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...