The EMBO Journal vol.12 no.3 pp.869-877, 1993 Brefeldin A reversibly blocks early but not late protein transport steps in the secretory pathway

Todd R.Graham2, Peggy A.Scott1 and Kex2-dependent cleavage of the mating pheromone a-factor, Scott D.Emr1 occur in sequential compartments of this organelle (Graham and Emr, 1991). The general protein transport factor Howard Hughes Medical Institute and IDivision of Cellular and Secl8/NSF appears to be required in vivo at each Molecular Medicine, University of California, San Diego, School of intercompartmental step in the secretory pathway (Graham Medicine, La Jolla, CA 92093-0668, USA 2Present address: Department of Molecular Biology, Vanderbilt and Emr, 1991), apparently in the process of transport University, Nashville, TN 37235, USA vesicle targeting or fusion with the target membrane (Block 2Corresponding author et al., 1988; Malhotra et al., 1988). We have also presented Communicated by D.I.Meyer evidence that the sorting of vacuolar proteins from proteins destined to be secreted is restricted to a late Golgi We have found that brefeldin A (BFA) inhibited the compartment, most likely the compartment that contains the growth of an isel mutant of Saccharomyces cerevisiae. Kex2 endopeptidase (Graham and Emr, 1991; Robinson Genetic complementation and mapping studies etal., 1991). demonstrated that isel was allelic to erg6, a gene required The Golgi complex of the mammalian cell is thought to for the biosynthesis of the principal membrane sterol of be comprised of distinct cis, medial, trans and trans-Golgi yeast, ergosterol. Treatment of isel cells with BFA network cisternae (reviewed in Farquhar and Palade, 1981; resulted in an immediate block in protein transport Farquhar, 1985; Pfeffer and Rothman, 1987). In addition, through the secretory pathway. Vacuolar a transitional compartment between the ER and Golgi carboxypeptidase Y (CPY) and the secreted pheromone complex has been proposed to exist (Schweizer et al., 1988). a-factor accumulated as both the core glycosylated (ER) A unique morphology, composition and/or function are and al,6 mannosylated (early Golgi) forms in drug- criteria that have been used to characterize distinct treated cells. The modification of a-factor with al,6 compartments of the secretory pathway. Due to the dynamic mannose in BFA-treated cells did not appear to result nature of the Golgi complex, the boundaries of the Golgi from retrograde transport of the al,6 mannosyl- subcompartments are difficult to define. In addition, the transferase into the ER. We found that transport of CPY differences in morphology and resident Golgi protein from medial and late Golgi compartments to the vacuole distribution between different cell types has further was unaffected by BFA, nor was secretion of al,3 complicated this issue (Roth et al., 1986). This has led to mannosylated a-factor or invertase blocked by BFA. The questions about the accuracy of current models of Golgi effects of BFA on the secretory pathway were also structure (reviewed in Mellman and Simons, 1992). A reversible after brief exposure (<40 min) to the drug. simplified model has been recently proposed that describes We suggest that the primary effect of BFA in S.cerevisiae the mammalian Golgi complex as only three functionally is restricted to the ER and the a(1,6 mannosyltransferase distinct compartments, the cis-Golgi network, medial Golgi compartment of the Golgi complex. and trans-Golgi network (Mellman and Simons, 1992). It Key words: brefeldin AlisellSaccharomyces cerevisiael is of considerable interest to gain a better view of the secretory pathway compartmental structure of the Golgi complex and to understand the mechanisms for maintaining the identity of these compartments, in spite of the constant flow of membrane and protein that passes through them. Introduction Treatment of cultured mammalian cells with BFA has been We are interested in the compartmental structure of the Golgi shown to perturb the structure and function of the ER and complex of Saccharomyces cerevisiae and how this structure Golgi complex, and therefore shows great promise as an relates to the function of the organelle in protein transport experimental tool for addressing how these compartments and sorting. Although the yeast Golgi complex is not as normally maintain their separate identities. This drug has amenable to ultrastructural analysis as is the Golgi complex been shown to inhibit export of newly synthesized proteins of plant or mammalian cells (Preuss et al., 1992), a at an early step in protein transport (Takatsuki and Tamura, functional description of the compartmental organization of 1985; Misumi et al., 1986) and to also cause a reversible this organelle has been inferred from an analysis of protein redistribution of Golgi resident proteins into the ER transport in sec mutants (Franzusoff and Schekman, 1989; (Lippincott-Schwartz et al., 1989). The morphology of the Graham and Emr, 1991), immunofluorescence localization Golgi complex changes dramatically within minutes after of Golgi marker proteins (Franzusoff et al., 1991; Redding addition of BFA to cells. Tubules were observed to extend et al., 1991) and subcellular fractionation (Cunningham and from distorted Golgi cisternae and appear to fuse with Wickner, 1989; Bowser and Novick, 1991). We have membranes of the ER (Lippincott-Schwartz et al., 1990). suggested that the following post-translational modifications The mixing of these compartments results in the modification attributed to the yeast Golgi: addition of a 1,6 mannose and of resident ER proteins by Golgi enzymes (Uchida et al., ca 1,6 mannose to N-linked oligosaccharides, and the 1985; Doms et al., 1989; Lippincott-Schwartz et al., 1989; Oxford University Press 869 T.R.Graham, P.A.Scott and S.D.Emr Ulmer and Palade, 1989). BFA also causes lysosomes to A. undergo morphological changes (Lippincott-Schwartz et al., 1991) and appears to induce a mixing of the trans-Golgi ,ug/ml BFA network with the endosomal system (Wood et al., 1991). 1.5 In addition, 3COP and ARF (ADP ribosylation factor) 75 Wild-type dissociate from the Golgi membranes in the presence of this 01 drug (Donaldson et al., 1990, 1991). These two proteins are 25 isel peripherally associated with Golgi membranes and are part 1.0 -0--O 75 of a coatomer complex (Waters et al., 1991) that surrounds the transport vesicles that shuttle proteins between compartments of the Golgi complex (Duden et al., 1991; Serafini et al., 1991a,b). The mechanism by which BFA 0.5 causes this myriad of effects is unknown. However, this drug clearly perturbs functions that are required to maintain the integrity of the secretory pathway and the role of these organelles in protein transport. In this report we describe the effects of BFA on the isel 2 4 6 8 10 strain of S. cerevisiae. This mutant strain has lost the natural time in BFA (hours) resistance of wild-type S. cerevisiae to BFA and is also hypersensitive to a number of structurally unrelated drugs, apparently because a change in the chemical composition B. of the plasma membrane renders this strain more permeable 8 to these compounds (Winsor et al., 1987; Nitiss and Wang, 107 1988). We have found that BFA causes an immediate block 7 in protein transport from the ER and an early Golgi 10 compartment and that later protein transport events were relatively unaffected. These data indicate that in S. cerevisiae, 6 ; R~~~~~g/mlBFA the 10- primary effect of BFA is restricted to the ER and an 75 early Golgi compartment that is marked by an o 1,6 -.* Wild-type mannosyltransferase, and also provides the basis for a genetic -0-- 75 isel approach to identify the cellular target(s) of BFA.

Results C IO 10 Brefeldin A inhibits growth of the ise1 mutant The isel strain of S. cerevisiae has been described previously to exhibit a pleiotropic drug-sensitive phenotype, presumably 0 2 4 6 8 10 due to enhanced drug permeability (Winsor et al., 1987; tiiime in BFA (hours) Nitiss and Wang, 1988). This strain has been shown to be hypersensitive to cycloheximide, crystal violet, G418 and Fig. 1. Effect of BFA on growth of the isel strain. A. Cultures of strains FL100 (wild-type) and FL599 (isel) were started at 0.1 OD/ml camptothecin (Winsor et al., 1987; Nitiss and Wang, 1988). in We have tested the effect of a range of BFA YPDH with 0, 25 or 75 jig/ml BFA. Aliquots were taken at the concentrations time-points indicated and the OD6W was determined. B. Samples were on the growth of the isel mutant. Cultures of strain FL599 removed from the cultures at the time-points indicated and were were incubated at 30°C in rich media with BFA present at diluted at least 200-fold in water. Aliquots containing 100-500 cells 0, 25 or 75 ytg/ml and growth was monitored by the increase were spread onto YPD plates, then incubated for 48 h at 30°C and the in the OD6W of the culture (Figure IA). BFA inhibited the colonies were counted (B). growth of the isel mutant, but did not affect growth of the wild-type parental strain FL100. Growth inhibition was half- To determine whether the BFA-sensitivite phenotype was maximal at a BFA concentration of 25 ltg/ml and was nearly linked to the isel locus, we out-crossed strain FL599 and complete at 75 /tg/ml. To determine the effect of BFA on followed segregation of the drug-sensitive phenotypes. The cell viability, aliquots of cells were removed from the BFA sensitivity phenotype was recessive and segregated 2:2 cultures containing BFA at various time-points, they were in tetrads derived from the diploid strain TGY413 (Figure 2). then diluted 200-fold with water and spread onto YPD plates Hypersensitivity to crystal violet co-segregated with the in duplicate. The colonies that formed after two days at 30°C BFA-sensitive phenotype in all tetrads analyzed (Figure 2). were counted (Figure 1B). In three experiments, the number These strains were also hypersensitive to cycloheximide, of viable isel cells after 1 h of BFA treatment ranged from although segregation patterns suggested that the FL599 strain 72-80% of the initial inoculum, but rapidly decreased may carry more than one mutant locus that confers thereafter such that by 9.5 h > 99% of the cells were dead. hypersensitivity to cycloheximide. These data indicate that Therefore the toxic effects of BFA on the isel strain of a mutation in a single gene (isel) results in the BFA-sensitive S. cerevisiae are reversible, but only after a brief exposure phenotype. to the drug. Because of this result we restricted our analysis We found a low frequency of tetratype tetrads when to the initial effect of BFA on the isel cells, at times when following the segregation of isel relative to the centromere- the majority of the cells were still viable. linked genes TRP1 (CENIII) and URA3 (CENV) that were 870 Effect of brefeldin A on the yeast secretory pathway

A\ IK CM I) \ 1i i.) Effect of BFA on transport of CPY through the yeast secretory pathway The relative mobility of CPY forms by SDS-PAGE is diagnostic of the location of the protein in the secretory -t pathway (reviewed in Klionsky et al., 1990). CPY is synthesized as a proenzyme that is core glycosylated in the ER (pl CPY, 67 kDa). In the Golgi complex, each of the four core oligosaccharides present on CPY is modified with YPD4BA.1i. additional mannose residues that increase the mass of the precursor by 2 kDa (p2 CPY, 69 kDa). Upon arrival in the vacuole, the propiece is proteolytically removed to generate the mature form of the enzyme (mCPY, 61 kDa). To test whether protein transport in the yeast secretory pathway is affected by BFA, we analyzed the transport of newly synthesized CPY to the vacuole in isel cells treated with -1I ) this drug. Cultures of strain FL599 were labeled with [35S]methionine in the presence or absence of BFA and at the times indicated, aliquots of cells were processed for immunoprecipitation with CPY-specific antisera as described YPD + CrvXstal V i(ole in Materials and methods (Figure 3A). Drug-treated cells specifically accumulated the core glycosylated, p1 precursor Fig. 2. The BFA-sensitive phenotype co-segregates with the isel locus of CPY, suggesting that protein transport from the ER was in genetic crosses. Tetrads derived from strain TGY413 were replica- inhibited (Figure 3A, +BFA). To test whether the p1 CPY plated onto YPDH or YPDH with 50 jg/ml BFA (YPD + BFA) and had been aberrantly secreted from the BFA-treated cells, an incubated for 2 days at 30°C. Segregants 2A-2D were spread onto a YPD plate containing 1 lsg/ml crystal violet (YPD + Crystal Violet) aliquot of cells was taken after 40 min of chase in the and incubated for 2 days at 30°C. presence of BFA, then enzymatically converted to spheroplasts and centrifuged to obtain a cellular and extracellular fraction. CPY was immunoprecipitated from Table I. Linkage analysis of the isel allele these fractions and subjected to SDS -PAGE. We found that <5% of pl CPY was found in the extracellular fraction Gene pair TGY413-8B xSF402-3B indicating that the CPY was not secreted from the BFA- treated cells (data not shown). PD NPD TT The conversion of pl CPY to p2 CPY is primarily due 2 isel sec59 21 0 to the addition of c 1,3 mannose residues in a medial Golgi PD, parental ditype; NPD, non-parental ditype; TT, tetratype. compartment (Ballou et al., 1990; Graham and Emr, 1991). CPY receives only a limited number of ca1,6 mannose residues in the early Golgi, insufficient to cause a significant also present in the cross (data not shown). This indicates change in the mobility of p1 CPY in an that the ISEJ gene is tightly linked to a centromere. In SDS -polyacrylamide gel (Franzusoff and Schekman, 1989). addition, only -50% of the tetrads from this cross gave To determine whether the p1 CPY that accumulated in BFA- four viable spores. Analysis of surviving segregants from treated cells had reached the early Golgi, we tested for the incomplete tetrads indicated that the isel trpJAl spores were presence of a 1,6 mannose residues on this form of the inviable. This incompatibility with trpl is also a phenotype enzyme. CPY was immunoprecipitated from BFA-treated exhibited by erg6 mutants (Gaber et al., 1989). The erg6 cells that had been labeled and chased for 0 or 40 min (as mutants are defective for sterol methylation and therefore shown in Figure 3A). The samples were split in half and are unable to synthesize ergosterol, the major membrane re-immunoprecipitated with either antisera to e1,6 mannose sterol of yeast (McCammon et al., 1984). The altered residues or again with antisera to CPY. After SDS -PAGE membrane structure ofthese mutants results in a temperature- the amount of CPY in each immunoprecipitate was sensitive defect in the transport of tryptophan across the quantitated as described in the Materials and methods. We plasma membrane (Gaber et al., 1989). In addition, erg6 found that the percentage of pl CPY that was precipitated strains are sensitive to BFA (E. Sztul, personal with the antisera to ai1,6 mannose increased from 2% at 0 communication). The ERG6 gene is tightly linked to secS9 min to 12% at 40 min. This represents a minimal estimate near the centromere on the right arm of chromosome XLI (a 6-fold increase) of the amount of CPY that had been (Gaber et al., 1989). We found that isel is also tightly linked modified with al,6 mannose because we do not know how to secS9 (Table I). The common phenotypes and genomic many of these residues are required for CPY to be position suggested that erg6 and isel may be allelic. To test quantitatively precipitated with the antisera to this this, we crossed an isel strain (TGY413-3D) with a Aerg6 carbohydrate epitope. These data suggest that CPY strain (1803-8D) and tested the diploid strain for accumulates in both the ER and in an early Golgi complementation of the BFA-sensitive phenotype. The compartment in BFA-treated cells. isel/Aerg6 diploid strain was sensitive to BFA (data not The data described above suggest that the early shown). The similar position in the yeast genome and the compartments of the secretory pathway that contain p1 CPY, lack of complementation indicate that isel and erg6 are the ER and the a 1,6 mannosyltransferase compartment, are alleles of the same gene. targets of the inhibitory effect of BFA. The potential effect 871 T.R.Graham, P.A.Scott and S.D.Emr

A FAt I1F -BFA +BFA -BFA t-BFA; 0' 1)' 40' 0' 1(0 4()

1n%llY - 4 0Y 4 0', --B FIA -{EiF'A

B in *111"'_.*

(.lhlasc B f Fig. 4. Reversibility of the BFA induced block in transport of CPY. Cultures of strain FL599 were pulse labeled for 5 min and chased for 10 min in the presence (+BFA, 75 mg/ml) or absence of BFA (-BFA) at 30°C, as described in Materials and methods. TCA was I I added to the culture without BFA to stop the chase and the cells were -40' processed for immunoprecipitation with CPY antisera. The culture with BFA was split into three equal samples and processed as follows. The first sample was immediately stopped with TCA (15 min +BFA). The I()' 4A second sample was incubated for an additional 40 min before the addition of TCA (40 rain +BFA). The third sample was centrifuged at 8000 g for 1 min and the cells were washed once with chase media lacking BFA. These cells were resuspended to the original volume in chase media and incubated for 40 min in the absence of BFA before the addition of TCA (40 min -BFA). CPY was recovered from the samples by immunoprecipitation and subjected to SDS-PAGE. Fig. 3. Effect of BFA on the transport and processing of CPY. A. Cultures of strain FL599 were pre-incubated for 10 min in the whether this was the case in S. cerevisiae, we labeled and presence (+BFA, 75 gg/ml) or absence of BFA (-BFA) at 20°C, chased isel cells in the presence of BFA for 15 min. We then pulse labeled for 10 min and chased for 40 min, as described in Materials and methods. Aliquots were removed at the time-points then took half of the culture and washed the cells once, indicated and stopped by the addition of TCA. CPY was recovered resuspended them in media lacking BFA and continued the from the samples by immunoprecipitation and subjected to incubation for 40 min. We found that -90% of the CPY SDS-PAGE (Materials and methods). B. Cells were pulse labeled and was processed to the mature form in the cells that had been chased as above except that BFA was added to the culture 10 min washed free of BFA indicating that protein after initiating the chase. Samples were collected at the time of BFA (Figure 4) addition (10 min) and 30 min later (40 min) and stopped by the transport to the vacuole could resume upon removal of the addition of TCA. drug. The capacity of the cell to reverse the block in protein transport induced by BFA was gradually lost upon longer incubations in BFA not We have also found of BFA on later compartments of the yeast Golgi complex (data shown). that the isel cells in BFA for 40 min or would not be seen in this experiment. To characterize further incubating longer resulted in the accumulation of a portion of the affected organelles in BFA-treated cells, we did the before labeling as the precursor following experiment. Strain FL599 was labeled and chased CPY unglycosylated cytoplasmic (Stevens in the absence of drug to populate the pre-vacuolar et al., 1982), suggesting a failure to be translocated across compartments of the secretory pathway with labeled the ER membrane (data not shown). A similar block in et was precursor forms of CPY. BFA was then added to the culture translocation of the Kar2 protein (Rose al., 1989) not These data suggest that and cells were collected at the time of drug addition and 30 also observed (data shown). long- min later (Figure 3B). We found that BFA prevented further term treatment of cells with BFA induces secondary modification of the pl form of CPY present at the time of phenotypes that include a partial disruption of protein drug addition. This effect was very rapid as the amount of translocation into the ER. p1 CPY was the same at 0 and 30 min. This indicated that the addition of BFA resulted in an immediate block ofprotein Effect of BFA on transport of a-factor through the transport from early compartments ofthe secretory pathway. yeast secretory pathway In contrast, the p2 CPY present at the time of drug addition We have also examined the effect of BFA on the transport was completely processed to mature CPY, indicating efficient of the yeast mating pheromone a-factor through the secretory delivery to the vacuole. This suggests that protein transport pathway, as ca-factor serves as a useful marker to monitor from later compartments of the yeast Golgi that contain p2 protein transport through the Golgi complex and late CPY, minimally the 1,3 mannosyltransferase and Kex2p exocytotic events (Julius et al., 1984; Fuller et al., 1988; compartments (Graham and Emr, 1991), are relatively Graham and Emr, 1991). As with CPY, a-factor is insensitive to the initial effect of BFA. synthesized as a high molecular weight precursor that is In mammalian cells, the effect of BFA on the secretory subject to core glycosylation and signal sequence cleavage pathway is reversible (Misumi et al., 1986; Lippincott- in the ER (Julius et al., 1984; Waters et al., 1988). The core Schwartz et al., 1989; Ulmer and Palade, 1989). To test oligosaccharides are extended in the Golgi complex by the 872 Effect of brefeldin A on the yeast secretory pathway

with BFA and accumulated intracellularly as both the core A. - B F. glycosylated ER form and the hyperglycosylated Golgi form. We could not detect any mature a-factor peptide, indicating V. . that the block in protein transport induced by BFA preceded the compartment that contained the Kex2 protease. The mature a-factor peptide is sensitive to proteolysis (Graham and Emr, 1991) and was not quantitatively recovered in the immunoprecipitates from the untreated cells (Figure 5A, -BFA). In addition, the loss in the amount of pro-a-factor recovered over time from the BFA-treated cells is apparently due to specific intracellular proteolysis (Figure SA, +BFA), as the amount of CPY protein immunoprecipitated from the same 0 and 40 min samples were equivalent (Figure 3A). The Kar2 protein was also stable in BFA-treated cells (data not shown). To determine the extent of glycosylation of the et-factor that was accumulated in the BFA-treated cells, we tested for the presence of a 1,6 and a 1,3 mannose epitopes. B. a-factor was immunoprecipitated from cells that had been BF.- l-.\ + I labeled for 5 min in the absence of drug or from cells labeled I1- At.) (f I UU-1f1. cf.1'- 1:.- 5 min and chased for 40 min in the presence of BFA. The 'I . _ ;1 l) a-factor was then eluted from the primary antibody and was split into three equal samples. These samples were subjected

1* :,f, i;, Leg to a second immunoprecipitation with antisera to either a- .- I t- I cf- I -,-, t I '.., f.-=i,I.,- I 7 factor, ac1,6 mannose residues, or a 1,3 mannose residues (Figure 5B). We found that the hyperglycosylated a-factor F that was accumulated in BFA-treated cells had only received (c I -(6 al ,6 mannose residues (Figure SB, 1-6). None of the precursor form modified with a 1,3 mannose residues J't)1'C accumulated in the BFA-treated cells (Figure SB, 1-3). This result indicates that the BFA block precedes the compartment that contains the a 1,3 mannosyltransferase and is consistent with the results obtained using CPY as the marker protein. Fig. 5. Effect of BFA on the transport and processing of a-factor. A. To determine whether BFA induced an immediate block Half of the samples prepared for the experiment shown in Figure 3A in protein transport from the a 1,6 mannosyltransferase were removed and centrifuged briefly to separate the cells (C) from compartment to the a 1,3 mannosyltransferase compartment, the media (M). ae-factor (af) was recovered from these samples by in immunoprecipitation and was subjected to SDS-PAGE. B. A culture we performed an experiment similar to that shown of strain FL599 was labeled for 5 min at 20°C in the absence of BFA Figure 3B, except that c-factor was immunoprecipitated to and stopped with TCA to prepare labeled ca-factor precursors (-BFA). monitor the effect of BFA on protein transport. Cultures of A second culture was pre-incubated for 10 min in BFA at 200C, then strain FL599 were labeled for 5 min without BFA and then labeled for 5 min and chased for 40 min before stopping with TCA chased for 10 min in the presence of either BFA or the (+BFA). a-factor was recovered from the samples by immunoprecipitation and was eluted from the primary antibody, as energy poisons sodium azide and sodium fluoride (Figure 6). described in Materials and methods. The eluates were split into three After immunoprecipitation, the az-factor was eluted from the equal aliquots and each was subjected to a second immunoprecipitation primary antibody, divided in half and subjected to a second with antisera to either ca-factor, a(1,6 mannose residues (1-6) or a1,3 immunoprecipitation with the carbohydrate-specific antisera mannose residues (1 -3). The position of the core glycosylated, a1,6 above We found that the a-factor mannosylated (al,6, the precursor form that is precipitated by the described (Figure 6). a1,6 mannose-specific antisera, but not the a1,3 mannose-specific precursor that was modified with a(1,6 mannose, but not antisera) and al ,3 mannosylated precursor forms (al<,3) are noted on ca1,3 mannose, was completely blocked from further the left margin. modification after the addition of BFA (Figure 6, BFA). Maturation of most of the a-factor precursor that was addition of a 1,6, a 1,2 and a 1,3 linked mannose residues. modified with cl1,3 mannose was not inhibited by BFA. Less In contrast to CPY, the N-linked oligosaccharides of a-factor than 35 % of this precursor form remained in the cell after are extensively and heterogeneously modified resulting in 10 min of chase in the presence of BFA. The remainder of a smeared migration pattern in SDS -PAGE (extending from the a 1,3 mannosylated precursor was processed to the 26 to - 120 kDa). The pro-a-factor polypeptide contains mature form and was secreted from the cell (data not shown). four tandem repeats of the mature 13 amino acid peptide In contrast, nearly all of the a 1,3 mannosylated ca-factor (Kurjan and Herskowitz, 1982). These peptides are excised precursor was blocked from further transport by the addition from the precursor by the Kex2 endopeptidase in a late Golgi of the energy poisons sodium azide and sodium fluoride compartment and are subsequently secreted from the cell (Figure 6, N3Fl). After 10 min of chase with no inhibitor (reviewed in Fuller et al., 1988). A portion of the cultures present, - 90% of the pro-a-factor was matured and secreted used in the experiment shown in Figure 3A were removed from the cell (data not shown). We have also examined the at the time-points indicated, centrifuged to divide each sample secretion of labeled invertase upon addition of BFA. We into a cell and media fraction and then processed for found that the majority of a 1,3 mannosylated invertase was immunoprecipitation with antisera to a-factor (Figure SA). secreted from the cell after addition of BFA, while the core We found that a-factor was not secreted from cells treated glycosylated form was blocked from further modification 873 T.R.Graham, P.A.Scott and S.D.Emr

fl siflY

-L .1.

v i ," .c _; _ 1. (I~~~~~ -j-..; It" "- ., (i~~~~~~~~~~~~~~~~~f.:.W1: I>U .:. F m

...... -.

f- .. I ..B .- \ ! It

1. 1, A_l. .1 Fig. 6. Immediate .. effect of BFA on the transport of a-factor through , the yeast Golgi complex. A culture of strain FL599 was labeled for 5 i min at 15°C and then split into three equal samples. The first sample was stopped immediately by adding TCA (0 min). A chase solution (Materials and methods) was added to the second sample along with sodium azide and sodium fluoride to a final concentration of 20 mM each and the culture was incubated for 10 min, then stopped with TCA (N3Fl). Chase solution and BFA (75 jg/ml final concentration) was added to the third culture, which was incubated for 10 min and then stopped with TCA (BFA). a-factor was recovered from the samples by immunoprecipitation and was eluted from the primary antibody as described in Materials and methods. The eluates were divided in half and were subjected to a second immunoprecipitation -waiaw, with the carbohydrate-specific antisera described above (al,6, al ,3). The ca-factor precursor forms are described in the legend to Figure 5.

Fig. 7. Modification of a-factor with al ,6 mannose residues in cells (data not shown). These data suggest that BFA induces an treated with BFA requires a functional Secl8 protein. A. Cultures of a immediate block in protein transport from both the ER and secl8 isel strain were pre-incubated in the presence (+BFA) or from an early Golgi compartment in which a-factor is absence of BFA (-BFA) for 10 min at 24°C, then half of each culture was shifted to the non-permissive temperature and the modified with al ,6 mannose, but later protein (34°C) transport other half was left at the permissive temperature (24°C). The cells events in the secretory pathway are unaffected. were immediately labeled for 10 min and chased for 30 min. B. One explanation for the data presented in Figure 5 would Aliquots were removed at the time-points indicated and the ae-factor be that BFA imposed blocks in protein transport from both was recovered by immunoprecipitation and subjected to SDS-PAGE. the ER to the a 1,6 mannosyltransferase compartment and from a1,6 mannosyltransferase compartment to the ac1,3 the BFA treatment at the permissive temperature, we might mannosyltransferase compartment. The a-factor that escaped expect the newly synthesized a-factor to be modified with the ER block was a then trapped in the 1,6 a 1,6 mannose even when trapped in the ER at the non- mannosyltransferase compartnent by the second BFA block. permissive temperature for the secl8 mutant. If Golgi Another explanation for this data would be that BFA induced modification of a-factor in the presence of BFA required a complete block in ER to Golgi protein transport, but also forward transport, we would expect only the core induced retrograde movement of the a 1,6 mannosyl- glycosylated form to be present at the non-permissive transferase into the ER where it could modify the core temperature. Aliquots of cells were taken at 0 and 30 min glycosylated a-factor in the ER. We designed an experiment of chase and processed for immunoprecipitation with antisera to test whether the addition of ai1,6 mannose residues to a- to a-factor (Figure 7). We found that in the presence of factor in BFA-treated cells was the result of retrograde BFA, az-factor was modified with al ,6 mannose at the transport of the a 1,6 mannosyltransferase from the Golgi permissive temperature (+BFA, 24°C), but we could not to the ER. We prepared a secl8 isel strain (TGY312-9B) detect this modification at the non-permissive temperature that allowed us to inhibit transport of proteins from the ER for the secl8 mutant (+BFA, 34°C). This result suggests to the Golgi by the to shifting cells the non-permissive that the a 1,6 mannosylation of a-factor that we observed temperature of the secl8 mutant (Novick et al., 1980, 1981; in BFA-treated cells required anterograde transport into the Graham and Emr, 1991). At the permissive temperature, Golgi complex. We also carried out this experiment with protein transport in this strain is comparable to wild-type a 30 min pre-incubation with BFA at the permissive yeast (data not shown). We pre-incubated the secl8 isel temperature and obtained the same result (data not shown). strain for 10 min with BFA at the permissive temperature In addition, we found that a resident ER glycoprotein, protein to induce the potential retrograde movement of Golgi disulfide isomerase (LaMantia et al., 1991), did not receive enzymes into the ER. The culture was split and each was significant modification with al1,6 mannose in BFA-treated labeled either at the permissive temperature (24°C) or at the cells, further supporting our contention that proteins in the non-permissive temperature (34°C) in the presence of BFA. ER of BFA-treated cells did not receive this Golgi If the al ,6 mannosyltransferase moved into the ER during modification (data not shown). These data do not rule out 874 Effect of brefeldin A on the yeast secretory pathway the possibility that BFA induces retrograde transport of Golgi only modified with core oligosaccharides, but a significant enzymes into the yeast ER because these proteins may not fraction of each protein was also modified with a 1,6 function in the ER, but it does indicate that the Golgi mannose residues (Figures 3 and 5). The addition of a 1,6 modification of a-factor that we detected in these mannose residues to glycoproteins is normally restricted to experiments required forward transport into the Golgi. an early Golgi compartment (Baker et al., 1988). It was We have also examined the effect of BFA on the possible that treatment of isel cells with BFA caused the intracellular localization of the Kex2 endopeptidase and a ER and early Golgi to fuse, resulting in proteins receiving vacuolar membrane protein, alkaline phosphatase by this Golgi modification in the ER. Analysis of the a1,6 immunofluorescence microscopy. Cells stained with antibody mannosylation of pro-a-factor in BFA-treated secl8 isel to the Kex2p exhibit small, punctate structures within the cells indicated that forward transport of pro-a-factor into cytoplasm that are excluded from the vacuole (Redding the early Golgi was required to receive this modification, et al., 1991). In contrast, cells stained with antibody to rather than indicating that this modification was occurring alkaline phosphatase exhibit large rings of fluorescence that in the ER (Figure 6). In addition, we could not detect Golgi corresponds to the vacuolar membrane (Roberts et al., modification of a resident ER glycoprotein, protein disulfide 1991). Cells (FL599) were incubated for 30 min in YPDH isomerase, in the presence of BFA. These data do not rule containing 75 atg/ml BFA and then processed for treatment out the possibility that BFA induces Golgi enzymes to be with affinity purified antibody to either Kex2p or alkaline transported back into the ER in S.cerevisiae, but we have phosphatase as previously described (Redding et al., 1991). not been able to detect such an event. The effect of BFA We could not detect a qualitative difference between BFA- on the yeast secretory pathway was reversible; cells treated treated and untreated cells in the fluorescent staining pattern with BFA remained viable for at least 1 h (Figure 1) and of these two proteins (data not shown). This result suggests CPY that was accumulated in the ER of BFA-treated cells that at the level of resolution possible with this technique, was transported to the vacuole upon removal of the drug the late Golgi compartment that contains the Kex2p and the (Figure 4). vacuole maintain their structural integrity. Addition of BFA to cells in which the ER and Golgi had been populated with labeled pl and p2 CPY precursors, respectively, resulted in a rapid block in the transport and Discussion modification of only pl CPY. The transport and sorting of p2 CPY to the vacuole was unaffected by BFA (Figure 3). Strain FL599 (isel) of S. cerevisiae had previously been Analysis of a-factor transport in this experiment shown to be hypersensitive to a number of drugs such as demonstrated that protein transport from both the ER and cycloheximide, camptothecin and crystal violet (Nitiss and the al1,6 mannosyltransferase compartment was blocked by Wang, 1988; Rose et al., 1989). We have found that this BFA addition (Figure SC), but transport from the al ,3 strain is also sensitive to brefeldin A (Figure 1). The BFA- mannosyltransferase compartment to the cell surface was not sensitive phenotype segregated as a single recessive locus inhibited. These results suggest that later compartments of and co-segregated with sensitivity to crystal violet, indicating the yeast Golgi are relatively insensitive to the effects of that the isel allele is responsible for the BFA-sensitive BFA. It is possible that BFA blocks a late transport event phenotype (Figure 2). From tetrad analysis, we noticed that to the vacuole and the conversion of p2 CPY to mature CPY isel was centromere-linked and was incompatible with the in our experiment (Figure 3) occurred artifactually, perhaps trpl allele present in the cross, in that isel trplJJ segregants as the result of the vacuole fusing with earlier compartments were never recovered. These two phenotypes, along with of the secretory pathway. There are two lines of evidence sensitivity to cycloheximide, have also been described for that suggest that this is not the case. (i) The morphology erg6 strains (Gaber et al., 1989). We have demonstrated that of the vacuole and late Golgi were unchanged by BFA erg6 and isel are allelic by complementation analysis and treatment as assessed by immunolocalization of alkaline genetic mapping (Table I). erg6 cells are unable to methylate phosphatase and the Kex2p, respectively. These data indicate ergosterol precursors at C-28; therefore, the physical that the BFA did not induce the fusion of the vacuole with properties of the plasma membrane of these cells are changed compartments of the late Golgi. (ii) The conversion of p2 (Gaber et al., 1989). One of the pleiotropic phenotypes CPY to mCPY in BFA-treated cells occurred efficiently with exhibited by these cells is a reduction in tryptophan transport apparently normal kinetics. In contrast, p2 CPY can be across the plasma membrane (Gaber et al., 1989), which trapped in pre-vacuolar compartments with minimal explains why the isel (erg6) mutants will not grow if conversion to mCPY by the addition of energy poisons auxotrophic for tryptophan. Possible reasons why the isel (sodium azide and sodium fluoride) (Vida et al., 1990). The strain is sensitive to BFA are that the plasma membrane is physical characteristics of the yeast endosome and the role more permeable to the drug or because a transport system of this organelle in the transport of vacuolar enzymes has used to detoxify the yeast cell cannot function properly in not yet been characterized; therefore, we cannot rule out the the altered membrane. The high concentrations required to possibility that CPY is matured in the endosome of BFA- inhibit the growth of the isel cells (50-75 ,ug/ml), relative treated cells. to the concentrations used with mammalian cells (1 /tg/ml), From earlier work, we have found that protein transport may indicate that other mechanisms exist to reduce the from the a1,3 mannosyltransferase compartment to the Kex2 intracellular concentration of the drug in the isel cells. compartment and from the Kex2 compartment to the plasma As with mammalian cells, we found that BFA is a potent membrane, required Sec 18/NSF (Graham and Emr, 1991). inhibitor of protein transport in S. cerevisiae. Cells treated These vesicle mediated transport steps appear to be with BFA accumulated the vacuolar enzyme CPY and the insensitive to BFA. There are conflicting reports concerning normally secreted pheromone a-factor intracellularly as the inhibition of protein transport from distal Golgi precursor forms. Most of the accumulated precursors were compartments in mammalian cells treated with BFA. While 875 T.R.Graham, P.A.Scott and S.D.Emr

BFA appears to block both constitutive and regulated secretion of proteins from the trans-Golgi network of core paf plCPy BHK-21 or PC 12 cells (Miller et al., 1992), it does not SEC23 appear to affect late protein transport steps along the O secretory pathway in rat pancreatic acinar cells (Hendricks BFA) et al., 1992). This discrepancy is most easily explained by flr 1 SEC18 the different cell types being used in these studies. al-6 The data presented in this work provide important insights Man paf plCPY into the potential target of BFA. Because the initial effect of BFA in S.cerevisiae appears to be restricted to protein transport from the ER to the a 1,6 mannosyltransferase compartment and from this latter compartment to the e 1,3 mannosyltransferase compartment, we suggest that al-3 components common to all vesicle-mediated transport steps within the Golgi, such as the Sec 18/NSF protein and | SEC28 potentially coatomer proteins (Waters et al., 1991), are unlikely to be the primary target of BFA action. The primary Kex2 Ci maf p2CPY ) target of BFA could be a transport factor(s) that is only required for protein transport from the ER to the a 1,6 mannosyltransferase compartment and from this latter .EC18 0 compartment to the c 1,3 mannosyltransferase compartment. At least one protein transport factor of this type has been maf described in yeast. We have previously shown that the sec23 secreted mutant exhibits a temperature-sensitive defect that is specific mCPY 1 I00o- for protein transport from the ER and the a 1,6 i mannosyltransferase compartment and does not affect later Fig. 8. Model for BFA-sensitive protein transport steps in S. cerevisiae. protein transport events (Graham and Emr, 1991). The Sec23 The model for compartmental organization of the yeast Golgi and the protein appears to be required for the budding of transport intercompartmental protein transport events that require SEC18 and vesicles from these compartments (Novick et al., 1981; SEC23 have been previously described (Graham and Emr, 1991). Kaiser and Schekman, 1990). The similar phenotypes Intercompartmental protein transport events that are inhibited by BFA are noted. Three possible mechanisms for the inhibitory action of BFA exhibited by isel cells treated with BFA or by sec23 cells on protein transport in Saccharomyces are as follows. (i) Factors shifted to the non-permissive temperature (Graham and Emr, required to bud transport vesicles only from the ER and cal,6 1991) suggest that a factor required for vesicle mediated mannosyltransferase compartment (such as Sec23p) could be a target of protein transport, such as the Sec23 protein, could be a target BFA. (ii) Factors required to target or fuse transport vesicles with the of BFA. Another model that could our results is that al,6 mannosyltransferase and cal,3 mannosyltransferase compartments explain could be a target of BFA. (iii) The al,6 mannosyltransferase BFA specifically disrupts the early Golgi compartment in compartment could be disrupted by BFA such that transport vesicles which a-factor is modified with a 1,6 mannose such that could no longer fuse with, or bud from, this compartment. transport vesicles derived from the ER can no longer recognize or fuse with this compartment, nor can transport TGY413-8B, MA4Ta isel leu2-3,112 lys2-801 suc2-A9; SF402-3B, MlATa vesicles bud from this compartment. These possibilities are secS9 (provided by R.Schekman, University of California at Berkeley); presented in Figure 8. 1803-8D, MA Ta ura3-52 leu2-3,112 trpl-289 Aerg6::LEU2. TGY312-9B and TGY312-17C are haploid segregants from the TGY312 There is a good correlation between the effect of different (FL599/SEY5187) diploid strain. TGY413 is a SEY6210/TGY312-17C BFA concentrations on growth and on the transport of diploid strain. proteins in the secretory pathway (data not shown). The most Standard rich media for yeast (Sherman et al., 1979) was used. YPDH likely explanation for why BFA inhibits growth of the isel media is YPD with 50 mM HEPES pH 7.0. Plates containing crystal violet cells is the block in and cycloheximide were prepared as previously described by Nitiss and protein transport. These observations Wang (1988). The minimal proline media of Wickerham (WiMP) provide the basis for selecting mutants, or multicopy (Wickerham, 1946) was supplemented with 0.2% yeast extract (WiMPYE) suppressors, in the isel (erg6) background that are and other supplements as needed. specifically resistant to BFA. Several mutants have been Materials isolated, but await detailed characterization. These mutants BFA was from Epicentre Technologies (Madison, WI) and was stored in may define new genes required for protein transport in the at 10 mg/ml, Trans35S-label was from ICN radiochemicals (Irvine, secretory pathway or for maintaining the integrity of the ER CA), protein-A Sepharose was from Boehringer Mannheim (Mannheim, and Golgi complex. A genetic analysis of BFA resistant Germany) and 0.2-0.3 mm glass beads were from Glen Mills Inc. mutants may also help to identify the target of BFA. (Maywood, NJ). All other chemicals were purchased from Sigma Chemical Co. (St Louis, MO). A rabbit antisera to a-factor was prepared using a 13-galactosidase-c-factor fusion protein purified from Escherichia coli as previously described by Rothblatt and Meyer (1986). Antisera to CPY has Materials and methods been described (Klionsky et al., 1988). Antisera to (a1,6 mannose linkages and al,3 mannose were provided by R.Schekman (Franzusoff and Strains and media Schekman, 1989); antisera to protein disulfide isomerase was provided by The yeast stains used were: SEY6210, MAiTa ura3-52 leu2-3,112 his3-A200 W.Lennarz (LaMantia et al., 1991); and antisera to Kar2p was provided trpl-A901 lys2-801 suc2-A9 (Robinson et al., 1988); SEY5187, MATa by M.Rose (Rose et al., 1989). secl8-1 suc2-A9 leu2-3,112 ura3-52; FL100, MA Ta; FL599, MATa isel; TGY312-9B, MA4Ta isel secl8-1 leu2-3,112 ura3-52; TGY312-17C, MA4Ta Cell labeling and immunoprecipitations isel secl8-1 leu2-3,112; TGY413-3D, AMTa isel leu2-3,112 lys2-801 Yeast cells were grown to mid-logarithmic phase in WiMPYE, washed twice suc2-A9; TGY413-6D, MA4Ta isel leu2-3,112 ura3-52 his3-A200 suc2-A9; with sterile water and then resuspended to 5 OD/ml in WiMP, 50 mM 876 Effect of brefeldin A on the yeast secretory pathway

Na-HEPES pH 7.0, 1 mg/ml BSA and 10 mg/ml a2-macroglobulin Kaiser,C.A. and Schekman,R. (1990) Cell, 61, 723-733. (Boehringer Mannheim) and pre-incubated for 15 min. Except where noted, Klionsky,D.J., Banta,L.M. and Emr,S.D. (1988) Mol. Cell. Biol., 8, BFA was added 10 min prior to labeling. To initiate labeling, Trans35S- 2105-2116. label was added to a final concentration of 150 MCi/ml. Cells were chased Klionsky,D.J., Herman,P.K. and Emr,S.D. (1990) Microbiol. Rev., 54, by adding a 50xchase solution (50 mM methionine, 10 mM cysteine and 266-292. 10% yeast extract) to a 1 x final concentration, and the chase was Kurjan,J. and Herskowitz,I. (1982) Cell, 30, 933-943. subsequently terminated by adding TCA to a final concentration of 5%. Laemmli,U.K. (1970) Nature, 227, 680-685. The TCA precipitates were washed twice with , dried, resuspended LaMantia,M., Miura,T., Tachikawa,H., Kaplan,H.A., Lennarz,W.J. and in 100 jl of breaking buffer (50 mM Tris-Cl pH 7.5, 6 M urea, 1% SDS Mizunaga,T. (1991) Proc. Natl. Acad. Sci. USA, 88, 4453-4457. and 1 mM EDTA) and vortexed 1 min in the presence of glass beads (80% Lippincott-Schwartz,J., Yuan,L.C., Bonifacino,J.S. and Klausner,R.D. volume). Samples were then boiled for 4 min and 900 1l of IP-Tween buffer (1989) Cell, 56, 801-813. (50 mM Tris-CI pH 7.5, 0.5% Tween-20, 150 mM NaCl, 0.1 mM EDTA) Lippincott-Schwartz,J., Donaldson,J.G., Schweizer,A., Berger,E.G., were added and the lysate cleared by centrifuging in a microcentrifuge for Hauri,H.P., Yuan,L.C. and Klausner,R.D. (1990) Cell, 60, 821-831. 15 min. The appropriate antisera and 90 il of a 20% (vol/vol) suspension Lippincott-Schwartz,J., Yuan,L., Tipper,C., Amherdt,M., Orci,L. and of protein A - Sepharose was added to the supematant and the samples were Klausner,R.D. (1991) Cell, 67, 601-616. rocked overnight at 4°C. Immunoprecipitates were washed once with IP- Malhotra,V., Orci,L., Glick,B.S., Block,M.R. and Rothman,J.E. (1988) Tween buffer with 2 M urea, twice with IP-Tween buffer and resuspended Cell, 54, 221-227. in 50 ml of Laemmli sample buffer (Laemmli, 1970). Samples to be re- McCammon,M.T., Harunann,M.A., Bottema,C.D. and Parks,L.W. (1984) immunoprecipitated were dissociated from the first antibody by boiling for J. Bacteriol., 157, 475-483. 4 min. in 100 dl of 1% SDS, 50 mM Tris-Cl pH 7.5, then subjected to Mellman,I. and Simons,K. (1992) Cell, 68, 829-840. a second immunoprecipitation as described above. CPY and ca-factor samples Miller,S.G., Carnell,L. and Moore,H.-P.H. (1992) J. Cell Biol., 118, were electrophoresed as previously described by Laemmli (1970), in 9% 267-283. and 15 % SDS-polyacrylamide gels, respectively. A Molecular Dynamics Misumi,Y., Misumi,Y., Miki,K., Takatsuki,A., Tamura,G. and Ikehara,Y. Phosphorlmager was used to quantitate the amount of 35S present within (1986) J. Biol. Chem., 261, 11398-11403. defined areas ofthe polyacrylamide gels. For Figure 6, the region containing Nitiss,J. and Wang,J.C. (1988) Proc. Natl. Acad. Sci. USA, 85,7501-7505. the hyperglycosylated a-factor was divided into 24 vertical boxes and Novick,P., Field,C. and Schekman,R. (1980) Cell, 21, 205-215. quantitated. Two peaks were obtained that corresponded to the a 1,6 and Novick,P., Ferro,S. and Schekman,R. (1981) Cell, 25, 461-469. al1,3 modified a-factor. The values given for each box in a peak were added Pfeffer,S.R. and Rothman,J.E. (1987) Annu. Rev. Biochem., 56, 829-852. to obtain a sum total value for the ca1,6 modified form and the ca1,3 modified Preuss,D., Mulholland,J., Franzusoff,A., Segev,N. and Botstein,D. (1992) form. The values obtained for the +BFA lane were divided by the Mol. Biol. Cell, 3, 789-803. corresponding values from the N3F lane to obtain the percentage of each Redding,K., Holcomb,C. and Fuller,R.S. (1991) J. Cell Biol., 113, form that was blocked by BFA. 527-538. Roberts,C.J., Raymond,C.K., Yamashiro,C.T. and Stevens,T.H. (1991) Methods Enzymol., 194, 644-661. Acknowledgements Robinson,J.S., Graham,T.R. and Emr,S.D. (1991) Mol. Cell. Biol., 12, We thank MaryLynne LaMantia and William Lennarz for supplying antibody 5813-5824. to protein disulfide isomerase, Kevin Redding and Robert Fuller for affinity Rose,M.D., Misra,L.M. and Vogel,J.P. (1989) Cell, 57, 1211-1221. purified antibody to Kex2p, Mary Seeger and Greg Payne for affinity punfied Roth,J., Taatjes,D.J., Weinstein,J., Paulson,J.C., Greenwell,P. and antibody to alkaline phosphatase, Mark Rose for antibody to Kar2, Randy Watkins,W.M. (1986) J. Biol. Chem., 261, 14307-14312. Schekmnan for the carbohydrate-specific antibodies, Richard Gaber for the Rothblatt,J.A. and Meyer,D.I. (1986) Cell, 44, 619-628. Aerg6 strain, and John Nitiss for strains FL100 and FL599. We would like Schweizer,A., Fransen,J.A.M., Bachi,T., Ginsel,L. and Hauri,H.-P. 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