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

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.

Sec24 Is a Coincidence Detector that Simultaneously Binds Two Signals to Drive ER Export

“…We tested whether Erv14 is a folding chaperone for Yor1 by monitoring susceptibility to limited proteolysis, which reflects the folding status of the assembled protein [32, 33](Figure S1C). We treated membranes isolated from wild-type and erv14Δ erv15Δ strains expressing Yor1-HA with increasing concentrations of trypsin and examined the profile of HA-tagged protein fragments that resulted.…”

Characterization of Two Second-Site Mutations Preventing Wild Type Protein Aggregation Caused by a Dominant Negative PMA1 Mutant

“…More recently, a similar intragenic suppressor screen was performed for YOR1 -I1084P, a mutation that induces misfolding and ER-retention of the transmembrane protein Yor1. The suppressor mutations found in that case were able to partially rescue the assembly defect caused by the primary change and restored the capacity of the protein to exit the ER [45]. Nonetheless, we did observe a change regarding the subcellular localization of the co-expressed wild type Pma1 that was now able to reach the plasma membrane, providing an explanation for the suppression of the inability to grow on galactose of the original mutant.…”

Candida Efflux ATPases and Antiporters in Clinical Drug Resistance

“…However, biochemical studies from other similar transporters have provided evidence that ICLs function as communication link between NBDs and TMDs (Sauna et al 2008;Ananthaswamy et al 2010). There are many instances where the drug-susceptibility phenotype resulting due to a point mutation in one of the TMSs could be repressed by chromosomal suppressor with a mutation in NBD (Sauna et al 2008;Pagant et al 2010). Considering the importance of inter-domain communication in yeast ABC transporters, detailed knowledge of the communication interface is also essential in designing inhibitors/modulators for similar transporters.…”

“…ABC transport proteins are classified into nine families (A to I) according to the nomenclature adopted by the human genome organization (HUGO) (Dean et al 2001). Of these, yeast proteins belonging to ABCB (MDR), ABCC (MRP) and ABCG (PDR) transporters are most often associated with the antifungal resistance (Thornewell et al 1997;Decottignies et al 1998;Sanguinetti et al 2006;Lamping et al 2010;Pagant et al 2010;Prasad and Goffeau 2012). ECL3 ECL4 ECL5 ECL6 ICL1 ICL2 ICL3/CCL ICL5 OUT IN ICL4 G165 P17O P175 S177 E178 A203 R184 R215 G219 A231 S232 D235 G264 P261 P257 G256 L246 F277 G133 M132 E429 G472 S495 Y369 Y365 V364 Y360 E351 G391 G515 P512 S505 V496 1 …”