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

The Saccharomyces cerevisiae genome encodes 15 full-size 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 up-regulated 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. Overexpressed Yor1p was photolabeled by [gamma-32P]2', 3'-O-(2,4,6-trinitrophenyl)-8-azido-ATP (K0.5 = 45 microM) and inhibited by ATP (KD = 0.3 mM) in plasma membranes. Solubilization and partial purification on sucrose gradient allowed to detect significant Yor1p ATP hydrolysis activity (approximately 100 nmol of Pi.min-1.mg-1). This activity was phospholipid-dependent and sensitive to low concentrations of vanadate (I50 = 0.3 microM) and oligomycin (I50 = 8.5 microg/ml). In vivo, we observed a correlation between the amount of Yor1p in the plasma membrane and the level of resistance to oligomycin. We also demonstrated that Yor1p drives an energy-dependent, proton uncoupler-insensitive, cellular extrusion of rhodamine B. Furthermore, cells lacking both Yor1p and Pdr5p (but not Snq2p) showed increased accumulation of the fluorescent derivative of 1-myristoyl-2-[6-(NBD)aminocaproyl]phosphatidylethanolamine. 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 inventory of all ion and drug ATPases encoded by the yeast genome.

The 5,885 members of the yeast proteome have been screened for amino acid sequence signatures of either P-type ATPases or ABC transporters. A total of 16 P-type ATPases have been classified into six phylogenetic families which each seem to transport a specific class of substrates. In addition, a total of 16 ABC transporters comprising two nucleotide binding folds and two membrane domains were classified in two distinct phylogenetic families. Two ABC transporters of Family I (Pdr5p and Snq2p) share overlapping promiscuity for numerous hydrophobic drugs with a member of Family II (Yor1p). In this case, substrate specificity seems to have differentiated more slowly during evolution than typical phylogenetic traits reflected by amino acid sequence similarity or predicted membrane topography

[Yeast, 4 years later]

La levure, 4 ans après. La levure, organisme modèle de longue date, gardera ce statut à l'ère post-génomique : quatre ans après la fin de son séquençage par un consortium de laboratoires travaillant en réseau, elle devient en effet l'organisme eucaryote le mieux compris