A Simple Chemical Method for Rendering Wild‐Type Yeast Permeable to Brefeldin A That Does Not Require the Presence of an erg6 Mutation

Abstract

The present work aims to develop a growth medium to render a wild-type strain of Saccharomyces cerevisiae permeable to the antifungal drug Brefeldin A. In the current study, a synthetic medium containing 0.1% L-proline and supplemented with 3.0 × 10 −3 % SDS is employed. When Brefeldin A is added to this medium, a wild-type strain shows increased growth sensitivity and a diminished transport of the amino acid L-leucine. Since Brefeldin A exerts its effect on the endoplasmic reticulum and the Golgi apparatus, the medium permits the study of the drug effect on the intracellular traffic of L-leucine permeases. INTRODUCTIONBefore their delivery to the plasma membrane (PM), the different permeases involved in amino acid transport, like most of the membrane proteins, enter the membrane of the endoplasmic reticulum (ER). They then proceed through the protein secretory pathway of the ER, via the Golgi complex (GC) and exocytic vesicles, until they finally reach the PM [1].A very useful agent for investigating permease transport through the secretory pathway is the antifungal agent, Brefeldin A (BFA), which reversibly blocks the transport of proteins from the ER to the Golgi [2,3,4]. This drug can be used to create a temporary block in transport, allowing accumulation of permeases in the ER and depletion of these permeases downstream. In addition, when the BFA block is present, loss of permease molecules from the PM through endocytosis can be studied independent of their replacement via the secretory pathway. Moreover, release of the BFA block would permit the investigation of the dynamics of replacing the permeases in the depleted membrane. Because wild-type yeast has a very low apparent permeability to BFA, previous investigations have used strains bearing the erg6 mutation that blocks the final methylation reaction in ergosterol biosynthesis. The lack of ergosterol in the PM changes the permeability properties of the membrane and renders cells sensitive to several inhibitors, including BFA and the dye, crystal violet (CV) [2]. These changes appear to be at least partly due to decreases in activity of multidrug resistance pumps such as Pdr5p [5].There are several disadvantages of using the erg6 mutation to obtain BFA sensitivity. The mutation itself causes a marked increase in permeability to sodium and lithium ions [6]. Efficiency of genetic transformation is lowered dramatically, and sexual conjugation is also greatly reduced. Moreover, transport of tryptophan is lowered substantially [7].We have developed a simple method for obtaining BFA sensitivity without requiring the introduction of erg6. Because the method requires no genetic manipulation, it can be applied to wild-type cells and to strains already bearing various mutations related to secretion, to altered amino acid transport, and to modified permease turnover. The method depends upon the use of an SDS-supplemented synthetic growth medium in which the wild-type strain MMY2 presents increased sensitivity to BFA. At appropriate concentrations, BFA inhibits grow...

Brefeldin a decreases the activity of the general amino acid permease (GAP1) and the more specific systems for L-leucine uptake in Saccharomyces cerevisiae

“…The values of the initial velocity decreased when the cells were incubated with BFA and concentrations of external L-leucine up to 2 mM. This confirms the results of our previous study, in which only one external amino acid concentration was used [14]. In order to compare the values of the kinetic parameters obtained under different conditions, we performed a data adjustment using Sigmaplot Version 7.0.…”

“…Although an erg6 mutation increases the permeability of S. cerevisiae to many different drugs, including BFA, the transport and processing of amino acid permeases are nevertheless affected in these mutants [9]. For this reason, in a previous study, we performed a protocol that allowed BFA to permeate into the yeast wild type strain MMY2 [14] when its cells were incubated in a medium containing L-proline as the only nitrogen source, and a low concentration of SDS. A feature of this technique is that it does not require mutations in ergosterol biosynthesis.…”

“…Samples containing 5 mg dw/ml (final cell concentration) of MMY2 cells were then incubated with L-14 C-leucine and aliquots were collected at 0, 1.5 and 3 min. The initial velocity for each amino acid concentration was calculated using a linear regression program with the values of L-14 C-leucine in μmol/g dw normalized according to cell viability [14]. The V vs [S] of L-leucine uptake for the wild type strain MMY2 is represented in Fig.…”

Yeast-to-Hyphal Transition Triggers Formin-dependent Golgi Localization to the Growing Tip inCandida albicans

“…BFA (Sigma-Aldrich, St. Louis, MO) was added to a final concentration of 80 g/ml (from a 5 mg/ml stock solution in ethanol) along with 0.06% SDS, whose addition renders the cells more permeable to BFA (Pannunzio et al, 2004). Corresponding volumes of ethanol and SDS were added to the control culture, and both were incubated for an additional 30 min at 37°C with shaking.…”

Proteasome Inhibition in Wild-Type Yeast Saccharomyces Cerevisiae Cells

“…Furthermore, in some cases, the ERG6 or PDR5 genes must be deleted in another mutant background, a technically cumbersome step, to establish the involvement of the 26S proteasome in a particular process (e.g., transcription or telomere maintenance) (9,10). Recently, a method was developed involving brefeldin A, an antifungal agent often used to study protein trafficking from the endoplasmic reticulum (ER) to the Golgi apparatus; this method allowed the efficient uptake of brefeldin A in wild-type yeast cells (11). The key elements of this strategy are the use of L-proline instead of ammonium sulfate as the sole nitrogen source in the growth medium and the addition of a small amount of sodium dodecyl sulfate (SDS; 0.003%).…”