Tenreiro, C
Tenreiro, C. in maintaining membrane asymmetry (24, 32, 34, 37, 44). Human Pgp/contributes to stabilize the cholesterol-rich microdomains by mediating cholesterol redistribution within the cell membrane (16). The acquisition of the MDR phenotype in certain mammalian cell lines is not only due to overexpression of the drug efflux pumps but is also accompanied by an upregulation of genes required for normal lipid metabolism that constitute membrane rafts (26). In yeasts as well, we have previously shown that efflux pump proteins, particularly of the ABC superfamily, are influenced by imbalances in membrane lipid composition (32, 34, 37, 44). The presence of detergent-resistant membranes (DRMs) within the yeast PM DLin-KC2-DMA has recently been exhibited (29, 46). In order to critically evaluate the role of the DRM lipid constituents in the localization of the efflux pumps, in this study, we have overexpressed green fluorescent protein (GFP)-tagged CaCdr1p and CaMdr1p in different lipid mutant backgrounds of or or or or DH-5. was cultured in Luria-Bertani medium (Difco, BD Biosciences), to which ampicillin was added (100 g/ml). The yeast strains used in this study are listed in Table ?Table1.1. The strains (AD1-8u? [10], PSCDR1-GFP [AD1-8u? derivative expressing CaCdr1p-GFP] [43], RPCaMDR1-GFP [AD1-8u? derivative expressing CaMdr1p-GFP] [35]) and the deletion mutants expressing either CaCdr1p-GFP or CaMdr1p-GFP were grown in yeast extract-peptone-dextrose (YEPD) broth (Bio101, Vista, CA), complete synthetic medium (CSM), or in SD Ura drop-out media (0.67% yeast nitrogen base, 0.2% drop-out mix, and 2% glucose; Difco). G418-resistant yeast colonies were selected on YEPD/G418 medium or CSM/G418 medium. For agar plates, 2.5% (wt/vol) Bacto agar (Difco, BD Biosciences) was added to the medium. TABLE 1. List of yeast strains used in this study strain expressing CaCdr1p-GFP at PDR5 locusThis studystrain expressing CaCdr1p-GFP at PDR5 locusThis studystrain expressing CaCdr1p-GFP at PDR5 locusThis studystrain expressing CaCdr1p-GFP at PDR5 locusThis studystrain expressing CaCdr1p-GFP at PDR5 locusThis studystrain expressing CaCdr1p-GFP at PDR5 locusThis studystrain expressing CaMdr1p-GFP at PDR5 locusThis studystrain expressing CaMdr1p-GFP at PDR5 locusThis studystrain expressing CaMdr1p-GFP at PDR5 locusThis studystrain expressing CaMdr1p-GFP at PDR5 locusThis studystrain expressing CaMdr1p-GFP at PDR5 locusThis study Open in a separate window Disruption of ergosterol and sphingolipid biosynthetic genes. For disruption of ergosterol biosynthesis DLin-KC2-DMA genes, which include (YNL280C), (YML008C), and (YGL012W), and sphingolipid biosynthesis DLin-KC2-DMA genes, which include (YLR372W), (YCR034W), and (YDR072C), the corresponding disruption cassettes (deletion mutant fused with in a Beckman TLS55 rotor at 4C. Six equal fractions were collected from the Smcb top of each gradient, where the proteins were precipitated with trichloroacetic acid (final concentration, 10%) and collected by centrifugation at 4C. This step was required DLin-KC2-DMA to prevent proteolysis by residual endogeneous proteases. The pellets were neutralized by and dissolved in 10 l of 1 1 M Tris base and 25 l of dissociation buffer (0.1 M Tris-HCl, pH 6.8, 4 mM EDTA, 4% SDS, 20% glycerol, 2% 2-mercaptoethanol, 0.02% bromphenol blue). The samples were incubated at 37C for 15 min and analyzed by SDS-PAGE and immunoblotting as described above. RESULTS Overexpression of GFP-tagged CaCdr1p and CaMdr1p. In this study, we exploited the well-established and extensively used expression system of for the overexpression of CaCdr1p and CaMdr1p (10, 25, 35, 42, 43). The strategy of GFP tagging and cloning of CaCdr1p and CaMdr1p in the plasmid pSK-PDR5PPUS was described previously (35, 43). The rimmed appearance of strains expressing GFP-tagged CaCdr1p (PSCDR1-GFP) and CaMdr1p (RPCaMDR1-GFP) under confocal microscopy and Western blot analysis of the PM fractions of the strains confirmed their proper expression and surface localization (Fig. ?(Fig.1A1A and B). Spot assays for drug susceptibility revealed that both the proteins are fully functional (Fig. ?(Fig.1C1C). Deletion of ergosterol and sphingolipid biosynthetic genes. PSCDR1-GFP and RPCaMDR1-GFP (AD1-8u? derivatives expressing CaCdr1p and CaMdr1p, respectively) were tested for their sensitivity to the drug G418, which is the selectable marker of the yeast knockout (YKO) collection of (Open Biosystems Mata haploid set) (Fig. ?(Fig.1D).1D). Both PSCDR1-GFP and RPCaMDR1-GFP strains were sensitive to G418 (Fig. ?(Fig.1D).1D). The.