Mapping of interdomain interfaces required for the functional architecture of Yor1p, a eukaryotic ATP-binding cassette (ABC) transporter.
ATP-binding cassette (ABC) transporters are a large superfamily of proteins that mediate substrate translocation across biological membranes. Our goal was to define the intramolecular interactions that contribute to quaternary assembly of a eukaryotic ABC transporter and determine how the architecture of this protein influences its biogenesis within the secretory pathway. We used chemical cross-linking approaches to map interdomain interactions in the yeast ABC transporter, Yor1p, which functions as a pleiotropic drug pump at the plasma membrane. We have defined interactions between the two nucleotide-binding domains (NBDs) and between the NBDs and specific intracellular loops (ICLs) that are consistent with current structural models of bacterial ABC exporters. Furthermore, we detected relatively weak NBD-NBD and ICL-ICL interactions that may correspond to transient sites of cross-talk between domains required for coupling of ATP hydrolysis with substrate translocation. Mutation of a key residue in ICL2 caused misassembly of the altered protein, leading to increased sensitivity to the mitochondrial poison, oligomycin. We identified intragenic suppressing mutations that rescued the oligomycin resistance associated with this aberrant protein and demonstrated that the suppressing mutations restored multiple interdomain interfaces. Together, our biochemical and genetic approaches contribute to a greater understanding of the architecture of this important class of proteins and provide insight into the quality control surveillance that regulates their biogenesis and deployment within the eukaryotic cell.
Pagant
Silvere
S
Department of Biological Sciences, Columbia University, New York, New York 10027, USA.
Brovman
Ethan Y
EY
Halliday
John J
JJ
Miller
Elizabeth A
EA
D018528:ATP-Binding Cassette Transporters / Q000235:genetics / Q000378:metabolism*; D019943:Amino Acid Substitution; D001692:Biological Transport / Q000235:genetics; D002462:Cell Membrane / Q000235:genetics / Q000378:metabolism*; D020125:Mutation, Missense; D010449:Peptide Mapping; D020836:Protein Structure, Quaternary / Q000235:genetics; D017434:Protein Structure, Tertiary / Q000235:genetics; D012441:Saccharomyces cerevisiae; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*
D018528:ATP-Binding Cassette Transporters; D029701:Saccharomyces cerevisiae Proteins; C097077:YOR1 protein, S cerevisiae