Functional significance of conserved cysteines in the extracellular loops of the atp binding cassette transporter pdr11p

Lyubomir Dimitrov Stanchev, Magdalena Marek, Feng Xian, Mara Klöhn, Daniele Silvestro, Gunnar Dittmar, Rosa Laura López-Marqués, Thomas Günther Pomorski*

*Corresponding author for this work

Research output: Contribution to journalArticleResearchpeer-review

1 Citation (Scopus)

Abstract

The pleiotropic drug resistance (PDR) transporter Pdr11p is expressed under anaerobic growth conditions at the plasma membrane of the yeast 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.

Original languageEnglish
Article number2
Pages (from-to)1-16
Number of pages16
JournalJournal of Fungi
Volume7
Issue number1
DOIs
Publication statusPublished - Jan 2021

Keywords

  • ABC transport proteins
  • ATPase activity
  • Disulfide bonds
  • Protein trafficking
  • Sterol uptake

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