ATPase and Multidrug Transport Activities of the Overexpressed Yeast ABC Protein Yor1p

Abstract

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 ...

The Rim101 Pathway Is Involved in Rsb1 Expression Induced by Altered Lipid Asymmetry

“…In yeast, Pdr5 and Yor1 are thought to be involved in the flop of glycerophospholipids (Decottignies et al, 1998;Pomorski et al, 2003). PDR5 and YOR1 are regulated by the transcription factor Pdr1, and their up-regulation in a gain-of-function PDR1-3 mutant causes a reduction in the accumulation of labeled PE, probably due to increased efflux (Decottignies et al, 1998). A Pdr5/Yor1-dependent increase in endogenous PE on the cell surface was later confirmed (Pomorski et al, 2003).…”

“…Because up-regulation of Pdr5 and Yor1 in a gain-of-function PDR1-3 mutant results in a reduction in the amount of PE exposed on the cell surface (Decottignies et al, 1998), we considered that altered lipid asymmetry resulting from the pdr5⌬ and pdr5⌬ yor1⌬ mutations induced the Rsb1 expression. However, to date the effects of the pdr5⌬ and yor1⌬ mutations have not been examined in a wild-type background (PDR1 ϩ ) or in our strain background.…”

“…In mammals, translocation/transport of glycerophospholipids (with little selectivity) by ABCB1 (MDR1) has been reported (van Helvoort et al, 1996), as has that of PC by ABCB4 (human MDR3, mouse mdr2; Smit et al, 1993), PS by ABCA1 (Abramova et al, 2001), and N-retinylidene-PE by ABCA4 (Weng et al, 1999). In yeast, Pdr5 and Yor1 are thought to be involved in the flop of glycerophospholipids (Decottignies et al, 1998;Pomorski et al, 2003). PDR5 and YOR1 are regulated by the transcription factor Pdr1, and their up-regulation in a gain-of-function PDR1-3 mutant causes a reduction in the accumulation of labeled PE, probably due to increased efflux (Decottignies et al, 1998).…”

Putative role for ABC multidrug exporters in yeast quorum sensing

“…We used a set of isogenic strains [6] derived from US50-18c (a strain possessing the PDR1-3 allele) and containing deletions in the transporter genes PDR5, SNQ2 and YOR1.…”

Involvement of FpTRP26, a thioredoxin‐related protein, in oxalic acid‐resistance of the brown‐rot fungus Fomitopsis palustris

“…The cDNA library thus prepared was introduced into a Saccharomyces cerevisiae AD (1-8) strain (Dyor1, Dsnq2, Dpdr5, Dpdr10, Dpdr11, Dycf1, Dpdr3, Dpdr) [14] by the lithium acetate method. Transformants selected on SD plates lacking uracil, SD(-Ura), were replicated on a 12 mM OA-containing SD(-Ura) plate and grown for 5 days at 30°C (first screening).…”