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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 sublethal concentration of rapamycin causes a defect in Gap1p sorting to the plasma membrane. Strains were grown for 18 h to exponential phase in ammonia medium with empty drug vehicle or with 5 ng/ml rapamycin. (A) Gap1p activity was measured by assaying the rate of [14C]citrulline uptake in wild-type (CKY759) and gdh1Δ (CKY762), both containing PADH1-GAP1-HA. The absolute Gap1p activity of wild-type with no rapamycin is 2,864 pmol/min/OD600. (B) The wild-type strain in A was harvested, and cell extracts were subjected to isopycnic fractionation on 20–60% sucrose density gradients. Pma1p, Dpm1p, and GDPase fractionated in a similar manner in the presence or absence of rapamycin.
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fig7: A sublethal concentration of rapamycin causes a defect in Gap1p sorting to the plasma membrane. Strains were grown for 18 h to exponential phase in ammonia medium with empty drug vehicle or with 5 ng/ml rapamycin. (A) Gap1p activity was measured by assaying the rate of [14C]citrulline uptake in wild-type (CKY759) and gdh1Δ (CKY762), both containing PADH1-GAP1-HA. The absolute Gap1p activity of wild-type with no rapamycin is 2,864 pmol/min/OD600. (B) The wild-type strain in A was harvested, and cell extracts were subjected to isopycnic fractionation on 20–60% sucrose density gradients. Pma1p, Dpm1p, and GDPase fractionated in a similar manner in the presence or absence of rapamycin.

Mentions: To test this prediction, we measured Gap1p activity in a strain containing PADH1-HA-GAP1 grown for 18 h in ammonia medium with a sublethal concentration of 5 ng/ml rapamycin. At this low rapamycin concentration, the doubling time is ∼90% of the doubling time seen in ammonia medium without rapamycin (unpublished data). Cells grown in the sublethal concentration of rapamycin showed 6% of the Gap1p activity of cells without rapamycin (Fig. 7 A), and a corresponding decrease in the amount of Gap1p localized to the plasma membrane (Fig. 7 B). As with lst8-1, the Gap1p sorting defect caused by the low level of rapamycin could be partially suppressed by gdh1Δ (Fig. 7 A). Furthermore, cells grown in ammonia medium plus 5 ng/ml rapamycin for 18 h showed a 2.6-fold increase in total amino acid content relative to cells grown without rapamycin (Table I). Thus, like mutation of lst8, impairment of the TOR pathway by growth with a sublethal rapamycin concentration causes increased amino acid levels and decreased Gap1p sorting to the plasma membrane.


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

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

A sublethal concentration of rapamycin causes a defect in Gap1p sorting to the plasma membrane. Strains were grown for 18 h to exponential phase in ammonia medium with empty drug vehicle or with 5 ng/ml rapamycin. (A) Gap1p activity was measured by assaying the rate of [14C]citrulline uptake in wild-type (CKY759) and gdh1Δ (CKY762), both containing PADH1-GAP1-HA. The absolute Gap1p activity of wild-type with no rapamycin is 2,864 pmol/min/OD600. (B) The wild-type strain in A was harvested, and cell extracts were subjected to isopycnic fractionation on 20–60% sucrose density gradients. Pma1p, Dpm1p, and GDPase fractionated in a similar manner in the presence or absence of rapamycin.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172900&req=5

fig7: A sublethal concentration of rapamycin causes a defect in Gap1p sorting to the plasma membrane. Strains were grown for 18 h to exponential phase in ammonia medium with empty drug vehicle or with 5 ng/ml rapamycin. (A) Gap1p activity was measured by assaying the rate of [14C]citrulline uptake in wild-type (CKY759) and gdh1Δ (CKY762), both containing PADH1-GAP1-HA. The absolute Gap1p activity of wild-type with no rapamycin is 2,864 pmol/min/OD600. (B) The wild-type strain in A was harvested, and cell extracts were subjected to isopycnic fractionation on 20–60% sucrose density gradients. Pma1p, Dpm1p, and GDPase fractionated in a similar manner in the presence or absence of rapamycin.
Mentions: To test this prediction, we measured Gap1p activity in a strain containing PADH1-HA-GAP1 grown for 18 h in ammonia medium with a sublethal concentration of 5 ng/ml rapamycin. At this low rapamycin concentration, the doubling time is ∼90% of the doubling time seen in ammonia medium without rapamycin (unpublished data). Cells grown in the sublethal concentration of rapamycin showed 6% of the Gap1p activity of cells without rapamycin (Fig. 7 A), and a corresponding decrease in the amount of Gap1p localized to the plasma membrane (Fig. 7 B). As with lst8-1, the Gap1p sorting defect caused by the low level of rapamycin could be partially suppressed by gdh1Δ (Fig. 7 A). Furthermore, cells grown in ammonia medium plus 5 ng/ml rapamycin for 18 h showed a 2.6-fold increase in total amino acid content relative to cells grown without rapamycin (Table I). Thus, like mutation of lst8, impairment of the TOR pathway by growth with a sublethal rapamycin concentration causes increased amino acid levels and decreased Gap1p sorting to the plasma membrane.

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