Implication of the ER quality control in the degradation of the yeast plasma membrane Pdr5 L183P ABC transporter

In yeast, overexpression of the Pdr5 ABC transporter in the plasma membrane results in Multiple Drug Resistance. We found that the substitution of a proline for leucine residue at position 183, within the first nucleotide-binding domain of Pdr5p, impairs cycloheximide resistance of pdr1-3 cells overexpressing the mutated ABC-transporter. Indeed, we showed that Pdr5L183P fails to reach the plasma membrane and accumulates in intracellular membranes where it is targeted for degradation by the proteasome, through the ER Associated Degradation pathway (ERAD). We showed that CDC48 and UBC7 encode two proteins required for Pdr5L183P ERAD. CDC48 and UBC7 genetically interact as we found that the UBC7 deletion suppresses the growth defect of the cdc48-1 strain at 18°C. However, loss of Ubc7p did not suppress the ts phenotype of cdc48-3 cells at 37°C, although it did with ufd1-2ts cells. In contrary to ufd1 cells, supplementing cdc48 cells with oleic acid does not restore growth. These results suggest that ubiquitylated proteins at the ER membrane impair Cdc48p to act on the cell cycle. We found that additional substitutions, Ser115Asn and Thr363Ile, in Pdr5L183P partially restored drug resistance to the pdr1-3 strain. These did not improve the subcellular localisation and turnover of Pdr5L183P, suggesting that Ser115, Leu183 and Thr363 are involved in drug transport and/or ATPase activity. We found also that the unfolded protein response (UPR) is not required for the degradation of Pdr5L183P, although it is stimulated by the overexpression of the mutated transporter, probably as a consequence of the perturbation of the secretory pathway. However, Pdr5L183P degradation is dependent on Sec12p GDP/GTP exchange factor that activates the COPII budding machinery. Our results indicate that, even if it does not leave the ER, Pdr5L183P requires a COPII dependent transport to sub-ER compartments dedicated to proteasome-dependent degradation. Finally we showed that the two truncated transporters lacking their last 354 and 468 amino acids, respectively, are slowly degraded and retained in the ER. Consistently, their overexpression fails to confer drug resistance to pdr1-3 cells. Their retention in the ER might result from the lack of residues that interact with COPII coat proteins before ER exit.Doctorat en sciences agronomiques et ingénierie biologique (AGRO 3)--UCL, 200