Candida drug resistance protein 1, a major multidrug ATP binding cassette transporter of Candida albicans, translocates fluorescent phospholipids in a reconstituted system. Academic Article uri icon

Overview

MeSH

  • Base Sequence
  • Biological Transport
  • DNA Primers
  • Flow Cytometry
  • Fluorescence
  • Liposomes

MeSH Major

  • ATP-Binding Cassette Transporters
  • Candida albicans
  • Fungal Proteins
  • Membrane Transport Proteins
  • Phospholipids

abstract

  • Candida albicans drug resistance protein 1 (Cdr1p), an ATP-dependent drug efflux pump, contributes to multidrug resistance in Candida-infected immunocompromised patients. Previous cell-based assays suggested that Cdr1p also acts as a phospholipid translocator. To investigate this, we reconstituted purified Cdr1p into sealed membrane vesicles. Comparison of the ATPase activities of sealed and permeabilized proteoliposomes indicated that Cdr1p was asymmetrically reconstituted such that approximately 70% of the molecules had their ATP binding sites accessible to the extravesicular space. Fluorescent glycerophospholipids were incorporated into the outer leaflet of the proteoliposomes, and their transport into the inner leaflet was tracked with a quenching assay using membrane-impermeant dithionite. We observed ATP-dependent transport of the fluorescent lipids into the inner leaflet of the vesicles. With approximately 6 molecules of Cdr1p per vesicle on average, the half-time to reach the maximal extent of transport was approximately 15 min. Transport was reduced in vesicles reconstituted with Cdr1p variants with impaired ATPase activity and could be competed out to different levels by a molar excess of drugs such as fluconazole and miconazole that are known to be effluxed by Cdr1p. Transport was not affected by ampicillin, a compound that is not effluxed by Cdr1p. Our results suggest a direct link between the ability of Cdr1p to translocate fluorescent phospholipids and efflux drugs. We note that only a few members of the ABC superfamily of Candida have a well-defined role as drug exporters; thus, lipid translocation mediated by Cdr1p could reflect its cellular function.

publication date

  • October 30, 2007

has subject area

  • ATP-Binding Cassette Transporters
  • Base Sequence
  • Biological Transport
  • Candida albicans
  • DNA Primers
  • Flow Cytometry
  • Fluorescence
  • Fungal Proteins
  • Liposomes
  • Membrane Transport Proteins
  • Phospholipids

Research

keywords

  • Journal Article

Identity

Language

  • eng

PubMed Central ID

  • PMC2386675

Digital Object Identifier (DOI)

  • 10.1021/bi700453e

PubMed ID

  • 17924650

Additional Document Info

start page

  • 12081

end page

  • 12090

volume

  • 46

number

  • 43