Functional significance of conserved cysteines in the extracellular loops of the ATP binding cassette transporter Pdr11p
- The pleiotropic drug resistance (PDR) transporter Pdr11p is expressed under anaerobic growth conditions at the plasma membrane of the yeast \(\textit {Saccharomyces cerevisiae}\), where it facilitates the uptake of exogenous sterols. Members of the fungal PDR family contain six conserved cysteines in their extracellular loops (ECL). For the functional analysis of these cysteine residues in Pdr11p, we generated a series of single cysteine-to-serine mutants. All mutant proteins expressed well and displayed robust ATPase activity upon purification. Mass-spectrometry analysis identified two cysteine residues (C582 and C603) in ECL3 forming a disulfide bond. Further characterization by cell-based assays showed that all mutants are compromised in facilitating sterol uptake, protein stability, and trafficking to the plasma membrane. Our data highlight the fundamental importance of all six extracellular cysteine residues for the functional integrity of Pdr11p and provide new structural insights into the PDR family of transporters.
| Author: | Lyubomir Dimitrov StanchevORCiDGND, Magdalena MarekGND, Feng XianORCiDGND, Mara KlöhnORCiDGND, Daniele SilvestroGND, Gunnar DittmarORCiDGND, Rosa Laura López-MarquésORCiDGND, Thomas Günther-PomorskiORCiDGND |
|---|---|
| URN: | urn:nbn:de:hbz:294-79948 |
| DOI: | https://doi.org/10.3390/jof7010002 |
| Parent Title (English): | Journal of Fungi |
| Publisher: | MDPI |
| Place of publication: | Basel |
| Document Type: | Article |
| Language: | English |
| Date of Publication (online): | 2021/04/12 |
| Date of first Publication: | 2020/12/22 |
| Publishing Institution: | Ruhr-Universität Bochum, Universitätsbibliothek |
| Tag: | ABC transport proteins; ATPase activity; disulfide bonds; protein trafficking; sterol uptake |
| Volume: | 7 |
| Issue: | 1, Article 2 |
| First Page: | 2-1 |
| Last Page: | 2-16 |
| Institutes/Facilities: | Lehrstuhl Biochemie II, Arbeitsgruppe Molekulare Neurobiochemie |
| Protein Research Department | |
| open_access (DINI-Set): | open_access |
| faculties: | Fakultät für Chemie und Biochemie |
| Licence (English): |  Creative Commons - CC BY 4.0 - Attribution 4.0 International |
