Communication Vol. 268, No. THE 8, Issue of March OF 15 ... · Communication Vol. 268, No. 8, Issue of March 15, pp. 5345-5348,1393 Printed in U.S.A. THE JOURNAL OF BIOLOGICAL CHEMISTRY

Communication Vol. 268, No. 8, Issue of March 15, pp. 5345-5348,1393 Printed in U.S.A.

THE JOURNAL OF BIOLOGICAL CHEMISTRY

Brefeldin A Reversibly Inhibits Secretion in Saccharomyces cerevisiae”

(Received for publication, December 17, 1992)

Nicky Shah$ and Richard D. Klausners From the Cell Biology and Metabolism Branch, National Institute of Child Health and Human Deuelopment, National Institutes of Health, Bethesda, Maryland 20892

Brefeldin A has proven to be a useful pharmacologic tool, which, when added to mammalian cells, results in a block in secretion as well as the structural disruption of specific intracellular organelles. In spite of our un- derstanding of some of the biochemistry underlying the action of brefeldin A, the most proximal molecular target(s) of the drug remain elusive. In attempting to address this problem, a genetic approach will undoubt- edly prove useful and complementary to the biochem- ical identification of such a site(s). As a result of the relatively resistant nature of wild-type Saccharomy- ces cerevisiae to brefeldin A, an approach utilizing yeast genetics has not been possible. We report the selective sensitivity of three drug-sensitive strains of S. cerevisiae (ise-1, ISE-2, and erg6) with enhanced membrane permeability allowing uptake of brefeldin A. Upon addition of the drug, growth is dramatically inhibited and invertase secretion is rapidly, specifi- cally, and reversibly blocked at the level of the endo- plasmic reticulum. In addition, only structural ana- logues of brefeldin A effective in mammalian cells are active in these yeast strains.

Brefeldin A (BFA),l first described by Singleton et al. (1958), is a heterocyclic lactone which is synthesized from palmitate (C16) via a series of reaction mechanisms akin to the production of prostaglandins from arachidonate (Bu Lock et al., 1969). In a wide variety of mammalian cells, BFA typically exerts three types of effects (reviewed by Klausner et al., 1992). First, BFA inhibits specific traffic pathways such as movement beyond the ER-Golgi system and the transcy- tosis of receptors. In addition, a stereotyped production or enhancement of membrane tubules are seen emanating from the Golgi apparatus, the trans-Golgi network, endosomes, and lysosomes. Finally, BFA causes the apparent disassembly of several organelles including the Golgi apparatus and the trans-Golgi network. All of these effects in a given cell are rapidly reversible, occur at the same doses of BFA (approxi- mately 100 nM), demonstrate identical requirements for spe- cific structural features of the drug, and are antagonized by

* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ Supported by the Howard Hughes Medical Institute-National Institutes of Health Research Scholars Program.

§ To whom all correspondence should be addressed: Cell Biology and Metabolism Branch, Bldg. 18T, Rm. 101, NICHD/NIH, 9000 Rockville Pike, Bethesda, MD 20892.

’ The abbreviations used are: BFA, brefeldin A, ER, endoplasmic reticulum; ARF, ADP-ribosylation factor.

specific diterpines, such as forskolin. Thus, although phar- macologic studies suggest similar, specific sites of action for BFA’s varied effects, the nature and number of molecular targets for BFA remain unknown. Certain cell lines such as PtKl (Kistakis et al., 1991) and Madin-Darby canine kidney (Hunziker et al., 1991) are quite resistant to the effects of BFA on the Golgi apparatus but still retain sensitivity to the drug in peripheral organelles, suggesting possible heteroge- neity in the target sites of BFA.

Significant advances have been made in identifying the biochemical effects of BFA. We initially observed that within 30 s after the addition of BFA, there was a redistribution of a 110-kDa peripheral membrane protein (Allen and Kreis, 1986) from a perinuclear, Golgi-like pattern to a more diffuse, cytosolic pattern (Donaldson et al., 1990). This protein, now known as @-COP (Duden et al., 1991), has been found to be part of a larger molecular weight complex of several proteins known as coatomer (Waters et al., 1991). The coatomer is likely to play an essential role in membrane traffic by deter- mining the production of transport structures (Malhotra et al., 1989). This complex was proposed to undergo a rapid cycle of binding to and release from target membranes (Orci et al., 1991). Using permeabilized cells and following the distribu- tion of the @-COP subunit of the Golgi-associated coatomer, GTP was shown to be required for the binding of this protein to membranes and GTP hydrolysis was necessary for its release. BFA acts by inhibiting the binding step of the coat cycle (Donaldson et al., 1991a, 1991b).

Similar to its effects on @-COP, BFA was found to inhibit, both in vitro and in uiuo, the cyclic interaction of another protein associated with the Golgi apparatus, ADP-ribosyla- tion factor (ARF) (Donaldson et al., 1991a). ARFs comprise a family of low molecular weight GTP-binding proteins (Kahn et al., 1991) that has been implicated in the secretory pathway of S. cereuisiae (Stearns et al., 1990a, 199Ob). The identifica- tion of two BFA-sensitive, GTP-driven assembly cycles sug- gested that the interaction of ARF with Golgi membranes may drive the membrane association of coatomer and thereby explain the drug sensitivities observed. Indeed, it has been demonstrated that ARF is required for the binding of coato- mer to membranes. BFA inhibits this proximal interaction and not, per se, the binding of coatomer to membranes directly (Donaldson et al., 1992a). More recently, it has been eluci- dated that BFA acts specifically by inhibiting the action of a Golgi-associated membrane guanine nucleotide exchange ac- tivity directed toward ARF (Donaldson et al., 1992b; Helms and Rothman, 1992).

As a resuIt of reports emphasizing the anti-microbial effects of BFA concomitant with the rapid advances in the molecular genetics of S. cerevisiae, yeast became in principle, the model of choice to genetically dissect the mechanism(s) of action of the drug. This effort proved to be problematic, however, when wild-type S. cerevisiue was found to be relatively insensitive to BFA? In this study, we report the use of drug-sensitive strains of S. cerevisiae to demonstrate that this organism is indeed sensitive to BFA and provide evidence for the similar- ity of the effects with those observed in mammalian cells.

* N. Shah and R. D. Klausner, unpublished results.

5345

5346 Brefeldin A Reversibly Inhibits Secretion in S. cerevisiae

EXPERIMENTAL PROCEDURES

Materials-Yeast strains used in this study were JN218 (MATa urd-52, leu2, hisl-7, canlR), JN284 (MATa urd-52, leu2 (-3,-112), his7-2, isel), JN362a (MATa urd-52, leu2 (-3,-112), his7-2, adel, trpl, tyrl, fSE2) (provided by James Wang (Harvard University) and John Nittus (Children's Hospital of Los Angeles)), RSY269 (MATa urd-52, his4-619, secl7-1) (provided by Randy Schekman, Univer- sity of California, Berkeley), STX429-2A (MATa erg 6-5 gal 2) (Yeast Genetics Stock Center, Berkeley, CA), and Sc252 (MATa urd-52, leu2 (-2,-112), adel) (provided by James E. Hopper, Milton S. Hershey Medical Center) (Nittus and Wang, 1988; .Kaiser and Schekman, 1990; McCamlnon et al., 1984; Johnston and Hopper, 1982). The yeast multicopy pRB58 plasmid containing the S. cerevis- iae SUC2 gene (Carlson and Botstein, 1982) as well as the polyclonal anti-invertase antibody were generously provided by Susan Ferro- Novick (Yale University). Yeast transformations were performed using the standard lithium acetate method (Ito, 1983) using 50 pg of boiled, sonicated, single-stranded calf thymus DNA (Sigma) as carrier (Gietz et al., 1992) and selecting for transformants on plates of SC medium lacking uracil. Derivatives of BFA were synthesized by Andrew Greene (Joseph Fourier University, Grenoble, France). All other reagents were purchased from commercial sources.

Media and Growth Conditions-Standard Wickersham's synthetic complex (SC) medium (Bacto-yeast nitrogen base without amino acids; Difco) (Wickersham, 1946) was used. Plates containing varying concentrations of BFA were prepared by adding the drug to the liquid form of the SC, 2% agar plates just prior to pouring. Growth curves were obtained by measuring the optical density (600 nm) at various time points of cultures grown in the presence of brefeldin A or methanol (vehicle control).

Invertase Assays-Yeast was grown to exponential phase in SC medium with 4% glucose. Approximately 2 units of Am cells/time point were washed with SC without glucose, split (for external or total activity), and resuspended in SC with 0.1% glucose (in order to induce invertase production) ? 300 p~ BFA. Both external and total invertase activity (units/ml) was measured up to 1 h after induction (as described by Johnson et al. (1987)). At time intervals, samples were chilled, washed in ice-cold 10 mM sodium azide, and resuspended in 75 pl of 0.1 M sodium acetate (pH 5.5). External invertase activity was assayed using the method of Goldstein and Lampen (1975). External (periplasmic) activity was measured directly in intact cells. Total invertase was measured by adding Triton X-100 up to 1% (w/ v) and lysed by three freeze-thaw cycles in a dry ice/ethanol water bath. These lysed cells as well as the intact cells were incubated with 25 pl of 0.5 M sucrose for 10 min at room temperature to determine enzymatic activity (as described by Pelham et al. (1988)). 100 pl of potassium phosphate (pH 7.0) was added, and the mixture heated for 2 min at 100 "C (for inactivation) and pelleted. An aliquot of the supernatant containing the hydrolyzed sucrose was assayed for the presence of D-glucose by adding 500 p1 of 100 mM potassium phos- phate (pH 7.0), 10 pg/ml horseradish peroxidase, 8.4 units/ml glucose oxidase (Boehringer Mannheim), and 0.6 mg/ml 0-dianisidine (Sigma). After 10 min at room temperature, the reaction was quenched with a final concentration of 6 M HCI and the optical density at 540 nm was measured.

Metabolic Labeling, Immunoprecipitation, and Gel Electrophore- sis-Cells were grown to exponential phase in minimal medium without sulfate supplemented with 100 p~ ammonium sulfate and 2% glucose. The RSY269 strain was incubated for another hour a t 37 "C. Am cells (5-10 units) were pelleted, washed in the same medium without any sulfate, and resuspended to equal density (-1 unit of A,,/ml) in minimal medium without sulfate containing 0.1% glucose f 300 p~ BFA (Sigma), and 150 pCi/ml Tran35S-label(-1000 Ci/mmol; ICN Ftadiochemicals, Inc., Irvine, CA). The cells were labeled at 30 'C (or 37 "C for RSY269) for 20 min, after which an equal volume of ice-cold 20 mM sodium azide (+ 300 p M BFA) was added. The cells were pelleted and washed again in ice-cold 10 mM sodium azide (& 300 p~ BFA) and resuspended in 120 pl of sphero- plast medium (-80-100 units of Am/ml) containing 1.2 M sorbitol, 50 mM potassium phosphate (pH 7.5), 10 mM sodium azide, 40 mM 8-mercaptoethanol, and 20 units of lyticase (Boehringer Mannheim)/ Am (? 300 p M BFA). After incubating for 45 min at 30 "C, the spheroplasts were pelleted at 1000 X g for 5 min. Because the effects of BFA on transport are reversible, all washing and spheroplasting was done in the presence of the same concentration of BFA. The cell pellets were extracted with 0.5 ml of ice-cold lysis buffer (0.5% (w/v) Triton X-100, 0.3 M NaCl, 50 m M Tris-HC1 buffer (pH 7.41, 0.1 mM phenylmethylsulfonyl fluoride, 10 pg/ml aprotinin, 10 pg/ml leupep-

tin, and 1 mM iodoacetamide), and the extracts incubated for 1 h at 4 "C with the polyclonal anti-invertase antibody bound to protein A- Sepharose beads. The spheroplast lysates were diluted 10-fold in lysis buffer and immunoprecipitated in an identical fashion. All samples were resolved by SDS-polacrylamide gel electrophoresis and visual- ized using fluorography.

RESULTS AND DISCUSSION

Two of the more likely explanations for the apparent insen- sitivity of S. cereuisiue to BFA are that either the drug is not able to penetrate the cell membrane of some fungi or, alter- natively, that sdme fungi do not possess the molecular tar- get(s) of BFA.

In order to address the underlying basis for BFA insensitiv- ity in S. cereuisiue, we made use of mutant yeast strains, which had been isolated for pleiotropic drug sensitivity. Two mutant strains isolated with this phenotype are called isel and ISE2. The laboratory of Lacroute was the first to identify the isel mutant (strain FL599) while screening for "inhibitor- sensitive" ( ise) strains. These strains were found to take up crystal violet dye readily, suggesting a defect in cell membrane permeability (Bard et al., 1978). The isel strain has been shown to be deficient in the C-24 methylation of membrane sterols. This lack of methyltransferase activity, which is es- sential for the production of ergosterol by yeast cells, may be the biochemical basis for the altered permeability (Winsor et al., 1987). This conclusion is consistent with a mutation that was previously described in the C-24 ergosterol methyltrans- ferase gene (erg6); however, complementation studies with isel have not been done to identify the precise genetic lesion (McCammon et al., 1984). Another mutant, ISEZ, with a similar phenotype was identified by screening for sensitivity to cycloheximide and aphidicolin, an inhibitor of DNA repli- cation (Nittus and Wang, 1988). All three of these strains were employed to test for BFA sensitivity.

Because mutant strains of yeast that are unable to secrete are also unable to grow (Novick and Schekman, 1980), we used this phenotype as a simple initial assay for BFA sensi- tivity. We first tested a range of concentrations of BFA on a wild-type strain of S. cereuisiue (Sc252), as well a strain containing the isel mutation (JN284) by a plate assay de- signed to measure BFA sensitivity as an inhibition of growth. BFA had minimal effect on the growth of the wild-type strain (Sc252) at concentrations exceeding 400 p~ (Fig. 1, a and 6). Generally, wild-type strains show BFA sensitivity at ex- tremely high concentrations approaching 1 mM. The ise2 strain, however, exhibited dramatically inhibited growth on SC medium plates containing as little as 80 PM BFA (Fig. 16). JN364 (ISE2) and STX429-2A (erg6) were found to be BFA- sensitive at 100 p ~ ; data not shown. Although the erg6 as well as ISE2 strains were both found to be much more sensitive than wild-type cells to BFA, isel was at least %fold more sensitive than these others and as a consequence was the strain selected for use in this study.

In order to more quantitatively assess the inability of isel to grow we tested growth in liquid culture. At 400 pM BFA, the growth rate of the wild-type strain was minimally affected (Fig. IC); however, that of the isel strain was completely inhibited (Fig. Id). In addition, titration of BFA demonstrated that this assay mimicked the effects on cells at the identical concentrations to those that inhibited the plate growth (data not shown). A number of structural variants of BFA have been synthesized by Andrew Greene (Joseph Fourier Univer- sity, Grenoble, France), and have been used in both mam- malian cells and in the in uitro assays for BFA described previously to establish structure-function relationships for the drug (Orci et al., 1991). Those variants that are inactive in mammalian cells are likewise unable to inhibit the growth

Brefeldin A Reversibly Inhibits Secretion in S. cerevisiae 5347

C

I

L ...

d -." I I

-a- JN284-RFA - JN284+RFA - JN284+R.%

n zn 40 60 so o 2 0 4 0 6 0 Rn Inn

FIG. 1. Brefeldin A inhibits the growth of a drug sensitive strain of S. cereuisiae. Ability to grow was assessed on SC plates impregnated with (from top to bottom, left to right) a methanol carrier control and 4,40,80, and 400 p~ BFA, as indicated on the plates (in pglml). All cells were incubated at 30 "C for 3 days. Rate of growth in SC liquid culture was determined by seeding cultures from a single colony in the presence of a methanol carrier control, 300 p~ BFA, or 300 p~ B36 (an inactive derivative (B36; a diacylated, reduced, epoxide derivative) of BFA). Panels a and c, a wild-type strain of S. cereuisiae (Sc252). Panels b and d, a drug-sensitive strain (JN284).

Time (hours)

of sensitive strains of S. cerevisiae. The effect of one of these, B36 (a diacetylated, reduced, epoxy derivative of BFA), is shown in Fig. Id.

One hallmark effect of BFA on most mammalian cells is the tight inhibition of the secretory pathway. We assessed the secretory status of these BFA-sensitive cells by examining the release of newly synthesized invertase. Invertase is the prod- uct of the SUC2 gene, which is constitutively produced as a cytoplasmic protein. In addition, this gene utilizes an alter- native promoter that is subject to catabolite repression by glucose to produce the same protein with an N-terminal leader sequence (Carlson and Botstein, 1982). We induced invertase synthesis by transfer of cells from medium containing 4% glucose to medium with 0.1% glucose. External and total invertase was measured by exploiting substrate (sucrose) ac- cessibility to each pool. Invertase that had been transported to the cell surface was assessed by enzymatic assay of whole cells. The fraction secreted was determined by assaying total cell associated invertase after cell lysis. In wild-type cells newly synthesized invertase was rapidly secreted and 70% of total cell activity was detected in the periplasmic space a t all times. This secretion was not affected by the presence of BFA, consistent with the insensitivity of these cells to the drug (data not shown). Likewise, in untreated isel cells, newly synthesized invertase is rapidly and efficiently transported to the cell surface (Fig. 2u). However, in contrast to wild-type

n I O 20 30 4 0 5 0 6 0

, " , . . . , . , . , n IO 20 30 4 0

Time lminl 50 0

FIG. 2. Brefeldin A rapidly and reversibly inhibits the se- cretion of invertase in the ise-1 mutant. External and total invertase levels were determined a t sequential time points up to 60 min after induction of the gene with 0.1% glucose ( t = 0 ) as described previously (see "Experimental Procedures") in the presence of meth- anol (panel a ) , 300 FM BFA (panel b ) , or 300 PM B36 (panel c). One unit corresponds to the amount of enzyme that hydrolyzes sucrose to produce 1 pmol of glucose/min/Aeoo unit of cells a t pH 5.5 and 25 "C. Panel d, percent secreted a t each time point was determined as ([external]/[total]) X 100. Brefeldin A was washed out after 30 min in order to assess reversibility.

cells, the addition of BFA during the induction resulted in a near-complete inhibition of expression of external enzyme (Fig. 2b). Accompanying this inhibition was a 50-60% inhi- bition in the total accumulation of invertase; whether this observation represented an inhibition in synthesis or, alter- natively, an enhanced degradation of retained enzyme was not determined. Treatment of sensitive cells with an inactive analogue of BFA, B36, resulted in no inhibition of invertase secretion (Fig. 2c). The inhibition of growth and the inhibition of secretion had an identical dose-response curve to BFA (data not shown). Removal of BFA from the yeast cultures resulted in a rapid and complete recovery of the secretory block (Fig. 2d) (as well as the growth inhibition; data not shown) to a level of secretion comparable to the wild-type cells.

In order to further characterize the secretory block induced by BFA, we decided to examine the fate of newly synthesized, metabolically labeled invertase by immunoprecipitation with a polyclonal antibody. The same strains used for the enzy- matic invertase assay were transformed with a high copy invertase plasmid (pRB58; see "Experimental Procedures"). BFA treatment was identical to that in the enzymatic inver- tase assay in that BFA was added to the cells simultaneously with the labeled methionine upon induction of the gene with 0.1% glucose. Following a 20-min labeling period, the fraction of invertase secreted was determined by comparing the amount of protein released into the periplasmic space with that retained intracellularly. In the absence of BFA, the vast majority of the labeled invertase was found to be secreted

5348 Brefeldin A Reversibly Inhibits Secretion in S. cerevisiae

(Fig. 3a). Furthermore, the external protein displayed a het- erogeneous apparent molecular weight indicative of post-ER carbohydrate modification. In contrast, the small amount of intracellular invertase showed little polymannan addition. These results were in marked contrast to those observed in the presence of BFA. Upon BFA addition, there was essen- tially no post-ER carbohydrate processing and the majority of the protein remained intracellular as a partially glycosy- lated invertase precursor. Endoglycosidase H digestion was performed to exclude the possibility of proteolysis or differ- ences in core protein structure. Cleavage of the carbohydrate moieties revealed identical apparent molecular weights, sug- gesting that the altered mobility was indeed due to a differ- ential degree of glycosylation (data not shown). The fact that some protein was scored as secreted but showed no processing may indicate either some cell lysis or the transport of unproc- essed protein. The effect of BFA is similar to the phenotype of the temperature-sensitive secl 7-1 mutant at nonpermissive temperatures in which ER to Golgi transport is blocked (Es- mon et al., 1981) (Fig. 3b). The lack of Golgi-associated carbohydrate processing in the presence of BFA is in contrast to the effects of the drug in mammalian cells in which both a block to exit from the ER and Golgi-associated processing of ER retained glycoproteins are seen (Lippincott-Schwartz et al., 1989). This phenomenon is the result of the disassembly of the Golgi and its mixing with the ER. However, even in mammalian cells, some cells respond to the addition of BFA with an inhibition of transport out of the ER without apparent Golgi-ER mixing (Hendricks et al., 1992). Whether BFA leads to the actual disassembly of the Golgi complex in S. cereuisiae will have to be determined by future studies using immuno- electron microscopy.

These studies establish the sensitivity of S. cereuisiae to the drug BFA. The similarity of its effects on yeast to those previously described for mammalian cells suggest that S. cereuisiae may provide valuable genetic approaches to under- standing the molecular target(s) for this drug as well as the biochemical components with which that target interacts. The similarities in BFA action in both S. cereuisiae and mamma- lian cells include: l) an inhibition of growth, 2) a block in secretion at the level of the ER, 3) rapid reversibility, and 4) identical effects of structural alteration of the drug. The requirement for a 10'- to 103-fold higher concentration of BFA for these effects in yeast as opposed to mammalian cells is likely to be due to the inability of the drug to enter the yeast cell. Alternatively, this reflects a lower binding affinity

-BFA +BFA 25'C 37'C a ) E I E I b ) E I E I

M, x 1 0 3

91.4 -

FIG. 3. Brefeldin A induces the accumulation of a partially glycosylated invertase precursor in the ise-2 mutant, similar to the sec 27-2 mutant at the restrictive temperature. a, log phase JN284 (isel) yeast cells were metabolically labeled for 20 min a t 30 "C in the absence or presence of BFA containing 0.1% glucose to induce the invertase gene. b, log phase RSY269 (secl7-2) cells were similarly metabolically labeled for 20 min at either 25 or 37 "C after being shifted to this restrictive temperature for 1 h. External ( E ) and internal invertase (I) were then separated for all samples by spheroplasting and subsequently immunoprecipitated and run on a 12.5% SDS-polyacrylamide gel (see "Experimental Procedures").

of the target site(s) in yeast for BFA. Ultimately, however, these observations point to very similar biochemical targets in yeast and mammalian cells and remarkable conservation of the molecular recognition of the structure of BFA among eukaryotes. Our best understanding of the effect of BFA relevant to the mammalian secretory pathway is its inhibition of a membrane guanine nucleotide exchange activity toward ARF, although the precise molecular target has yet to be demonstrated. Certainly ARF plays an essential function in the secretory pathway in yeast and its remarkable conserva- tion between yeast and mammals has been demonstrated by the ability to complement an ARF deletion in S. cereuisiae with a human ARF (Stearns et al., 1990a, 1990b). The recent description of a yeast coatomer of similar compostion to that of mammalian cells suggests that ARF may be driving the association of this coat with membranes, analogous to mam- malian cells (Hosobuchi et al., 1992). Thus it is not unreason- able to imagine that BFA's effects in yeast will also involve its inhibition of ARF exchange activity resulting in an inhi- bition of yeast coatomer binding, a hypothesis that will be addressed in future studies.

Acknowledgments-We thank John Nittus and James C. Wang for generously providing the JN218, JN284, and JN362A strains, Randy Schekman for providing the RSY269 strain, and James E. Hopper for providing the Sc252 strain. We also thank Susan Ferro-Novick for the kind gift of pRB58 plasmid as well as the polyclonal anti- invertase antibody. We are indebted to Carolyn Suzuki, Juan Boni- facino, Andy Dancis, John Nittus, and Susan Ferro-Novick for their many stimulating discussions and critical review of the manuscript.

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