred as soon as 2.5 min after addition of BFA, and was complete by 10-15 min. Delivery of Golgi enzymes to the ER was energy dependent and occurred only at temperatures greater than or equal to 20 degrees C. BFA also induced retrograde transport of G protein from the medial Golgi to the ER. Golgi enzymes were completely recovered from the ER 10 min after removal of BFA. These findings demonstrate that BFA induces retrograde transport of both resident and itinerant Golgi proteins to the ER in a fully reversible manner.     

Accumulation of polyunsaturated free fatty acids coincident with the fusion of rough endoplasmic reticulum membranes.

The accumulation of polyunsaturated free fatty acids (PUFAs) was observed coincident with GTP-dependent fusion of liver rough microsomes. Whereas 0.5 mM NADPH led to a parallel reduction (greater than 50%) in membrane fusion and PUFA accumulation, indomethacin (50 microM) either had little effect or slightly augmented both processes. CTP was observed to stimulate accumulation of PUFAs and diacylglycerol (DAG). Therefore PUFAs may be relevant for GTP-dependent membrane fusion and together with DAG may play a role in fusion stimulated in the presence of CTP.     

Domains of rough endoplasmic reticulum (a review)

The rough endoplasmic reticulum isolated from several eukaryotic cell lines can be separated into subfractions. These subfractions possess different properties indicating that they represent separate domains of the endoplasmic reticulum system.     

Translocation of UDP-N-acetylglucosamine into vesicles derived from rat liver rough endoplasmic reticulum and Golgi apparatus

A mixture of UDP-N-acetylglucosamine labeled with different radioisotopes in the uridine and glucosamine was used to show that the intact sugar nucleotide was translocated across the membrane of vesicles derived from rat liver rough endoplasmic reticulum (RER) and Golgi apparatus. Translocation was dependent on temperature, saturable at high concentrations of sugar nucleotide, and inhibited by treatment of vesicles with proteases, suggesting protein carrier mediated transport. Translocation of UDP-GlcNAc by RER-derived vesicles appeared to be specific since these vesicles were unable to translocate UDP-galactose, in contrast to those derived from the Golgi apparatus. Preliminary results suggest that the mechanism of UDP-GlcNAc translocation into RER-derived vesicles is via a coupled exchange with lumenal nucleoside monophosphate. This is similar to the recently postulated mechanism for translocation of sugar nucleotides into vesicles derived from the Golgi apparatus.     

Brefeldin A-regulated retrograde transport into the endoplasmic reticulum of internalised wheat germ agglutinin

The effects of the fungal metabolite brefeldin A (BFA) on the endocytic routes of internalised wheat germ agglutinin (WGA) were studied in human HepG2 hepatoma cells, drawing particular attention to the application times in relation to the membrane dynamics occurring at the trans Golgi face during endocytosis. As shown in previous studies, transport of internalised WGA into the Golgi apparatus can be classified in three stages being characterised by predominance of vesicular endosomes (stage I), formation of an extended endocytic trans Golgi network (stage II) and uptake of WGA into the stacked Golgi cisternae (stage III). BFA treatment of the cells led to rapid tubular-reticular transformations of the Golgi stacks. Retrograde transport and further destinations of internalised WGA depended on the time of BFA application. When BFA was administered during stages I or II, WGA was localised within the BFA-induced tubules and networks, but never was found within the endoplasmic reticulum. By contrast, BFA treatment during stage III led to a redistribution of internalised WGA into cisternae of the endoplasmic reticulum. These results show that BFA administered according to a precise time schedule can be used as a regulatory agent that allows to control retrograde traffic of internalised molecules into the endoplasmic reticulum.     

Ultrastructural cryoinjury and cryoprotection of rough endoplasmic reticulum

One phase of a study on cryosurvival and cryoprotection of mammalian cells, in terms of ultrastructural alteration of rough endoplasmic reticulum (RER) within rat pancreatic acinar cells, is presented. Small (2–3 mm) squares of tissue, 0.7–0.9 mm in thickness, were compared as unfrozen controls, with (w) and without (wo) glycerol pretreatment (15% $\text{v}\text{v}$ in mammalian Ringer's solution) at 0 °C and 22 °C (to regulate glycerol permeability); as well as parallel frozen-thawed samples, after combinations of slow (3.8 °C/min) freezing (SF) and rapid (38 °C/sec) freezing (RF) with either slow (1.5 °C/min) thawing (ST) or rapid (8 °C/sec) thawing (RT). Regimens compared were SFRT, SFST, RFRT, and RFST, all w and wo glycerol pretreatment at 0 °C and 22 °C. Tissue from each treatment was prepared for electron microscopic observations. The results on rates of freezing and thawing and relative cryoprotection of intracellular and extracellular glycerol under conditions described are intended to serve as a correlative basis for subsequent parallel studies on function (protein synthesis) and ultrastructure of the frozen state. They now indicate the following: (1) Cryoinjury of RER, which occurred during all treatments compared, was manifested in irregularity, dilatation, vesiculation, and altered matrix density of cisternae, and ribosomal derangement or disjunction. Least injury was shown by some disorientation and dilatation with increasing degrees of damage involving accentuation of these and other alterations. Such ultrastructural alterations to RER are not unique to cryoinjury, since they have been induced by treatments and agents other than freeze-thawing in experimental pathology. (2) Cryoinjury is unique, however, in that it can be regulated to demonstrate a spectrum of degrees of injury to cells and their organelles, immediately after cryoexposure. Controlled cryoinjury is suggested as a research tool for studies on injury, in general, on an ultrastructural-functional level. (3) Glycerol is injurious or toxic during pretreatment. Toxicity, which resembles cryoinjury, is greater during 22 ° C (intracellular) than 0 °C (extracellular) glycerol pretreatment, especially with respect to dilatation of cisternae. (4) Extra-cellular glycerol is cryoprotective during both slow and rapid freezing followed by either slow or rapid thawing, while little or no cryoprotection is afforded when glycerol is located simultaneously in the intracellular and extracellular location. (5) Rate of freezing is more important than rate of thawing as a factor in cryosurvival. Rapid freezing is more injurious than slow freezing, in the absence of glycerol or in the presence of extracellular glycerol, with slight or no differences seen as a function of thawing rate. Neither rate of freezing nor rate of thawing is of serious consequence when glycerol is intracellular. (6) Rate of thawing has importance after slow freezing, when slow thawing is more injurious than rapid, but not after rapid freezing, either in the presence or absence of extracellular glyeerol.     

A membrane preparation that contains proteins characteristic of the rough endoplasmic reticulum

We describe a procedure for disassembling rat liver rough microsomes, which allows the purification of the rough endoplasmic reticulum (ER) membrane. Membrane-bound ribosomes and adsorbed proteins are first detached by washing rough microsomes with 5 mM Na-pyrophosphate. In a second step, the vesicle membrane is opened by digitonin, with concomitant release of the luminal content. The purification is monitored at each step by electron microscopy, and by assaying chemical constituents (protein, phospholipid, RNA) and marker enzymes for the main subcellular organelles. The final membrane preparation is representative of the ER, since it contains 24.1% of the liver glucose 6-phosphatase with a relative specific activity of 14.2. Contaminants represent less than 5% of its protein content. SDS-polyacrylamide gel electrophoresis, followed by immunoblot analysis, reveals that the ribophorins I and II, two established markers of the rough (d) domain are still present in the final membrane preparation. It also contains the docking protein (or signal recognition particle receptor) and protein disulfide isomerase, and has conserved the functional capacity to remove co- and post-translationally the signal peptide of pre-secretory proteins. The membrane preparation is suitable for studies on the polypeptide composition of the d domain.     

Brefeldin A causes disassembly of the Golgi complex and accumulation of secretory proteins in the endoplasmic reticulum

The antiviral antibiotic brefeldin A (BFA) strongly inhibits the protein secretion in cultured rat hepatocytes (Misumi, Y., Misumi, Y., Miki, K., Takatsuki, A., Tamura, G., and Ikehara, Y. (1986) J. Biol. Chem. 261, 11398-11403). We have further examined the inhibitory effect of the drug on intracellular transport of albumin by an immunocytochemical technique with peroxidase-conjugated Fab fragments of anti-rat albumin IgG. In hepatocytes treated with BFA (2.5 micrograms/ml) for 1 h at 37 degrees C, no characteristic structures of the Golgi complex could be observed, and albumin was diffusely distributed in the endoplasmic reticulum (ER), nuclear envelope, and small vesicles around, in contrast to its condensed localization in the Golgi complex in the control cells. Such an unusual distribution of the secretory protein, however, was rearranged to the normal localization in the Golgi complex after 4 h even in the presence of the drug, possibly due to a metabolism of the drug to an inert form. Exposure of the cells to BFA with constant renewals (2.5 micrograms/ml at 1-h intervals) or at a higher concentration (10 micrograms/ml) caused a prolonged accumulation of albumin in the ER, resulting in its dilation. These results indicate that BFA primarily blocks the protein transport from the ER to the Golgi complex, consistent with the biochemical data previously reported.     

Association of methionyl-tRNA synthetase with detergent-insoluble components of the rough endoplasmic reticulum

Using fluorescent antibody staining, we have established the association of methionyl-tRNA synthetase with the endoplasmic reticulum in PtK2 cells. After Triton X-100 extraction, 70% of the recovered aminoacyl-tRNA synthetase activity was found in the detergent-insoluble fraction. This fraction of the enzyme remained localized with insoluble endoplasmic reticulum antigens and with ribosomes, which were stained with acridine orange. By both fluorescence microscopy and electron microscopy the organization of the detergent-insoluble residue was found to depend on the composition of the extracting solution. After extraction with a microtubule-stabilizing buffer containing EGTA, Triton X-100, and polyethylene glycol (Osburn, M., and K. Weber, 1977, Cell, 12:561-571) the ribosomes were aggregated in large clusters with remnants of membranes. After extraction with a buffer containing Triton X-100, sucrose, and CaCl2 (Fulton, A. B., K. M. Wang, and S. Penman, 1980, Cell, 20:849-857), the ribosomes were in small clusters and there were few morphologically recognizable membranes. In both cases the methionyl-tRNA synthetase and some endoplasmic reticulum antigens retained approximately their normal distribution in the cell. Double fluorochrome staining showed no morphological association of methionyl-tRNA synthetase with the microtubule, actin, or cytokeratin fiber systems of PtK2 cells. These observations demonstrate that detergent-insoluble cellular components, sometimes referred to as "cytoskeletal" preparations, contain significant amounts of nonfilamentous material including ribosomes, and membrane residue. Caution is required in speculating about intermolecular associations in such a complex cell fraction.     

Antibodies to the Golgi complex and the rough endoplasmic reticulum

Rabbits were immunized with membrane fractions from either the Golgi complex or the rough endoplasmic reticulum (RER) by injection into the popliteal lymph nodes. The antisera were then tested by indirect immunofluorescence on tissue culture cells or frozen, thin sections of tissue. There were may unwanted antibodies to cell components other than the RER or the Golgi complex, and these were removed by suitable absorption steps. These steps were carried out until the pattern of fluorescent labeling was that expected for the Golgi complex or RER. Electron microscopic studies, using immunoperoxidase labeling of normal rat kidney (NRK) cells, showed that the anti-Golgi antibodies labeled the stacks of flattened cisternae that comprise the central feature of the Golgi complex, many of the smooth vesicles around the stacks, and a few coated vesicles. These antibodies were directed, almost entirely, against a single polypeptide with an apparent molecular weight of 135,000. The endoplasmic reticulum (ER) in NRK cells is an extensive, reticular network that pervades the entire cell cytoplasm and includes the nuclear membrane. The anit-RER antibodies labeled this structure alone at the light and electron microscopic levels. They were largely directed against four polypeptides with apparent molecular weights of 29,000, 58,000, 66,000, and 91,000. Some examples are presented, using immunofluorescence microscopy, where these antibodies have been used to study the Golgi complex and RER under a variety of physiological and experimental condition . For biochemical studies, these antibodies should prove useful in identifying the origin of isolated membranes, particularly those from organelles such as the Golgi complex, which tend to lose their characteristic morphology during isolation.     

Topography of glycosylation reactions in the rough endoplasmic reticulum membrane

The translocation of UDP-glucose and GDP-mannose from an external to a luminal compartment has been examined in rat liver vesicles derived from the rough endoplasmic reticulum (RER). RER vesicles with the same topographical orientation as in vivo were incubated with a mixture of [3H]UDP-glucose and UDP-[14C]glucose to demonstrate that the intact sugar nucleotide was translocated into the lumen of the vesicles. The translocation of UDP-glucose was dependent on temperature and was saturable at high concentrations of the sugar nucleotide. The transfer of glucose to endogenous acceptors was dependent on the translocation of UDP-glucose into the lumen of the RER since leaky vesicles resulted in both a decrease in transport and transfer of glucose to endogenous acceptors. Preliminary results suggest that the mechanism of UDP-glucose transport into RER-derived vesicles is via a coupled exchange with luminal UMP. Using the same experimental approach to detect translocation of UDP-glucose into the lumen of RER vesicles, we were unable to detect transport of GDP-mannose. Incubation of leaky vesicles with GDP-mannose resulted in stimulation of the amount of mannose transferred to endogenous acceptors, in marked contrast to that observed for UDP-glucose and UDP-N-acetylglucosamine. These results suggest that whereas UDP-glucose is translocated across the RER membrane in vitro, GDP-mannose is not transported. In addition, these results tentatively suggest that there is asymmetric synthesis of the lipid-linked oligosaccharides within the membrane of the RER.     

A novel SNARE complex implicated in vesicle fusion with the endoplasmic reticulum.

Intracellular vesicular traffic is controlled in part by v- and t-SNAREs, integral membrane proteins which allow specific interaction and fusion between vesicles (v-SNAREs) and their target membranes (t-SNAREs). In yeast, retrograde transport from the Golgi complex to the ER is mediated by the ER t-SNARE Ufe1p, and also requires two other ER proteins, Sec20p and Tip20p, which bind each other. Although Sec20p is not a typical SNARE, we show that both it and Tip20p can be co-precipitated with Ufe1p, and that a growth-inhibiting mutation in Ufe1p can be compensated by a mutation in Sec20p. Furthermore, Sec22p, a v-SNARE implicated in forward transport from ER to Golgi, co-precipitates with Ufe1p and Sec20p, and SEC22 acts as an allele-specific multicopy suppressor of a temperature-sensitive ufe1 mutation. These results define a new functional SNARE complex, with features distinct from the plasma membrane and cis-Golgi complexes previously identified. They also show that a single v-SNARE can be involved in both anterograde and retrograde transport, which suggests that the mere presence of a particular v-SNARE may not be sufficient to determine the preferred target for a transport vesicle.     

Protein fatty acyltransferase is located in the rough endoplasmic reticulum

The fatty acid acylation of polypeptides was studied in vivo and in vitro by incorporation of radiolabeled palmitic acid into Semliki Forest viral polypeptides. Utilizing a cell-free system for acylation protein fatty acyltransferase was characterized as an integral membrane protein. No acylation activity was detected in the cytosol. During subcellular fractionation of a variety of mammalian or avian cells the enzyme was localized to the rough endoplasmic reticulum. Therefore this posttranslational hydrophobic modification starts earlier in the biosynthesis of acylated polypeptides than previously believed.     

Freeze-fracture analysis of the effects of intermediates of the phosphatidylinositol cycle on fusion of rough endoplasmic reticulum membranes.

While searching for the identity of the effector of the putative GTP-binding protein involved in fusion of rough endoplasmic reticulum (RER) cell-free incubation conditions were found permitting fusion in a GTP-independent manner. Membrane fusion was obtained using medium required to study synthesis of phosphatidylinositol (PI). We now report on the effects of various co-factors and intermediates of the PI cycle on the interaction of rough microsomes. By freeze-fracture, fusion of rough microsomes was defined as the appearance of fracture-planes of membrane larger than those of unincubated membrane. Cytosine triphosphate (CTP, 3 mM) in the presence of 2 mM MnCl2 was most effective in stimulating fusion. Guanosine triphosphate (GTP) at the same concentration, could substitute for CTP to stimulate fusion, ATP, ITP, UTP and guanosine 5'-[gamma-thio]triphosphate (GTP gamma S) could not. When combined together in the same medium CTP potentiated the effect of GTP. Arachidonic acid (20 micrograms/ml) also stimulated fusion in the presence of MnCl2. This led to the appearance of large fracture-planes of membrane with a heterogeneous distribution of intramembranous particles. Other saturated fatty acids at the same concentration did not stimulate fusion. Phosphatidylinositol (PI, 50 micrograms) and 2 mM MnCl2 had a similar effect as arachidonic acid and MnCl2 in stimulating fusion. The PI effect was largely augmented in the presence of CTP. Our results are consistent with the concept that metabolism of phospholipids may modulate GTP-dependent fusion of RER membranes.     

Identification of a G protein in rough endoplasmic reticulum of canine pancreas

Employing [32P]ADP-ribosylation by pertussis toxin we have identified a G protein that is located in the rough endoplasmic reticulum of canine pancreas and therefore termed it GRER. Identification of GRER is based on the following data. A 41-kDa polypeptide was the only polypeptide that was [32P]ADP-ribosylated by pertussis toxin in pancreas rough microsomes. Guanosine 5'-(gamma-thio)triphosphate (GTP gamma S) and 1 mM ATP, 6 mM MgCl2, 10 mM NaF (AMF) inhibited ADP-ribosylation of this polypeptide. The [32P]ADP-ribosylated 41-kDa polypeptide was immunoprecipitated by antisera which specifically recognized the C-terminal residues of the alpha subunits of Gi and transducin, indicating that the 41-kDa polypeptide is immunologically related to the alpha subunits of heterotrimeric G proteins. Treatment with GTP gamma S resulted in a reduction in the sedimentation rate of the [32P]ADP-ribosylated, detergent-solubilized GRER. It also induced the release of the [32P]ADP-ribosylated 41-kDa polypeptide from rough microsomes in the absence of detergent, unlike ADP-ribosylated alpha subunits of plasma membrane-associated G proteins. These data are consistent with an oligomeric nature of GRER. The codistribution of GRER with an endoplasmic reticulum marker protein during subcellular fractionation and the lack of plasma membrane contamination of the rough microsomal fraction, combined with the isodensity of GRER with rough microsomes as well as the isodensity of GRER with "stripped" microsomes after extraction of rough microsomes with EDTA and 0.5 M KCl, localized GRER to the rough endoplasmic reticulum. Preliminary experiments suggest that GRER appears not to be involved in translocation of proteins across the rough endoplasmic reticulum membrane.     

Heparin releases monosomes and polysomes from rough endoplasmic reticulum

Incubation of membrane-bound polyribosomes isolated from murine myeloma cells with heparin caused release of material which sedimented in the polysome, monosome and ribosomal subunit regions of linear sucrose gradients. The released material corresponded to approximately one half that which could be released by treatment with heparin plus Triton X-100. The action of heparin appeared to be related to its polyanionic nature. The use of heparin as a ribonuclease inhibitor in the separation and isolation of free and membrane-bound polysomes could cause artificial accumulation of detached polysomes in the free polysome fraction.