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

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dc.contributor.authorDecottignies, Anabelle
dc.contributor.authorGrant, Althea M
dc.contributor.authorNichols, J Wylie
dc.contributor.authorde Wet, Heidi
dc.contributor.authorMcIntosh, David B
dc.contributor.authorGoffeau, André
dc.date.accessioned2021-10-08T07:20:45Z
dc.date.available2021-10-08T07:20:45Z
dc.date.issued1998
dc.description.abstractThe 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.
dc.identifier.apacitationDecottignies, A., Grant, A. M., Nichols, J. W., de Wet, H., McIntosh, D. B., & Goffeau, A. (1998). ATPase and Multidrug Transport Activities of the Overexpressed Yeast ABC Protein Yor1p. <i>The Journal of Biological Chemistry</i>, 273(20), 12612 - 12622. http://hdl.handle.net/11427/34998en_ZA
dc.identifier.chicagocitationDecottignies, Anabelle, Althea M Grant, J Wylie Nichols, Heidi de Wet, David B McIntosh, and André Goffeau "ATPase and Multidrug Transport Activities of the Overexpressed Yeast ABC Protein Yor1p." <i>The Journal of Biological Chemistry</i> 273, 20. (1998): 12612 - 12622. http://hdl.handle.net/11427/34998en_ZA
dc.identifier.citationDecottignies, A., Grant, A.M., Nichols, J.W., de Wet, H., McIntosh, D.B. & Goffeau, A. 1998. ATPase and Multidrug Transport Activities of the Overexpressed Yeast ABC Protein Yor1p. <i>The Journal of Biological Chemistry.</i> 273(20):12612 - 12622. http://hdl.handle.net/11427/34998en_ZA
dc.identifier.issn0021-9258
dc.identifier.issn1083-351X
dc.identifier.ris TY - Journal Article AU - Decottignies, Anabelle AU - Grant, Althea M AU - Nichols, J Wylie AU - de Wet, Heidi AU - McIntosh, David B AU - Goffeau, André AB - 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. DA - 1998 DB - OpenUCT DP - University of Cape Town IS - 20 J1 - The Journal of Biological Chemistry LK - https://open.uct.ac.za PY - 1998 SM - 0021-9258 SM - 1083-351X T1 - ATPase and Multidrug Transport Activities of the Overexpressed Yeast ABC Protein Yor1p TI - ATPase and Multidrug Transport Activities of the Overexpressed Yeast ABC Protein Yor1p UR - http://hdl.handle.net/11427/34998 ER - en_ZA
dc.identifier.urihttp://hdl.handle.net/11427/34998
dc.identifier.vancouvercitationDecottignies A, Grant AM, Nichols JW, de Wet H, McIntosh DB, Goffeau A. ATPase and Multidrug Transport Activities of the Overexpressed Yeast ABC Protein Yor1p. The Journal of Biological Chemistry. 1998;273(20):12612 - 12622. http://hdl.handle.net/11427/34998.en_ZA
dc.language.isoeng
dc.publisher.departmentDivision of Chemical Pathology
dc.publisher.facultyFaculty of Health Sciences
dc.sourceThe Journal of Biological Chemistry
dc.source.journalissue20
dc.source.journalvolume273
dc.source.pagination12612 - 12622
dc.source.urihttps://dx.doi.org/10.1074/jbc.273.20.12612
dc.subject.otherATP-Binding Cassette Transporters
dc.subject.other-
dc.subject.othergenetics
dc.subject.otherisolation &
dc.subject.otherpurification
dc.subject.othermetabolism
dc.subject.otherAdenosine Triphosphatases
dc.subject.otherAdenosine Triphosphate
dc.subject.otheranalogs &
dc.subject.otherderivatives
dc.subject.otherDrug Resistance, Multiple
dc.subject.otherFungal Proteins
dc.subject.otherHydrolysis
dc.subject.otherKinetics
dc.subject.otherOligomycins
dc.subject.otherpharmacology
dc.subject.otherPhosphatidylethanolamines
dc.subject.otherProtein Binding
dc.subject.otherRhodamines
dc.subject.otherSaccharomyces cerevisiae Proteins
dc.subject.otherSubstrate Specificity
dc.titleATPase and Multidrug Transport Activities of the Overexpressed Yeast ABC Protein Yor1p
dc.typeJournal Article
uct.type.publicationResearch
uct.type.resourceJournal Article
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