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LST8 negatively regulates amino acid biosynthesis as a component of the TOR pathway.

Chen EJ, Kaiser CA - J. Cell Biol. (2003)

Bottom Line: Here, we report that the Gap1p sorting defect in the lst8-1 mutant results from derepression of Rtg1/3p activity and the subsequent accumulation of high levels of intracellular amino acids, which signal Gap1p sorting to the vacuole.Further, we show that a sublethal concentration of rapamycin mimics the Gap1p sorting defect of an lst8 mutant.Finally, the different effects of lst8 alleles on the activation of either the Rtg1/3p or Gln3p transcription factors reveal that these two pathways constitute distinct, genetically separable outputs of the Tor-Lst8 regulatory complex.

View Article: PubMed Central - PubMed

Affiliation: Dept. of Biology, Massachusetts Institute of Technology, 77 Massachusetts Ave., Cambridge, MA 02139, USA.

ABSTRACT
LST8, a Saccharomyces cerevisiae gene encoding a 34-kD WD-repeat protein, was identified by mutations that caused defects in sorting Gap1p to the plasma membrane. Here, we report that the Gap1p sorting defect in the lst8-1 mutant results from derepression of Rtg1/3p activity and the subsequent accumulation of high levels of intracellular amino acids, which signal Gap1p sorting to the vacuole. To identify the essential function of Lst8p, we isolated lst8 mutants that are temperature-sensitive for growth. These mutants show hypersensitivity to rapamycin and derepressed Gln3p activity like cells with compromised TOR pathway activity. Like tor2 mutants, lst8 mutants also have cell wall integrity defects. Confirming a role for Lst8p in the TOR pathway, we find that Lst8p associates with both Tor1p and Tor2p and is a peripheral membrane protein that localizes to endosomal or Golgi membranes and cofractionates with Tor1p. Further, we show that a sublethal concentration of rapamycin mimics the Gap1p sorting defect of an lst8 mutant. Finally, the different effects of lst8 alleles on the activation of either the Rtg1/3p or Gln3p transcription factors reveal that these two pathways constitute distinct, genetically separable outputs of the Tor-Lst8 regulatory complex.

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A model of how the Tor1/2 and Lst8 proteins affect amino acid biosynthesis and Gap1p sorting. Tor1/2p and Lst8p negatively regulate the activity of the Rtg1p and Rtg3p transcription factors, decreasing the expression of enzymes responsible for α-ketoglutarate synthesis and limiting the synthesis of α-ketoglutarate, glutamate, glutamine, and the other amino acids. Tor1/2p and Lst8p lso negatively regulate the activity of the Gln3p transcription factor, decreasing the expression of GDH1 and GLN1, further limiting amino acid biosynthesis (Mitchell and Magasanik, 1984; Daugherty et al., 1993). Thus, mutation of lst8 or other impairment of Tor1/2p activity (such as treatment with a sublethal concentration of rapamycin) causes increased amino acid levels, which act as a signal for sorting Gap1p to the vacuole.
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fig9: A model of how the Tor1/2 and Lst8 proteins affect amino acid biosynthesis and Gap1p sorting. Tor1/2p and Lst8p negatively regulate the activity of the Rtg1p and Rtg3p transcription factors, decreasing the expression of enzymes responsible for α-ketoglutarate synthesis and limiting the synthesis of α-ketoglutarate, glutamate, glutamine, and the other amino acids. Tor1/2p and Lst8p lso negatively regulate the activity of the Gln3p transcription factor, decreasing the expression of GDH1 and GLN1, further limiting amino acid biosynthesis (Mitchell and Magasanik, 1984; Daugherty et al., 1993). Thus, mutation of lst8 or other impairment of Tor1/2p activity (such as treatment with a sublethal concentration of rapamycin) causes increased amino acid levels, which act as a signal for sorting Gap1p to the vacuole.

Mentions: In this paper, we provide genetic and biochemical evidence that LST8 encodes a positively acting component of the TOR pathway. Lst8p associates with Tor1p and Tor2p, and is involved in the regulation of Rtg1/3p and Gln3p, and in maintenance of cell wall integrity. We can now explain the role of Lst8p in the regulation of Gap1p sorting by the three distinct regulatory processes diagramed in Fig. 9 : (1) Tor1/2p and Lst8p act together to negatively regulate both the Rtg1/3p and Gln3p transcription factors, limiting the synthesis of α-ketoglutarate, glutamate, and glutamine; (2) inactivation of the TOR pathway by mutation of LST8 causes an increase in the intracellular pools of glutamate and glutamine, as well as the other amino acids derived from glutamate and glutamine; and (3) because all amino acids can act as a signal to cause Gap1p sorting to the vacuole, mutation of LST8 causes Gap1p to be sorted to the vacuole. As validation of this model, we have shown that growth of wild-type cells in a low, sublethal concentration of rapamycin or that mutation of LST8 produces an increase in amino acid pools and a consequent decrease in Gap1p activity caused by sorting to the vacuole.


LST8 negatively regulates amino acid biosynthesis as a component of the TOR pathway.

Chen EJ, Kaiser CA - J. Cell Biol. (2003)

A model of how the Tor1/2 and Lst8 proteins affect amino acid biosynthesis and Gap1p sorting. Tor1/2p and Lst8p negatively regulate the activity of the Rtg1p and Rtg3p transcription factors, decreasing the expression of enzymes responsible for α-ketoglutarate synthesis and limiting the synthesis of α-ketoglutarate, glutamate, glutamine, and the other amino acids. Tor1/2p and Lst8p lso negatively regulate the activity of the Gln3p transcription factor, decreasing the expression of GDH1 and GLN1, further limiting amino acid biosynthesis (Mitchell and Magasanik, 1984; Daugherty et al., 1993). Thus, mutation of lst8 or other impairment of Tor1/2p activity (such as treatment with a sublethal concentration of rapamycin) causes increased amino acid levels, which act as a signal for sorting Gap1p to the vacuole.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172900&req=5

fig9: A model of how the Tor1/2 and Lst8 proteins affect amino acid biosynthesis and Gap1p sorting. Tor1/2p and Lst8p negatively regulate the activity of the Rtg1p and Rtg3p transcription factors, decreasing the expression of enzymes responsible for α-ketoglutarate synthesis and limiting the synthesis of α-ketoglutarate, glutamate, glutamine, and the other amino acids. Tor1/2p and Lst8p lso negatively regulate the activity of the Gln3p transcription factor, decreasing the expression of GDH1 and GLN1, further limiting amino acid biosynthesis (Mitchell and Magasanik, 1984; Daugherty et al., 1993). Thus, mutation of lst8 or other impairment of Tor1/2p activity (such as treatment with a sublethal concentration of rapamycin) causes increased amino acid levels, which act as a signal for sorting Gap1p to the vacuole.
Mentions: In this paper, we provide genetic and biochemical evidence that LST8 encodes a positively acting component of the TOR pathway. Lst8p associates with Tor1p and Tor2p, and is involved in the regulation of Rtg1/3p and Gln3p, and in maintenance of cell wall integrity. We can now explain the role of Lst8p in the regulation of Gap1p sorting by the three distinct regulatory processes diagramed in Fig. 9 : (1) Tor1/2p and Lst8p act together to negatively regulate both the Rtg1/3p and Gln3p transcription factors, limiting the synthesis of α-ketoglutarate, glutamate, and glutamine; (2) inactivation of the TOR pathway by mutation of LST8 causes an increase in the intracellular pools of glutamate and glutamine, as well as the other amino acids derived from glutamate and glutamine; and (3) because all amino acids can act as a signal to cause Gap1p sorting to the vacuole, mutation of LST8 causes Gap1p to be sorted to the vacuole. As validation of this model, we have shown that growth of wild-type cells in a low, sublethal concentration of rapamycin or that mutation of LST8 produces an increase in amino acid pools and a consequent decrease in Gap1p activity caused by sorting to the vacuole.

Bottom Line: Here, we report that the Gap1p sorting defect in the lst8-1 mutant results from derepression of Rtg1/3p activity and the subsequent accumulation of high levels of intracellular amino acids, which signal Gap1p sorting to the vacuole.Further, we show that a sublethal concentration of rapamycin mimics the Gap1p sorting defect of an lst8 mutant.Finally, the different effects of lst8 alleles on the activation of either the Rtg1/3p or Gln3p transcription factors reveal that these two pathways constitute distinct, genetically separable outputs of the Tor-Lst8 regulatory complex.

View Article: PubMed Central - PubMed

Affiliation: Dept. of Biology, Massachusetts Institute of Technology, 77 Massachusetts Ave., Cambridge, MA 02139, USA.

ABSTRACT
LST8, a Saccharomyces cerevisiae gene encoding a 34-kD WD-repeat protein, was identified by mutations that caused defects in sorting Gap1p to the plasma membrane. Here, we report that the Gap1p sorting defect in the lst8-1 mutant results from derepression of Rtg1/3p activity and the subsequent accumulation of high levels of intracellular amino acids, which signal Gap1p sorting to the vacuole. To identify the essential function of Lst8p, we isolated lst8 mutants that are temperature-sensitive for growth. These mutants show hypersensitivity to rapamycin and derepressed Gln3p activity like cells with compromised TOR pathway activity. Like tor2 mutants, lst8 mutants also have cell wall integrity defects. Confirming a role for Lst8p in the TOR pathway, we find that Lst8p associates with both Tor1p and Tor2p and is a peripheral membrane protein that localizes to endosomal or Golgi membranes and cofractionates with Tor1p. Further, we show that a sublethal concentration of rapamycin mimics the Gap1p sorting defect of an lst8 mutant. Finally, the different effects of lst8 alleles on the activation of either the Rtg1/3p or Gln3p transcription factors reveal that these two pathways constitute distinct, genetically separable outputs of the Tor-Lst8 regulatory complex.

Show MeSH
Related in: MedlinePlus