Dissociation of a 110-kD peripheral membrane protein from the Golgi apparatus is an early event in brefeldin A action

Brefeldin A (BFA) has a profound effect on the structure of the Golgi apparatus, causing Golgi proteins to redistribute into the ER minutes after drug treatment. Here we describe the dissociation of a 110-kD cytoplasmically oriented peripheral membrane protein (Allan, V. J., and T. E. Kreis. 1986. J. Cell Biol. 103:2229-2239) from the Golgi apparatus as an early event in BFA action, preceding other morphologic changes. In contrast, other peripheral membrane proteins of the Golgi apparatus were not released but followed Golgi membrane into the ER during BFA treatment. The 110-kD protein remained widely dispersed throughout the cytoplasm during drug treatment, but upon removal of BFA it reassociated with membranes during reformation of the Golgi apparatus. Although a 30-s exposure to the drug was sufficient to cause the redistribution of the 110-kD protein, removal of the drug after this short exposure resulted in the reassociation of the 110-kD protein and no change in Golgi structure. If cells were exposed to BFA for 1 min or more, however, a portion of the Golgi membrane was committed to move into and out of the ER after removal of the drug. ATP depletion also caused the reversible release of the 110-kD protein, but without Golgi membrane redistribution into the ER. These findings suggest that the interaction between the 110-kD protein and the Golgi apparatus is dynamic and can be perturbed by metabolic changes or the drug BFA.     

Perturbation of the morphology of the trans-Golgi network following Brefeldin A treatment: redistribution of a TGN-specific integral membrane protein, TGN38

Brefeldin A (BFA) has a dramatic effect on the morphology of the Golgi apparatus and induces a rapid redistribution of Golgi proteins into the ER (Lippincott-Schwartz, J., L. C. Yuan, J. S. Bonifacino, and R. D. Klausner. 1989. Cell. 56:801-813). To date, no evidence that BFA affects the morphology of the trans-Golgi network (TGN) has been presented. We describe the results of experiments, using a polyclonal antiserum to a TGN specific integral membrane protein (TGN38) (Luzio, J.P., B. Brake, G. Banting, K. E. Howell, P. Braghetta, and K. K. Stanley. 1990. Biochem. J. 270:97-102), which demonstrate that incubation of cells with BFA does induce morphological changes to the TGN. However, rather than redistributing to the ER, the majority of the TGN collapses around the microtubule organizing center (MTOC). The effect of BFA upon the TGN is (a) independent of protein synthesis, (b) fully reversible (c) microtubule dependent (as shown in nocodazole-treated cells), and (d) relies upon the hydrolysis of GTP (as shown by performing experiments in the presence of GTP gamma S). ATP depletion reduces the ability of BFA to induce a redistribution of Golgi proteins into the ER; however, it has no effect upon the BFA-induced relocalizations of the TGN. These data confirm that the TGN is an organelle which is independent of the Golgi, and suggest a dynamic interaction between the TGN and microtubules which is centered around the MTOC.     

100-kD proteins of Golgi- and trans-Golgi network-associated coated vesicles have related but distinct membrane binding properties

The 100-110-kD proteins (alpha-, beta-, beta'-, and gamma-adaptins) of clathrin-coated vesicles and the 110-kD protein (beta-COP) of the nonclathrin-coated vesicles that mediate constitutive transport through the Golgi have homologous protein sequences. To determine whether homologous processes are involved in assembly of the two types of coated vesicles, the membrane binding properties of their coat proteins were compared. After treatment of MDBK cells with the fungal metabolite Brefeldin A (BFA), beta-COP was redistributed to the cytoplasm within 15 s, gamma-adaptin and clathrin in the trans-Golgi network (TGN) dispersed within 30 s, but the alpha-adaptin and clathrin present on coated pits and vesicles derived from the plasma membrane remained membrane associated even after a 15-min exposure to BFA. In PtK1 cells and MDCK cells, BFA did not affect beta-COP binding or Golgi morphology but still induced redistribution of gamma-adaptin and clathrin from TGN membranes to the cytoplasm. Thus BFA affects the binding of coat proteins to membranes in the Golgi region (Golgi apparatus and TGN) but not plasma membranes. However, the Golgi binding interactions of beta-COP and gamma-adaptin are distinct and differentially sensitive to BFA. BFA treatment did not release gamma-adaptin or clathrin from purified clathrin-coated vesicles, suggesting that their distribution to the cytoplasm after BFA treatment of cells was due to interference with their rebinding to TGN membranes after a normal cycle of disassembly. This was confirmed using an in vitro assay in which gamma-adaptin binding to TGN membranes was blocked by BFA and enhanced by GTP gamma S, similar to the binding of beta-COP to Golgi membranes. These results suggest the involvement of GTP-dependent proteins in the association of the 100-kD coat proteins with membranes in the Golgi region of the cell.     

Forskolin inhibits and reverses the effects of brefeldin A on Golgi morphology by a cAMP-independent mechanism

Brefeldin A (BFA) causes rapid redistribution of Golgi proteins into the ER, leaving no definable Golgi apparatus, and blocks transport of proteins into post-Golgi compartments in the cell. In this study we follow the disassembly of the Golgi apparatus in BFA-treated, living cells labeled with NBD-ceramide and demonstrate that forskolin can both inhibit and reverse this process. Long, tubular processes labeled with NBD-ceramide were observed emerging from Golgi elements and extending out to the cell periphery in cells treated with BFA for 5 min. With longer incubations in BFA, the NBD label was dispersed in a fine reticular pattern characteristic of the ER. Treatment with forskolin inhibited these effects of BFA as well as BFA's earliest morphologic effect on the Golgi apparatus: the redistribution to the cytosol of a 110-kD Golgi peripheral membrane protein. In addition, forskolin could reverse BFA's block in protein secretion. Forskolin inhibition of BFA's effects was dose dependent and reversible. High concentrations of BFA could overcome forskolin's inhibitory effect, suggesting forskolin and BFA interact in a competitive fashion. Remarkably, in cells already exposed to BFA, forskolin could reverse BFA's effects causing the 110-kD Golgi peripheral membrane protein to reassociate with Golgi membrane and juxtanuclear Golgi complexes to reassemble. Neither membrane permeant cAMP analogues nor cAMP phosphodiesterase inhibitors could replicate or enhance forskolin's inhibition of BFA. 1,9-Dideoxyforskolin, which does not activate adenylyl cyclase, was equally as effective as forskolin in antagonizing BFA. A derivative of forskolin, 7-HPP-forskolin, that is less potent than forskolin at binding to adenylyl cyclase, was also equally effective as forskolin in antagonizing BFA. In contrast a similar derivative, 6-HPP-forskolin, that is equipotent with forskolin at binding to adenylyl cyclase, did not inhibit BFA's effects. These results suggest that forskolin acts as a competitive antagonist to BFA, using a cAMP-independent mechanism to prevent and reverse the morphologic effects induced by BFA.     

Gap junction turnover, intracellular trafficking, and phosphorylation of connexin43 in brefeldin A-treated rat mammary tumor cells

Intercellular gap junction channels are thought to form when oligomers of connexins from one cell (connexons) register and pair with connexons from a neighboring cell en route to forming tightly packed arrays (plaques). In the current study we used the rat mammary BICR-M1Rk tumor cell line to examine the trafficking, maturation, and kinetics of connexin43 (Cx43). Cx43 was conclusively shown to reside in the Golgi apparatus in addition to sites of cell-cell apposition in these cells and in normal rat kidney cells. Brefeldin A (BFA) blocked Cx43 trafficking to the surface of the mammary cells and also prevented phosphorylation of the 42-kD form of Cx43 to 44- and 46-kD species. However, phosphorylation of Cx43 occurred in the presence of BFA while it was still a resident of the ER or Golgi apparatus yielding a 43-kD form of Cx43. Moreover, the 42- and 43-kD forms of Cx43 trapped in the ER/Golgi compartment were available for gap junction assembly upon the removal of BFA. Mammary cells treated with BFA for 6 h lost preexisting gap junction "plaques," as well as the 44- and 46-kD forms of Cx43 and functional coupling. These events were reversible 1 h after the removal of BFA and not dependent on protein synthesis. In summary, we provide strong evidence that in BICR-M1Rk tumor cells: (a) Cx43 is transiently phosphorylated in the ER/Golgi apparatus, (b) Cx43 trapped in the ER/Golgi compartment is not subject to rapid degradation and is available for the assembly of new gap junction channels upon the removal of BFA, (c) the rapid turnover of gap junction plaques is correlated with the loss of the 44- and 46-kD forms of Cx43.     

Capacity of the golgi apparatus for biogenesis from the endoplasmic reticulum

It is unclear whether the mammalian Golgi apparatus can form de novo from the ER or whether it requires a preassembled Golgi matrix. As a test, we assayed Golgi reassembly after forced redistribution of Golgi matrix proteins into the ER. Two conditions were used. In one, ER redistribution was achieved using a combination of brefeldin A (BFA) to cause Golgi collapse and H89 to block ER export. Unlike brefeldin A alone, which leaves matrix proteins in relatively large remnant structures outside the ER, the addition of H89 to BFA-treated cells caused ER accumulation of all Golgi markers tested. In the other, clofibrate treatment induced ER redistribution of matrix and nonmatrix proteins. Significantly, Golgi reassembly after either treatment was robust, implying that the Golgi has the capacity to form de novo from the ER. Furthermore, matrix proteins reemerged from the ER with faster ER exit rates. This, together with the sensitivity of BFA remnants to ER export blockade, suggests that presence of matrix proteins in BFA remnants is due to cycling via the ER and preferential ER export rather than their stable assembly in a matrix outside the ER. In summary, the Golgi apparatus appears capable of efficient self-assembly.     

PtK1 cells contain a nondiffusible, dominant factor that makes the Golgi apparatus resistant to brefeldin A

Brefeldin A (BFA) was shown in earlier studies of numerous cell types to inhibit secretion, induce enzymes of the Golgi stacks to redistribute into the ER, and to cause the Golgi cisternae to disappear. Here, we demonstrate that the PtK1 line of rat kangaroo kidney cells is resistant to BFA. The drug did not disrupt the morphology of the Golgi complex in PtK1 cells, as judged by immunofluorescence using antibodies to 58- (58K) and 110-kD (beta-COP) Golgi proteins, and by fluorescence microscopy of live cells labeled with C6-NBD-ceramide. In addition, BFA did not inhibit protein secretion, not alter the kinetics or extent of glycosylation of the vesicular stomatitis virus (VSV) glycoprotein (G-protein) in VSV-infected PtK1 cells. To explore the mechanism of resistance to BFA, PtK1 cells were fused with BFA-sensitive CV-1 cells that had been infected with a recombinant SV-40 strain containing the gene for VSV G-protein and, at various times following fusion, the cultures were exposed to BFA. Shortly after cell fusion, heterokaryons contained one Golgi complex associated with each nucleus. Golgi membranes derived from CV-1 cells were sensitive to BFA, whereas those of PtK1 origin were BFA resistant. A few hours after fusion, most heterokaryons contained a single, large Golgi apparatus that was resistant to BFA and contained CV-1 galactosyltransferase. In unfused cells that had been perforated using nitrocellulose filters, retention of beta-COP on the Golgi was optimal in the presence of cytosol, ATP, and GTP. In perforated cell models of the BFA-sensitive MA104 line, BFA caused beta-COP to be released from the Golgi complex in the presence of nucleotides, and either MA104 or PtK1 cytosol. In contrast, when perforated PtK1 cells were incubated with BFA, nucleotides, and cytosol from either cell type, beta-COP remained bound to the Golgi complex. We conclude that PtK1 cells contain a nondiffusible factor, which is located on or very close to the Golgi complex, and confers a dominant resistance to BFA. It is possible that this factor is homologous to the target of BFA in cells that are sensitive to the drug.     

A Rab1 mutant affecting guanine nucleotide exchange promotes disassembly of the Golgi apparatus

The Golgi apparatus is a dynamic organelle whose structure is sensitive to vesicular traffic and to cell cycle control. We have examined the potential role for rab1a, a GTPase previously associated with ER to Golgi and intra-Golgi transport, in the formation and maintenance of Golgi structure. Bacterially expressed, recombinant rab1a protein was microinjected into rat embryonic fibroblasts, followed by analysis of Golgi morphology by fluorescence and electron microscopy. Three recombinant proteins were tested: wild-type rab, mutant rab1a(S25N), a constitutively GDP-bound form (Nuoffer, C., H. W. Davidson, J. Matteson, J. Meinkoth, and W. E. Balch, 1994. J. Cell Biol. 125: 225- 237), and mutant rab1a(N124I) defective in guanine nucleotide binding. Microinjection of wild-type rab1a protein or a variety of negative controls (injection buffer alone or activated ras protein) did not affect the appearance of the Golgi, as visualized by immunofluorescence of alpha-mannosidase II (Man II), used as a Golgi marker. In contrast, microinjection of the mutant forms promoted the disassembly of the Golgi stacks into dispersed vesicular structures visualized by immunofluorescence. When S25N-injected cells were analyzed by EM after immunoperoxidase labeling, Man II was found in isolated ministacks and large vesicular elements that were often surrounded by numerous smaller unlabeled vesicles resembling carrier vesicles. Golgi disassembly caused by rab1a mutants differs from BFA-induced disruption, since beta- COP remains membrane associated, and Man II does not redistribute to the ER. BFA can still cause these residual Golgi elements to fuse and disperse, albeit at a slower rate. Moreover, BFA recovery is incomplete in the presence of rab1 mutants or GTP gamma S. We conclude that GTP exchange and hydrolysis by GTPases, specifically rab1a, are required to form and maintain normal Golgi stacks. The similarity of Golgi disassembly seen with rab1a mutants to that occurring during mitosis, may point to a molecular basis involving rab1a for fragmentation of the Golgi apparatus during cell division.     

A Secretory Golgi Bypass Route to the Apical Surface Domain of Epithelial MDCK Cells

Proteins leave the endoplasmic reticulum (ER) for the plasma membrane via the classical secretory pathway, but routes bypassing the Golgi apparatus have also been observed. Apical and basolateral protein secretion in epithelial Madin-Darby canine kidney (MDCK) cells display differential sensitivity to Brefeldin A (BFA), where low concentrations retard apical transport, while basolateral transport still proceeds through intact Golgi cisternae . We now describe that BFA-mediated retardation of glycoprotein and proteoglycan transport through the Golgi apparatus induces surface transport of molecules lacking Golgi modifications, possessing those acquired in the ER. Low concentrations of BFA induces apical Golgi bypass, while higher concentrations were required to induce basolateral Golgi bypass. Addition of the KDEL ER-retrieval sequence to model protein cores allowed observation of apical Golgi bypass in untreated MDCK cells. Basolateral Golgi bypass was only observed after the addition of BFA or upon cholesterol depletion. Thus, in MDCK cells, an apical Golgi bypass route can transport cargo from pre-Golgi organelles in untreated cells, while the basolateral bypass route is inducible.     

Rapid redistribution of Golgi proteins into the ER in cells treated with brefeldin A: evidence for membrane cycling from Golgi to ER

In cells treated with brefeldin A (BFA), movement of newly synthesized membrane proteins from the endoplasmic reticulum (ER) to the Golgi apparatus was blocked. Surprisingly, the glycoproteins retained in the ER were rapidly processed by cis/medial Golgi enzymes but not by trans Golgi enzymes. An explanation for these observations was provided from morphological studies at both the light and electron microscopic levels using markers for the cis/medial and trans Golgi. They revealed a rapid and dramatic redistribution to the ER of components of the cis/medial but not the trans Golgi in response to treatment with BFA. Upon removal of BFA, the morphology of the Golgi apparatus was rapidly reestablished and proteins normally transported out of the ER were efficiently and rapidly sorted to their final destinations. These results suggest that BFA disrupts a dynamic membrane-recycling pathway between the ER and cis/medial Golgi, effectively blocking membrane transport out of but not back to the ER.     

Disruption of endoplasmic reticulum to Golgi transport leads to the accumulation of large aggregates containing beta-COP in pancreatic acinar cells.

When transport between the rough endoplasmic reticulum (ER) and Golgi complex is blocked by Brefeldin A (BFA) treatment or ATP depletion, the Golgi apparatus and associated transport vesicles undergo a dramatic reorganization. Because recent studies suggest that coat proteins such as beta-COP play an important role in the maintenance of the Golgi complex, we have used immunocytochemistry to determine the distribution of beta-COP in pancreatic acinar cells (PAC) in which ER to Golgi transport was blocked by BFA treatment or ATP depletion. In controls, beta-COP was associated with Golgi cisternae and transport vesicles as expected. Upon BFA treatment, PAC Golgi cisternae are dismantled and replaced by clusters of remnant vesicles surrounded by typical ER transitional elements that are generally assumed to represent the exit site of vesicular carriers for ER to Golgi transport. In BFA-treated PAC, beta-COP was concentrated in large (0.5-1.0 micron) aggregates closely associated with remnant Golgi membranes. In addition to typical ER transitional elements, we detected a new type of transitional element that consists of specialized regions of rough ER (RER) with ribosome-free ends that touched or extended into the beta-COP containing aggregates. In ATP-depleted PAC, beta-COP was not detected on Golgi membranes but was concentrated in similar large aggregates found on the cis side of the Golgi stacks. The data indicate that upon arrest of ER to Golgi transport by either BFA treatment or energy depletion, beta-COP dissociates from PAC Golgi membranes and accumulates as large aggregates closely associated with specialized ER elements. The latter may correspond to either the site of entry or exit for vesicles recycling between the Golgi and the RER.     

Brefeldin A-induced disassembly of the Golgi apparatus is followed by disruption of the endoplasmic reticulum in plant cells

Brefeldin A (BFA), a fungal fatty acid derivative, is a potentagent for disrupting the Golgi apparatus in plant and animalcells. We have examined its action using marker antibodies whichrecognize an epitope in the plant Golgi apparatus (JIM 84),and for proteins held in the endoplasmic reticulum by the HDELER-retention signal (2E7), in combination with double immunolabelling.In maize root cells, disruption of the ER occurs after breakdownof the Golgi apparatus is initiated. The redistribution of theGolgi is shown to be predominantly separate from that of theER, and as with the Golgi, the action of BFA on the ER is alsoreversible. The mode of action of BFA on the ER and Golgi ofplant cells is compared with that described for animal cells. Key words: Zea mays L, Brefeldin A, plant cells, endoplasmic reticulum, Golgi apparatus     

Distinct biochemical requirements for the budding, targeting, and fusion of ER-derived transport vesicles

The transport of pro-alpha-factor from the ER to the Golgi apparatus in gently lysed yeast spheroplasts is mediated by diffusible vesicles. These transport vesicles contain core-glycosylated pro-alpha-factor and are physically separable from donor ER and target Golgi compartments. The formation of diffusible vesicles from the ER requires ATP, Sec12p, Sec23p, and GTP hydrolysis. The vesicles produced are functionally distinct from the ER: they transfer pro-alpha-factor to the Golgi apparatus faster and more efficiently than the ER, they do not require Sec12p or Sec23p to complete transfer, and transfer is resistant to GTP gamma S. Targeting of vesicles to the Golgi apparatus requires Ypt1p and Sec18p. Fusion of vesicles that have targeted requires calcium and ATP.     

Guanosine nucleotides modulate the inhibitory effect of brefeldin A on protein secretion

Brefeldin A (BFA) causes rapid redistribution of Golgi proteins into the endoplasmic reticulum (ER), leaving no definable Golgi-apparatus, and blocks transport of proteins from the ER to distal secretory compartments of the cell. Using pulse-chase experiments the present study shows that BFA (1 microgram/ml) inhibits basal and CCK-stimulated protein secretion in isolated pancreatic acinar cells by 65 +/- 6% and 84 +/- 5%, respectively. In isolated permeabilized cells higher concentrations of BFA (30 micrograms/ml) were necessary to obtain inhibition of protein secretion. In parallel experiments protein secretion was stimulated by GTP (1 mM). BFA had no inhibitory effect on protein secretion in the presence of GTP, indicating that BFA might act on a GTP-binding protein. Investigating the effect of BFA on small molecular weight GTP-binding proteins we observed that [alpha-32P]GTP binding to a 21 kDa protein in a subcellular fraction enriched in ER was increased in the presence of BFA. We conclude that this 21 kDa and possibly also other GTP-binding proteins may be the molecular target of Brefeldin A in pancreatic acinar cells.     

Microtubule-dependent retrograde transport of proteins into the ER in the presence of brefeldin A suggests an ER recycling pathway

Characteristics of brefeldin A (BFA)-induced redistribution of Golgi proteins into the endoplasmic reticulum (ER) and its relationship to an ER retrieval pathway were investigated. Retrograde movement of Golgi proteins into the ER occurred via long, tubulovesicular processes extending out of the Golgi along microtubules. Microtubule-disrupting agents (i.e., nocodazole), energy poisons, and reduced temperatures inhibited this pathway. In BFA-treated cells Golgi proteins appeared to cycle between the ER and an intermediate compartment marked by a 53 kd protein. Addition of nocodazole disrupted this dynamic cycle by preferentially inhibiting retrograde movement, causing Golgi proteins to accumulate in the intermediate compartment. In the absence of BFA, such an ER cycling pathway appeared to be followed normally by the 53 kd protein but not by Golgi proteins, as revealed by temperature shift experiments. We propose that BFA induces the interaction of the Golgi with an intermediate "recycling" compartment that utilizes a microtubule-dependent pathway into the ER.     

gp74 a membrane glycoprotein of the cis-Golgi network that cycles through the endoplasmic reticulum and intermediate compartment

A monoclonal antibody CC92 (IgM), raised against a fraction of rat liver enriched in Golgi membranes, recognizes a novel Endo H-resistant 74-kD membrane glycoprotein (gp74). The bulk of gp74 is confined to the cis-Golgi network (CGN). Outside the Golgi gp74 is found in tubulovesicular structures and ER foci. In cells incubated at 37 degrees C the majority of gp74 is segregated from the intermediate compartment (IC) marker p58. However, in cells treated with organelle perturbants such as low temperature, BFA, and [AIF4]- the patterns of the two proteins become indistinguishable. Both proteins are retained in the Golgi complex at 20 degrees C and in the IC at 15 degrees C. Incubation of cells with BFA results in relocation of gp74 to p58 positive IC elements. [AIF4]- induces the redistribution of gp74 from the Golgi to p58-positive vesicles and does not retard the translocation of gp74 to IC elements in cells treated with BFA. Disruption of microtubules by nocodazol results in the rapid disappearance of the Golgi elements stained by gp74 and redistribution of the protein into vesicle-like structures. The responses of gp74 to cell perturbants are in sharp contrast with those of cis/middle and trans-Golgi resident proteins whose location is not affected by low temperatures or [AIF4]-, are translocated to the ER upon addition of BFA, and stay in slow disintegrating Golgi elements in cells treated with nocodazol. The results suggest that gp74 is an itinerant protein that resides most of the time in the CGN and cycles through the ER/IC following the pathway used by p58.     

Recruitment of coat proteins onto Golgi membranes in intact and permeabilized cells: effects of brefeldin A and G protein activators.

Brefeldin A (BFA) causes a rapid redistribution of coat proteins (e.g., gamma-adaptin) associated with the clathrin-coated vesicles that bud from the trans-Golgi network (TGN), while the clathrin-coated vesicles that bud from the plasma membrane are unaffected. gamma-Adaptin redistributes with the same kinetics as beta-COP, a coat protein associated with the non-clathrin-coated vesicles that bud from the Golgi complex. Upon removal of BFA, however, gamma-adaptin recovers its perinuclear distribution more rapidly. Redistribution of both proteins can be prevented by pretreating cells with AlF4-. Recruitment of adaptors from the cytosol onto the TGN membrane has been reconstituted in a permeabilized cell system and is increased by addition of GTP gamma S and blocked by addition of BFA. These results suggest a role for G proteins in the control of the clathrin-coated vesicle cycle at the TGN and further extend the similarities between clathrin-coated vesicles and non-clathrin-coated vesicles.     

Two distinct populations of ARF bound to Golgi membranes

ADP-ribosylation factor (ARF) is a small molecular weight GTP-binding protein (20 kD) and has been implicated in vesicular protein transport. The guanine nucleotide, bound to ARF protein is believed to modulate the activity of ARF but the mechanism of action remains elusive. We have previously reported that ARF binds to Golgi membranes after Brefeldin A-sensitive nucleotide exchange of ARF-bound GDP for GTP gamma S. Here we report that treatment with phosphatidylcholine liposomes effectively removed 40-60% of ARF bound to Golgi membranes with nonhydrolyzable GTP, presumably by competing for binding of activated ARF to lipid bilayers. This revealed the presence of two different pools of ARF on Golgi membranes. Whereas total ARF binding did not appear to be saturable, the liposome-resistant pool is saturable suggesting that this pool of ARF is stabilized by interaction with a Golgi membrane-component. We propose that activation of ARF by a guanine nucleotide-exchange protein results in association of myristoylated ARF GTP with the lipid bilayer of the Golgi apparatus. Once associated with the membrane, activated ARF can diffuse freely to associate stably with a target protein or possibly can be inactivated by a GTPase activating protein (GAP) activity.     

A Golgi-related structure remains after the brefeldin A-induced formation of an ER-Golgi hybrid compartment.

Brefeldin A (BFA) has previously been shown to block protein transport from the endoplasmic reticulum (ER), to cause the redistribution of Golgi components to the ER, and to change profoundly the morphology of the Golgi apparatus. In order to quantitate the effects of this drug on the morphology of the ER and the Golgi apparatus in HeLa cells, the numerical, surface and volume densities of these organelles were determined by stereological means. We found that in cells treated with BFA (5 micrograms/ml) clusters of vesicles and tubules, often located near transitional elements of the ER, replaced the Golgi apparatus. The numerical density of these clusters in cells treated with BFA for 30 min or 4.5 h is similar to that of Golgi complexes and Golgi-related clusters in control cells. The surface density of the vesicles and tubules contained in these clusters is about 50% of that represented by Golgi elements in control cells. Concomitantly, a corresponding increase in the surface density of the ER-Golgi hybrid compartment was observed. This hybrid compartment contained Golgi-specific enzymes effecting modifications of N-linked oligosaccharides and the transfer of O-linked sugars. Antibodies recognizing different subcompartments of the Golgi apparatus or the intermediate compartment, labeled vesicles and tubules of the Golgi-related clusters. Applying low doses of BFA allowed for the dissection of the disassembly of the Golgi apparatus into at least two phases. At very low doses (10-20 ng/ml) the numerical density of vesicles in the clusters increased up to 4-fold above control, while the surface density did not markedly change, suggesting that vesiculation of the Golgi cisternae had occurred. Fusion of Golgi elements with the ER seemed to occur only at doses of BFA higher than 20 ng/ml. Contrary to observations on other cell types, removal of BFA from HeLa cell cultures resulted in a rather slow reformation (1-2 h) of the Golgi complex, which allowed us to observe several intermediate stages in this process. During this time period an ER was restored which no longer contained Golgi-specific O-glycosylation functions. Our results demonstrate that BFA does not simply cause the disappearance of the Golgi apparatus by fusion with the ER, but instead clusters of vesicles and tubules remain that contain Golgi-specific markers.     

Binding and hydrolysis of guanine nucleotides by Sec4p, a yeast protein involved in the regulation of vesicular traffic

The 23.5-kDa Sec4 protein is required for vesicular transport between the Golgi apparatus and the plasma membrane in Saccharomyces cerevisiae. In order to analyze its biochemical properties, we have purified the soluble pool of the wild-type protein from an overproducing yeast strain. At 30 degrees C, Sec4p bound [35S] guanosine 5'-O-(thiotriphosphate) (GTP gamma S) with a rate of 0.18 min-1 in a reaction requiring micromolar concentration of free magnesium ions. The protein had high affinity for guanine nucleotides with Kd values for GTP gamma S and GTP of 3.7 nM and 3.5 nM, respectively, and that for GDP of 77 nM. The dissociation of [3H] GDP from Sec4p occurred with a rate of 0.21 min-1 suggesting that the association of GTP gamma S was the result of exchange for prebound GDP. The release of GTP from Sec4p was slow and correlated with a low inherent GTPase activity of 0.0012 min-1. By analogy with other classes of GTP binding proteins, both the nucleotide exchange and hydrolysis activities of Sec4p may be modulated in vivo to facilitate its role in the regulation of intercompartmental membrane traffic.