The Saccharomyces cerevisiae genome encodes 15 fullsize ATP binding cassette transporters (ABC), of which PDR5, SNQ2, and YOR1 are known to be regulated by the transcription factors Pdr1p and Pdr3p (pleiotropic drug resistance). We have identified two new ABC transporter-encoding genes, PDR10 and PDR15, which were upregulated by the PDR1-3 mutation. These genes, as well as four other ABC transporter-encoding genes, were deleted in order to study the properties of Yor1p. The PDR1-3 gain-of-function mutant was then used to overproduce Yor1p up to 10% of the total plasma membrane proteins. Despite their different topologies, both Yor1p and Pdr5p mediated the ATP-dependent translocation of similar drugs and phospholipids across the yeast cell membrane. Both ABC transporters exhibit ATP hydrolysis in vitro, but Pdr5p ATPase activity is about 15 times higher than that of Yor1p, which may indicate mechanistic or regulatory differences between the two enzymes. The yeast YOR11 gene confers oligomycin resistance on overexpression in a 2-m plasmid (1). Its nucleotide sequence reveals an ORF of 1477 amino acids encoding an ABC protein highly homologous to mammalian transporters such as the multidrug resistance-conferring enzyme MRP (BLAST (see Ref. 2) sequence homology score: p ϭ e Ϫ228 ), the organic anion transporter cMOAT (p ϭ e Ϫ216 ), the sulfonylurea receptor (p ϭ e Ϫ164 ), and the cystic fibrosis transmembrane conductance regulator CFTR (p ϭ e Ϫ132 ). Yor1p is a "full-size" ABC transporter with the topology (TM-NBF) 2 (3, 4). It consists of two homologous halves, with each containing a putative ATP-binding domain (NBF) and a transmembrane domain of six membrane spans (TM). Cui et al. (5) showed that Yor1p confers resistance to a series of drugs including reveromycin A and suggested that Yor1p may be involved in the cellular efflux of organic anions including the fluorescent dye rhodamine B. They also showed that incubation with reveromycin A increases the YOR1 mRNA level. The transcription of YOR1 is controlled by the homologous pair of transcription factors Pdr1p/Pdr3p. The level of YOR1 transcription is decreased by the deletion of either PDR1 or PDR3 and increased in the presence of the gain-of-function PDR1 alleles (1).In this paper, we have investigated the transport activity of Yor1p. Building on previous studies, which indicated that the (TM-NBF) 2 -type Yor1p, together with the (NBF-TM) 2 -type Pdr5p and Snq2p ABC transporters, are overexpressed in the PDR1-3 mutant plasma membrane (6 -8), the PDR1-3 mutant has been used as a tool that enhances the Yor1p protein level. As another investigative tool, we constructed a set of isogenic strains, in the PDR1-3 mutant, with multiple deletions of homologous ABC genes since, in situations where two or more proteins located in the same subcellular compartment share a common substrate, a clear phenotype is only seen when all the corresponding genes are deleted, as illustrated by the work of Mahé et al. (9), who showed that Pdr5p and Snq2p have an overlapping transport ...

Cancer cell resistance to chemotherapy is often mediated by overexpression of P-glycoprotein, a plasma membrane ABC (ATP-binding cassette) transporter which extrudes cytotoxic drugs at the expense of ATP hydrolysis. P-glycoprotein (ABCB1, according to the human gene nomenclature committee) consists of two homologous halves each containing a transmembrane domain (TMD) involved in drug binding and efflux, and a cytosolic nucleotide-binding domain (NBD) involved in ATP binding and hydrolysis, with an overall (TMD-NBD)2 domain topology. Homologous ABC multidrug transporters, from the same ABCB family, are found in many species such as Plasmodiumfalciparum and Leishmania spp. protozoa, where they induce resistance to antiparasitic drugs. In yeasts, some ABC transporters involved in resistance to fungicides, such as Saccharomyces cerevisiae Pdr5p and Snq2p, display a different (NBD-TMD)2 domain topology and are classified in another family, ABCG. Much effort has been spent to modulate multidrug resistance in the different species by using specific inhibitors, but generally with little success due to additional cellular targets and/or extrusion of the potential inhibitors. This review shows that due to similarities in function and maybe in three-dimensional organization of the different transporters, common potential modulators have been found. An in vitro 'rational screening' was performed among the large flavonoid family using a four-step procedure: (i) direct binding to purified recombinant cytosolic NBD and/or full-length transporter, (ii) inhibition of ATP hydrolysis and energy-dependent drug interaction with transporter-enriched membranes, (iii) inhibition of cell transporter activity monitored by flow cytometry and (iv) chemosensitization of cell growth. The results indicate that prenylated flavonoids bind with high affinity, and strongly inhibit drug interaction and nucleotide hydrolysis. As such, they constitute promising potential modulators of multidrug resistance.

The sarcoplasmic reticulum (SR)1 Ca 2ϩ -ATPase belongs to a family of cation transport P-type ATPases that are phosphorylated by ATP on an aspartyl residue during the catalytic cycle. Ca 2ϩ translocation occurs in the first part of the cycle and is activated by Ca 2ϩ and MgATP binding to separate high affinity sites leading to phosphorylation and Ca 2ϩ occlusion within the protein (1-5). Phosphorylation appears to be facilitated by an ATP-induced conformational change which may align enzymatic groups in the transition state (6). A nucleotide-dependent conformational change in the presence of Ca 2ϩ is recorded by probes attached to Cys 674 (7-10) and is seen also by the increased reactivity of the latter (8, 11) and the exposure of a critical, reducible disulfide (12). A further conformational change of the phosphoenzyme permits Ca 2ϩ release to the lumen (13, 14). The cycle is completed with dephosphorylation and the associated transport of H ϩ counterions (15). The pump cycle is regulated by ATP. Ca 2ϩ binding (16 -20), Ca 2ϩ release to the lumen and the E1P to E2P transition (13, 21-23), and dephosphorylation (24 -27) are accelerated by ATP in different concentration ranges. These modulations result in a complex dependence of ATP hydrolysis on ATP concentration (1, 23, 28, 29). The regulatory ATP binding site has been the subject of intense study over many years, and there is increasing evidence, mainly from results obtained with probes covalently attached at the catalytic site (27, 30 -32), that at least part of the effect is due to ATP rebinding at or close to the catalytic site following phosphoenzyme formation and ADP dissociation.The identification and role of residues complexing ATP, in either a catalytic or a regulatory mode, are being elucidated by a variety of approaches. Chemical modification and photoaffinity labeling have implicated Lys 492 (31,(33)(34)(35)(36)(37) ) appear to be involved in binding ATP in a regulatory capacity following phosphorylation. However, some doubt has been cast on the role of Lys 492 in ATP binding because derivatization with 7-amino-4-methylcoumarin-3-acetic acid succinimidyl ester apparently has no effect on ATPase activity (37). The function of Lys 492 may be complex, because derivatization with TNP-8N 3 -ATP partially uncoupled Ca 2ϩ transport from hydrolysis of the tethered nucleotide (32) and cross-linking Lys 492 to Arg 678 with glutaraldehyde permitted ATP-dependent Ca 2ϩ occlusion but completely blocked Ca 2ϩ release to the lumen (14).Site-directed mutagenesis has been used to probe several conserved amino acids and segments for involvement in ATP binding. Defective ATP binding following mutation of Lys

No abstract

No abstract