title: Functional rescue of a misfolded eukaryotic ATP-binding cassette transporter by domain replacement.
abstract: ATP-binding cassette (ABC) transporters are integral membrane proteins that couple ATP binding/hydrolysis with the transport of hydrophilic substrates across lipid barriers. Deletion of Phe-670 in the first nucleotide-binding domain (NBD1) of the yeast ABC transporter, Yor1p, perturbs interdomain associations, reduces functionality, and hinders proper transport to the plasma membrane. Functionality of Yor1p-ΔF was restored upon co-expression of a peptide containing wild-type NBD1. To gain insight into the biogenesis of this important class of proteins, we defined the requirements for this rescue. We show that a misfolding lesion in NBD1 of the full-length protein is a prerequisite for functional rescue by exogenous NBD1, which is mediated by physical replacement of the dysfunctional domain by the soluble NBD1. This association does not restore trafficking of Yor1p-ΔF but instead confers catalytic activity to the small population of Yor1p-ΔF that escapes to the plasma membrane. An important coupling between the exogenous NBD1 and ICL4 within full-length aberrant Yor1p-ΔF is required for functional rescue but not for the physical interaction between the two polypeptides. Together, our genetic and biochemical data reveal that it is possible to modulate activity of ABC transporters by physically replacing dysfunctional domains.
authors.affiliation: Department of Biological Sciences, Columbia University, New York, New York 10027, USA.
authors.forename: Raymond J
authors.initials: RJ
authors.lastname: Louie
authors.forename: Silvere
authors.initials: S
authors.lastname: Pagant
authors.forename: Ji-Young
authors.initials: JY
authors.lastname: Youn
authors.forename: John J
authors.initials: JJ
authors.lastname: Halliday
authors.forename: Gregory
authors.initials: G
authors.lastname: Huyer
authors.forename: Susan
authors.initials: S
authors.lastname: Michaelis
authors.forename: Elizabeth A
authors.initials: EA
authors.lastname: Miller
mesh_terms: D018528:ATP-Binding Cassette Transporters / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites / Q000235:genetics; D002462:Cell Membrane / Q000378:metabolism*; D004721:Endoplasmic Reticulum / Q000378:metabolism; D005057:Eukaryotic Cells / Q000378:metabolism; D005816:Genetic Complementation Test / Q000379:methods; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D015151:Immunoblotting; D018613:Microscopy, Confocal; D009154:Mutation; D017510:Protein Folding; D021381:Protein Transport; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017384:Sequence Deletion
chemical_list: D018528:ATP-Binding Cassette Transporters; D029701:Saccharomyces cerevisiae Proteins; C097077:YOR1 protein, S cerevisiae; D049452:Green Fluorescent Proteins