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Negative regulation of the yeast ABC transporter Ycf1p by phosphorylation within its N-terminal extension

Christian M Paumi1, Matthew Chuk, Igor Chevelev

  • 1Department of Cell Biology, The Johns Hopkins School of Medicine, Baltimore, Maryland 21205, USA.

The Journal of Biological Chemistry
|August 1, 2008
PubMed

Insights

Phosphorylation of yeast vacuolar membrane protein Ycf1p at Ser-251 negatively regulates its function, impacting cellular detoxification. Kinases CKA1 and HAL5 were identified as negative regulators of Ycf1p activity.

Area of Science:

  • Cellular Biology
  • Molecular Transport Mechanisms
  • Biochemistry

Background:

  • Yeast vacuolar membrane protein Ycf1p and its mammalian homolog MRP1 are ABCC subfamily ATP-binding cassette (ABC) transporters.
  • These transporters are crucial for eliminating toxic endogenous and xenobiotic compounds from cells.
  • Ycf1p transports substrates as glutathione conjugates and is regulated by phosphorylation and guanine nucleotide exchange factors.

Purpose of the Study:

  • To investigate the role of phosphorylation in the N-terminal extension of Ycf1p.
  • To identify kinases that negatively regulate Ycf1p function.
  • To elucidate the complex regulatory mechanisms governing Ycf1p activity.

Main Methods:

  • Site-directed mutagenesis to create Ycf1p-S251A and Ycf1-S251E mutants.
  • In vivo cadmium resistance assays.
  • In vitro transport assays using [(3)H]estradiol-beta-17-glucuronide.
  • Integrated membrane yeast two-hybrid (iMYTH) screening to identify interacting kinases.

Main Results:

  • Phosphorylation of Ser-251 in the Ycf1p N-terminal extension negatively regulates its activity.
  • The Ycf1p-S251A mutant showed increased resistance to cadmium and enhanced transport activity compared to wild-type.
  • CKA1 and HAL5 were identified as kinases that negatively regulate Ycf1p function upon deletion.
  • Genetic evidence suggests Cka1p may directly or indirectly phosphorylate Ser-251.

Conclusions:

  • Phosphorylation of Ser-251 is a key negative regulatory mechanism for Ycf1p.
  • Both positive and negative regulation of Ycf1p, mediated by phosphorylation, contribute to its optimal function.
  • CKA1 is identified as a potential kinase involved in the negative regulation of Ycf1p via Ser-251 phosphorylation.

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