Limits...
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.

Show MeSH

Related in: MedlinePlus

Inclusion of sorbitol or SDS in the growth medium or deletion of cwh41 or fks1 suppresses the temperature-sensitive growth defect of lst8 mutants. (A) Wild-type (CKY443), lst8-6 (CKY770), and lst8-7 (CKY771) were streaked onto YPD or YPD + 1 M sorbitol and incubated at 37°C for 2 or 3 d, respectively. (B) The same strains as in A were streaked onto YPD or YPD + 0.0025% SDS and incubated at 34°C for 2 d. (C) Wild-type, lst8-6, cwh41Δ (Euroscarf), fks1Δ (Euroscarf), lst8-6 cwh41Δ (CKY786), and lst8-6 fks1Δ (CKY787) were streaked onto YPD and incubated at 24°C or 34°C for 3 or 2 d, respectively.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2172900&req=5

fig5: Inclusion of sorbitol or SDS in the growth medium or deletion of cwh41 or fks1 suppresses the temperature-sensitive growth defect of lst8 mutants. (A) Wild-type (CKY443), lst8-6 (CKY770), and lst8-7 (CKY771) were streaked onto YPD or YPD + 1 M sorbitol and incubated at 37°C for 2 or 3 d, respectively. (B) The same strains as in A were streaked onto YPD or YPD + 0.0025% SDS and incubated at 34°C for 2 d. (C) Wild-type, lst8-6, cwh41Δ (Euroscarf), fks1Δ (Euroscarf), lst8-6 cwh41Δ (CKY786), and lst8-6 fks1Δ (CKY787) were streaked onto YPD and incubated at 24°C or 34°C for 3 or 2 d, respectively.

Mentions: We found that inclusion of sorbitol in the growth medium fully rescued the temperature sensitivity of lst8-6 and lst8-7, indicating that these mutations were lethal at high temperature due to cell lysis (Fig. 5 A). However, inclusion of sorbitol in the growth medium did not restore growth to an lst8Δ mutant (unpublished data), indicating that the lst8Δ mutant fails to grow for reasons other than cell wall instability. We found that SDS in the growth medium or fks1Δ or cwh41Δ mutations could partially suppress the temperature sensitivity of lst8-6 (Fig. 5, B and C). Together, these data suggest that Lst8p may also participate with Tor2p in maintaining cell wall integrity.


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

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

Inclusion of sorbitol or SDS in the growth medium or deletion of cwh41 or fks1 suppresses the temperature-sensitive growth defect of lst8 mutants. (A) Wild-type (CKY443), lst8-6 (CKY770), and lst8-7 (CKY771) were streaked onto YPD or YPD + 1 M sorbitol and incubated at 37°C for 2 or 3 d, respectively. (B) The same strains as in A were streaked onto YPD or YPD + 0.0025% SDS and incubated at 34°C for 2 d. (C) Wild-type, lst8-6, cwh41Δ (Euroscarf), fks1Δ (Euroscarf), lst8-6 cwh41Δ (CKY786), and lst8-6 fks1Δ (CKY787) were streaked onto YPD and incubated at 24°C or 34°C for 3 or 2 d, respectively.
© Copyright Policy
Related In: Results  -  Collection

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

fig5: Inclusion of sorbitol or SDS in the growth medium or deletion of cwh41 or fks1 suppresses the temperature-sensitive growth defect of lst8 mutants. (A) Wild-type (CKY443), lst8-6 (CKY770), and lst8-7 (CKY771) were streaked onto YPD or YPD + 1 M sorbitol and incubated at 37°C for 2 or 3 d, respectively. (B) The same strains as in A were streaked onto YPD or YPD + 0.0025% SDS and incubated at 34°C for 2 d. (C) Wild-type, lst8-6, cwh41Δ (Euroscarf), fks1Δ (Euroscarf), lst8-6 cwh41Δ (CKY786), and lst8-6 fks1Δ (CKY787) were streaked onto YPD and incubated at 24°C or 34°C for 3 or 2 d, respectively.
Mentions: We found that inclusion of sorbitol in the growth medium fully rescued the temperature sensitivity of lst8-6 and lst8-7, indicating that these mutations were lethal at high temperature due to cell lysis (Fig. 5 A). However, inclusion of sorbitol in the growth medium did not restore growth to an lst8Δ mutant (unpublished data), indicating that the lst8Δ mutant fails to grow for reasons other than cell wall instability. We found that SDS in the growth medium or fks1Δ or cwh41Δ mutations could partially suppress the temperature sensitivity of lst8-6 (Fig. 5, B and C). Together, these data suggest that Lst8p may also participate with Tor2p in maintaining cell wall integrity.

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