Plasma Membrane ATP-binding Cassette Transporter Research Articles

Mutations in the nucleotide-binding domain of putative sterol importers Aus1 and Pdr11 selectively affect utilization of exogenous sterol species in yeast.

Sterol uptake in the yeast Saccharomyces cerevisiae is mediated by two plasma membrane ATP-binding cassette transporters, Aus1 and Pdr11. Their expression is regulated by oxygen and is triggered by anaerobic growth conditions. Under these conditions, internal ergosterol synthesis is arrested and utilization of exogenous sterol is vital for yeast cells. Here, we demonstrate that Aus1 is the major importer of non-yeast sterols, mammalian cholesterol, and plant sterols under anaerobic conditions. In contrast, uptake of yeast native sterol, ergosterol, is relatively low. This uptake could not be enhanced by overexpression of either of the transporters. Interestingly, overexpression of the minor importer Pdr11 resulted in a substantial import of non-yeast sterols. We show that mutation of the conserved residue in one of the ABC characteristic motifs-the H-loop in Aus1 and Pdr11-lowered their ATPase activity. The residual activity was sufficient to import exogenous sterols and to preserve cell viability. Importantly, the reduction of sterol import was dramatic for mammalian cholesterol and plant sterols, whereas import of yeast ergosterol was decreased only slightly indicating substrate selectivity of the sterol utilization process.

Pseudomonas aeruginosa Cif Protein Enhances the Ubiquitination and Proteasomal Degradation of the Transporter Associated with Antigen Processing (TAP) and Reduces Major Histocompatibility Complex (MHC) Class I Antigen Presentation

Cif (PA2934), a bacterial virulence factor secreted in outer membrane vesicles by Pseudomonas aeruginosa, increases the ubiquitination and lysosomal degradation of some, but not all, plasma membrane ATP-binding cassette transporters (ABC), including the cystic fibrosis transmembrane conductance regulator and P-glycoprotein. The goal of this study was to determine whether Cif enhances the ubiquitination and degradation of the transporter associated with antigen processing (TAP1 and TAP2), members of the ABC transporter family that play an essential role in antigen presentation and intracellular pathogen clearance. Cif selectively increased the amount of ubiquitinated TAP1 and increased its degradation in the proteasome of human airway epithelial cells. This effect of Cif was mediated by reducing USP10 deubiquitinating activity, resulting in increased polyubiquitination and proteasomal degradation of TAP1. The reduction in TAP1 abundance decreased peptide antigen translocation into the endoplasmic reticulum, an effect that resulted in reduced antigen available to MHC class I molecules for presentation at the plasma membrane of airway epithelial cells and recognition by CD8(+) T cells. Cif is the first bacterial factor identified that inhibits TAP function and MHC class I antigen presentation.

Vacuolar Import of Phosphatidylcholine Requires the ATP‐Binding Cassette Transporter Ybt1

ATP-binding cassette (ABC) transporters are well known for their roles as multidrug resistance determinants but also play important roles in regulation of lipid levels. In the yeast Saccharomyces cerevisiae, the plasma membrane ABC transporter proteins Pdr5 and Yor1 are required for normal rates of transport of phosphatidyethanolamine to the surface of the cell. Loss of these ABC transporters causes a defect in phospholipid asymmetry across the plasma membrane and has been linked with slowed rates of trafficking of other membrane proteins. Four ABC transporter proteins are found on the limiting membrane of the yeast vacuole and loss of one of these vacuolar ABC transporters, Ybt1, caused a major defect in the normal delivery of the phosphatidylcholine (PC) analog NBD-PC (7-nitro-2,1,3-benzoxadiazol-PC) to the lumen of the vacuole. NBD-PC accumulates on cytosolic membranes in an ybt1Δ strain. We demonstrated that Ybt1 is required to import NBD-PC into vacuoles in the presence of ATP in vitro. Loss of Ybt1 prevented vacuolar remodeling of PC analogs. Turnover of Ybt1 was reduced under conditions in which function of this vacuolar remodeling pathway was required. Our data describe a novel vacuolar route for lipid remodeling and reutilization in addition to previously described enzymatic avenues in the cytoplasm.

ABCC11 expression is regulated by estrogen in MCF7 cells, correlated with estrogen receptor expression in postmenopausal breast tumors and overexpressed in tamoxifen-resistant breast cancer cells

ABCC11 (Multidrug resistance protein 8; MRP8), a plasma membrane ATP-binding cassette transporter, has been implicated in drug resistance of breast cancer by virtue of its ability to confer resistance to fluoropyrimidines and to efflux methotrexate, and by its expression in this tumor. Expression of ABCC11 in breast, a hormonally regulated tissue, as well as the pump's ability to transport estrogen conjugates, suggest the possibility that expression of ABCC11 may be susceptible to regulation by estrogen. However, nothing is currently known about regulation of this gene. In this study, estradiol (E(2)) treatment reduced expression of ABCC11 mRNA in estrogen receptor (ER)-alpha-positive MCF7 cells, and E(2) antagonists such as ICI 182 780 and tamoxifen (TAM) abrogated E(2)-mediated downregulation. ABCC11 expression was positively correlated with ER-alpha expression in both breast cell lines, and two independent series of tumors from postmenopausal patients. In addition, expression of ABCC11 was upregulated in MCF7 cells exposed to TAM for 72 h, and was overexpressed in TAM-resistant cell lines. Drug sensitivity analysis of the TAM-resistant cells indicated that they were also resistant to 5-fluorouracil (5-FU), consistent with the reported ability of ABCC11 to confer resistance to this agent. These studies indicate that ABCC11 expression is negatively regulated by E(2), but that ABCC11 expression is high in high-expressing ER-alpha breast cancers. Our findings support the notion that expression of ABCC11 in ER-alpha-positive breast cancers may contribute to decreased sensitivity to chemotherapy combinations that include 5-FU. ABCC11 may be a potential predictive tool in the choice of anticancer therapies in ER-positive breast cancers resistant to TAM.

Novel stress responses facilitate Saccharomyces cerevisiae growth in the presence of the monocarboxylate preservatives

Certain yeasts are relatively resistant to the small number of monocarboxylic acids allowed in food preservation, with the result that these preservatives often have to be used in high concentrations in order to prevent spoilage. When grown at slightly acid pH, Saccharomyces cerevisiae acquires elevated resistance to these acids by means of discrete stress responses. Acquisition of resistance to acetic acid involves loss of Fps1p, the aquaglyceroporin of the plasma membrane that facilitates the passive diffusional entry of this acid into cells. Acetic acid stress transiently activates Hog1p mitogen-activated protein kinase, which then directly phosphorylates Fps1p in order to target this channel for endocytosis and degradation in the vacuole. Other carboxylate preservatives (propionate, sorbate or benzoate) are too large to traverse the Fps1p pore. Instead, being more lipophilic than acetic acid, they enter cells mainly by a process of non-facilitated diffusion across the plasma membrane. Once inside the cell, these acids activate War1p, a transcription factor that induces the gene for the Pdr12p plasma membrane ATP-binding cassette transporter. Pdr12p lowers the intracellular levels of propionate, sorbate or benzoate by catalysing the active efflux of the preservative anion from the cell. Still other mechanisms of weak acid resistance are found in Zygosaccharomyces, including a capacity for the oxidative degradation of sorbic and benzoic acids conferred by a mitochondrial monooxygenase, a system absent in S. cerevisiae.

Subcellular trafficking of the yeast plasma membrane ABC transporter, Pdr5, is impaired by a mutation in the N‐terminal nucleotide‐binding fold

The plasma membrane ATP-binding cassette (ABC) transporter, Pdr5p, mediates resistance to many different xenobiotic compounds in yeast. We have isolated several mutated forms that fail to confer resistance to cycloheximide and itraconazole. Here, we examined two variants, the expression of which was abnormally low when cells reach the stationary phase of growth. The Pdr5(1157) variant lacked the C-terminal transmembrane domain due to the presence of a nonsense mutation at codon 1158. The second variant, Pdr5(L183P), contained a Leu183Pro substitution close to the Walker A motif in the N-terminal nucleotide-binding domain. This substitution impaired UTPase activity as well as protein stability. The Pdr5(L183P) variant induced the unfolded protein response and was targeted to the proteasome for degradation. Fluorescence microscopy showed that the highly unstable Pdr5(L183P) was mislocalized to endoplasmic reticulum (ER)-associated compartments, whereas the truncated Pdr5(1157) protein was retained in the ER. When threonine 363 (located in the first nucleotide-binding domain, close to the Walker B motif) in Pdr5(L183P) was replaced with isoleucine, this double mutant conferred partial drug resistance. These results suggest that Pdr5p requires a properly folded nucleotide-binding domain for trafficking to the plasma membrane.

The multidrug transporter, P-glycoprotein, actively mediates cholesterol redistribution in the cell membrane.

P-glycoprotein (P-gp) is a plasma membrane ATP-binding cassette transporter, responsible for multidrug resistance in tumor cells. P-gp catalyzes the ATP hydrolysis-dependent efflux of numerous amphiphilic compounds of unrelated chemical structures. In the absence of any identified substrate, P-gp exhibits an apparently futile, basal ATPase activity. By using native membrane vesicles containing high amounts of P-gp, we show here that (i) this basal ATPase activity is tightly dependent on the presence of cholesterol in the membrane; (ii) the stimulation of P-gp ATPase activity by drugs transported by P-gp is higher in the absence than in the presence of cholesterol and, conversely, the stimulation of P-gp ATPase activity by cholesterol is higher in the absence than in the presence of known P-gp substrates; (iii) P-gp mediates the ATP-dependent relocation of cholesterol from the cytosolic leaflet to the exoplasmic leaflet of the plasma membrane; and (iv) the decrease of the cholesterol dependence of P-gp ATPase activity induced by known P-gp substrates is correlated with the inhibition of the ATP-dependent cholesterol redistribution within the membrane. These data are highly evocative of a coupling between the basal ATPase activity of P-gp and its intramembrane cholesterol-redistribution function, and they are fully consistent with the possibility that P-gp may actively translocate cholesterol in the membrane. Finally, this P-gp-mediated cholesterol redistribution in the cell membrane makes it likely that P-gp contributes in stabilizing the cholesterol-rich microdomains, rafts and caveolae, and that it is involved in the regulation of cholesterol trafficking in cells.

ERG6 and PDR5 regulate small lipophilic drug accumulation in yeast cells via distinct mechanisms

Diagnosis and circumvention of multi-drug resistance requires an understanding of the underlying cellular mechanisms. In the model organism Saccharomyces cerevisiae, deletions of PDR5 or ERG6 increase sensitivity to many small lipophilic drugs. Pdr5p is a plasma membrane ATP-binding cassette transporter that actively exports drugs, thereby lowering their intracellular levels. The mechanism by which ERG6, an enzyme in sterol biosynthesis, affects drug accumulation is less clear. We show here that ERG6 limits the rate of passive drug diffusion across the membrane, without affecting Pdr5p-mediated drug export. Consistent with their action by distinct mechanisms, PDR5 and ERG6 effects on drug accumulation are additive.

Prenyl-Flavonoids as Potent Inhibitors of the Pdr5p Multidrug ABC Transporter from Saccharomyces cerevisiae

The Pdr5p multidrug ABC ("ATP-binding cassette) transporter was highly overexpressed in plasma membranes from a yeast strain exhibiting both pdr1-3 gain-of-function mutation in the transcription factor-encoding gene PDR1 and disruption of genes encoding other plasma membrane ABC transporters. Solubilized and purified Pdr5p displayed a tryptophan-characteristic intrinsic fluorescence, whose quenching was used to monitor interactions with substrates and effectors. The transporter exhibited a magnesium-dependent binding affinity for ATP and its fluorescent analogue 2'(3')-N-methylanthraniloyl-ATP, producing a marked fluorescence resonance-energy transfer. It also bound a series of known drug substrates and modulators. Interestingly, yeast Pdr5p interacted with flavonoids recently found to bind to cancer cell P-glycoprotein and to the protozoan parasite multidrug transporter. The extent of high-affinity binding of prenyl-flavonoids to purified Pdr5p was correlated to their efficiency to inhibit energy-dependent quenching of rhodamine 6G fluorescence catalyzed by Pdr5p-enriched plasma membranes. The hydrophobic flavonoid derivative 6-(3, 3-dimethylallyl)galangin was the most efficient, with a K(i) of 0.18 microM for competitive inhibition of the MgATP-dependent quenching of rhodamine 6G fluorescence. In contrast, inhibition of either ATP or UTP hydrolysis occurred at much higher concentrations and appeared to be noncompetitive. Prenyl-flavonoids therefore behave as potent inhibitors of drug binding to the yeast Pdr5p ABC transporter.