Reference: Paumi CM, et al. (2008) Negative Regulation of the Yeast ABC Transporter Ycf1p by Phosphorylation within Its N-terminal Extension. J Biol Chem 283(40):27079-88

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Abstract


The yeast vacuolar membrane protein Ycf1p and its mammalian counterpart MRP1 belong to the ABCC subfamily of ATP-binding cassette (ABC) transporters that rid cells of toxic endogenous and xenobiotic compounds. Like most members of the ABCC subfamily, Ycf1p contains an N-terminal extension (NTE) in addition to its ABC "core" domain and transports substrates in the form of glutathione conjugates. Ycf1p is subject to complex regulation to ensure its optimal function. Previous studies showed that Ycf1p activity is stimulated by a guanine nucleotide exchange factor Tus1p and is positively regulated by phosphorylation in its ABC "core" domain at residues Ser908 and Thr911. Here we provide evidence that phosphorylation of Ser251 in the Ycf1p NTE negatively regulates activity. Mutant Ycf1p-S251A exhibits increased resistance to cadmium in vivo and increased Ycf1p-dependent transport of E2ss17G in vitro, as compared to wild-type Ycf1p. Activity is restored to the wild-type level for Ycf1-S251E. To identify kinase(s) that negatively regulate Ycf1p function we conducted an integrated membrane yeast two-hybrid (iMYTH) screen and identified two kinase genes, CKA1 or HAL5, deletion of which increase Ycf1p function. Genetic evidence suggests that Cka1p may regulate Ycf1p function through phosphorylation of Ser251, either directly or indirectly. Overall, this study provides compelling evidence that negative, as well as positive, regulation of Ycf1p is mediated by phosphorylation.

Reference Type
Journal Article
Authors
Paumi CM, Chuk M, Chevelev I, Stagljar I, Michaelis S
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