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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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Mutations in lst8 confer rapamycin hypersensitivity. Wild-type (CKY443), lst8-1 (CKY526), lst8-6 (CKY770), lst8-7 (CKY771), tor1Δ (Euroscarf), and gln3Δ (CKY778) were streaked onto YPD or YPD + 200 ng/ml rapamycin and incubated at 30°C for 2 or 4 d, respectively. The tor1Δ strain is included as a control for a rapamycin-hypersensitive strain, and the gln3Δ strain is included as an example of a rapamycin-resistant strain.
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fig2: Mutations in lst8 confer rapamycin hypersensitivity. Wild-type (CKY443), lst8-1 (CKY526), lst8-6 (CKY770), lst8-7 (CKY771), tor1Δ (Euroscarf), and gln3Δ (CKY778) were streaked onto YPD or YPD + 200 ng/ml rapamycin and incubated at 30°C for 2 or 4 d, respectively. The tor1Δ strain is included as a control for a rapamycin-hypersensitive strain, and the gln3Δ strain is included as an example of a rapamycin-resistant strain.

Mentions: We looked for a link between Lst8p function and the activity of the TOR pathway by testing the sensitivity of lst8 mutants to rapamycin. Like Lst8p and Mks1p, the TOR pathway negatively regulates Rtg1/3p, as shown by the ability of the TOR inhibitor rapamycin to induce the expression of Rtg1/3p-dependent genes in strains grown on glutamate or glutamine (Komeili et al., 2000). At the semi-permissive temperature of 30°C on rich medium, lst8 mutants were hypersensitive to rapamycin (Fig. 2) , like a tor1Δ mutant that was previously shown to be rapamycin hypersensitive (Chan et al., 2000), indicating that Lst8p might function positively in the TOR pathway.


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

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

Mutations in lst8 confer rapamycin hypersensitivity. Wild-type (CKY443), lst8-1 (CKY526), lst8-6 (CKY770), lst8-7 (CKY771), tor1Δ (Euroscarf), and gln3Δ (CKY778) were streaked onto YPD or YPD + 200 ng/ml rapamycin and incubated at 30°C for 2 or 4 d, respectively. The tor1Δ strain is included as a control for a rapamycin-hypersensitive strain, and the gln3Δ strain is included as an example of a rapamycin-resistant strain.
© Copyright Policy
Related In: Results  -  Collection

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

fig2: Mutations in lst8 confer rapamycin hypersensitivity. Wild-type (CKY443), lst8-1 (CKY526), lst8-6 (CKY770), lst8-7 (CKY771), tor1Δ (Euroscarf), and gln3Δ (CKY778) were streaked onto YPD or YPD + 200 ng/ml rapamycin and incubated at 30°C for 2 or 4 d, respectively. The tor1Δ strain is included as a control for a rapamycin-hypersensitive strain, and the gln3Δ strain is included as an example of a rapamycin-resistant strain.
Mentions: We looked for a link between Lst8p function and the activity of the TOR pathway by testing the sensitivity of lst8 mutants to rapamycin. Like Lst8p and Mks1p, the TOR pathway negatively regulates Rtg1/3p, as shown by the ability of the TOR inhibitor rapamycin to induce the expression of Rtg1/3p-dependent genes in strains grown on glutamate or glutamine (Komeili et al., 2000). At the semi-permissive temperature of 30°C on rich medium, lst8 mutants were hypersensitive to rapamycin (Fig. 2) , like a tor1Δ mutant that was previously shown to be rapamycin hypersensitive (Chan et al., 2000), indicating that Lst8p might function positively in the TOR pathway.

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