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The conserved Pkh-Ypk kinase cascade is required for endocytosis in yeast.

deHart AK, Schnell JD, Allen DA, Hicke L - J. Cell Biol. (2002)

Bottom Line: Ypk1 is required for both receptor-mediated and fluid-phase endocytosis, and is not necessary for receptor phosphorylation or ubiquitination.Ypk1 itself is phosphorylated by Pkh kinases, homologues of mammalian PDK1.The threonine in Ypk1 that is phosphorylated by Pkh1 is required for efficient endocytosis, and pkh mutant cells are defective in alpha-factor internalization and fluid-phase endocytosis.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Molecular Biology, and Cell Biology, Northwestern University, Evanston, IL 60208, USA.

ABSTRACT
Internalization of activated signaling receptors by endocytosis is one way cells downregulate extracellular signals. Like many signaling receptors, the yeast alpha-factor pheromone receptor is downregulated by hyperphosphorylation, ubiquitination, and subsequent internalization and degradation in the lysosome-like vacuole. In a screen to detect proteins involved in ubiquitin-dependent receptor internalization, we identified the sphingoid base-regulated serine-threonine kinase Ypk1. Ypk1 is a homologue of the mammalian serum- and glucocorticoid-induced kinase, SGK, which can substitute for Ypk1 function in yeast. The kinase activity of Ypk1 is required for receptor endocytosis because mutations in two residues important for its catalytic activity cause a severe defect in alpha-factor internalization. Ypk1 is required for both receptor-mediated and fluid-phase endocytosis, and is not necessary for receptor phosphorylation or ubiquitination. Ypk1 itself is phosphorylated by Pkh kinases, homologues of mammalian PDK1. The threonine in Ypk1 that is phosphorylated by Pkh1 is required for efficient endocytosis, and pkh mutant cells are defective in alpha-factor internalization and fluid-phase endocytosis. These observations demonstrate that Ypk1 acts downstream of the Pkh kinases to control endocytosis by phosphorylating components of the endocytic machinery.

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Ypk1 is required for fluid-phase endocytosis. (A and B) Lucifer yellow (LY) localization was assayed in ypk1G490R (LHY2543), ypk1Δ (LHY2536), and wild-type cells from the same tetrad as each mutant (LHY2761 and LHY2537, respectively). Cells were grown to early logarithmic phase in rich medium at 24°C, shifted to 37°C for 15 min, and then allowed to internalize LY at 37°C for 30 or 60 min (A) Images were taken using DIC optics (top) and fluorescence optics (bottom). (B) The percent of cells that accumulated LY in their vacuoles was quantified for the 30- and 60-min time points by blind counting of each sample. The number of cells counted is indicated to the right of the corresponding bar.
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fig2: Ypk1 is required for fluid-phase endocytosis. (A and B) Lucifer yellow (LY) localization was assayed in ypk1G490R (LHY2543), ypk1Δ (LHY2536), and wild-type cells from the same tetrad as each mutant (LHY2761 and LHY2537, respectively). Cells were grown to early logarithmic phase in rich medium at 24°C, shifted to 37°C for 15 min, and then allowed to internalize LY at 37°C for 30 or 60 min (A) Images were taken using DIC optics (top) and fluorescence optics (bottom). (B) The percent of cells that accumulated LY in their vacuoles was quantified for the 30- and 60-min time points by blind counting of each sample. The number of cells counted is indicated to the right of the corresponding bar.

Mentions: To investigate the role of Ypk1 in fluid-phase endocytosis, we assayed the ability of ypk1 cells to deliver Lucifer yellow (LY)* to the vacuole (Fig. 2, A and B) . Both ypk1G490R and ypk1Δ cells were significantly impaired in LY transport compared with their congenic wild-type strains. Ypk1 was also required for internalization of receptors carrying the linear peptide internalization signal NPFXD (Tan et al., 1996), instead of a ubiquitin signal (unpublished data). Ypk1 is not generally required for membrane trafficking, because carboxypeptidase Y was transported through the biosynthetic pathway to the vacuole with normal kinetics in ypk1G490R cells incubated at the restrictive temperature (unpublished data). These observations indicate that Ypk1 is necessary for fluid-phase endocytosis and for the internalization of plasma membrane proteins carrying different types of internalization signals.


The conserved Pkh-Ypk kinase cascade is required for endocytosis in yeast.

deHart AK, Schnell JD, Allen DA, Hicke L - J. Cell Biol. (2002)

Ypk1 is required for fluid-phase endocytosis. (A and B) Lucifer yellow (LY) localization was assayed in ypk1G490R (LHY2543), ypk1Δ (LHY2536), and wild-type cells from the same tetrad as each mutant (LHY2761 and LHY2537, respectively). Cells were grown to early logarithmic phase in rich medium at 24°C, shifted to 37°C for 15 min, and then allowed to internalize LY at 37°C for 30 or 60 min (A) Images were taken using DIC optics (top) and fluorescence optics (bottom). (B) The percent of cells that accumulated LY in their vacuoles was quantified for the 30- and 60-min time points by blind counting of each sample. The number of cells counted is indicated to the right of the corresponding bar.
© Copyright Policy
Related In: Results  -  Collection

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fig2: Ypk1 is required for fluid-phase endocytosis. (A and B) Lucifer yellow (LY) localization was assayed in ypk1G490R (LHY2543), ypk1Δ (LHY2536), and wild-type cells from the same tetrad as each mutant (LHY2761 and LHY2537, respectively). Cells were grown to early logarithmic phase in rich medium at 24°C, shifted to 37°C for 15 min, and then allowed to internalize LY at 37°C for 30 or 60 min (A) Images were taken using DIC optics (top) and fluorescence optics (bottom). (B) The percent of cells that accumulated LY in their vacuoles was quantified for the 30- and 60-min time points by blind counting of each sample. The number of cells counted is indicated to the right of the corresponding bar.
Mentions: To investigate the role of Ypk1 in fluid-phase endocytosis, we assayed the ability of ypk1 cells to deliver Lucifer yellow (LY)* to the vacuole (Fig. 2, A and B) . Both ypk1G490R and ypk1Δ cells were significantly impaired in LY transport compared with their congenic wild-type strains. Ypk1 was also required for internalization of receptors carrying the linear peptide internalization signal NPFXD (Tan et al., 1996), instead of a ubiquitin signal (unpublished data). Ypk1 is not generally required for membrane trafficking, because carboxypeptidase Y was transported through the biosynthetic pathway to the vacuole with normal kinetics in ypk1G490R cells incubated at the restrictive temperature (unpublished data). These observations indicate that Ypk1 is necessary for fluid-phase endocytosis and for the internalization of plasma membrane proteins carrying different types of internalization signals.

Bottom Line: Ypk1 is required for both receptor-mediated and fluid-phase endocytosis, and is not necessary for receptor phosphorylation or ubiquitination.Ypk1 itself is phosphorylated by Pkh kinases, homologues of mammalian PDK1.The threonine in Ypk1 that is phosphorylated by Pkh1 is required for efficient endocytosis, and pkh mutant cells are defective in alpha-factor internalization and fluid-phase endocytosis.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Molecular Biology, and Cell Biology, Northwestern University, Evanston, IL 60208, USA.

ABSTRACT
Internalization of activated signaling receptors by endocytosis is one way cells downregulate extracellular signals. Like many signaling receptors, the yeast alpha-factor pheromone receptor is downregulated by hyperphosphorylation, ubiquitination, and subsequent internalization and degradation in the lysosome-like vacuole. In a screen to detect proteins involved in ubiquitin-dependent receptor internalization, we identified the sphingoid base-regulated serine-threonine kinase Ypk1. Ypk1 is a homologue of the mammalian serum- and glucocorticoid-induced kinase, SGK, which can substitute for Ypk1 function in yeast. The kinase activity of Ypk1 is required for receptor endocytosis because mutations in two residues important for its catalytic activity cause a severe defect in alpha-factor internalization. Ypk1 is required for both receptor-mediated and fluid-phase endocytosis, and is not necessary for receptor phosphorylation or ubiquitination. Ypk1 itself is phosphorylated by Pkh kinases, homologues of mammalian PDK1. The threonine in Ypk1 that is phosphorylated by Pkh1 is required for efficient endocytosis, and pkh mutant cells are defective in alpha-factor internalization and fluid-phase endocytosis. These observations demonstrate that Ypk1 acts downstream of the Pkh kinases to control endocytosis by phosphorylating components of the endocytic machinery.

Show MeSH