Intragenic Suppressing Mutations Correct the Folding and Intracellular Traffic of Misfolded Mutants of Yor1p, a Eukaryotic Drug Transporter

Silvere Pagant, John J. Halliday, Christos Kougentakis, Elizabeth A. Miller (Lead / Corresponding author)

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22 Citations (Scopus)
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Abstract

ATP-binding cassette (ABC) transporters play pivotal physiological roles in substrate transport across membranes, and
defective assembly of these proteins can cause severe disease
associated with improper drug or ion flux. The yeast protein
Yor1p is a useful model to study the biogenesis of ABC transporters; deletion of a phenylalanine residue in the first nucleotide-binding domain (NBD1) causes misassembly and retention
in the endoplasmic reticulum (ER) of the resulting protein
Yor1p-F670, similar to the predominant disease-causing allele
in humans, CFTR-F508. Here we describe two novel Yor1p
mutants, G278R and I1084P, which fail to assemble and traffic
similar to Yor1p-F670. These mutations are located in the two
intracellular loops (ICLs) that interface directly with NBD1, and
thus disrupt a functionally important structural module. We
isolated 2 second-site mutations, F270S and R1168M, which
partially correct the folding injuries associated with the G278R,
I1084P, and F670 mutants and reinstate their trafficking. The
position of both corrective mutations at the cytoplasmic face of
a transmembrane helix suggests that they restore biogenesis by
influencing the behavior of the transmembrane domains rather
than by direct restoration of the ICL1-ICL4-NBD1 structural
module. Given the conserved topology of many ABC transporters, our findings provide new understanding of functionally
important inter-domain interactions and suggest new potential
avenues for correcting folding defects caused by abrogation of
those domain interfaces.
Original languageEnglish
Pages (from-to)36304-36314
Number of pages11
JournalJournal of Biological Chemistry
Volume285
Issue number47
DOIs
Publication statusPublished - 19 Nov 2010

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