Brefeldin a Reversibly Blocks Early but Not Late Protein Transport Steps in the Yeast Secretory Pathway

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

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