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

Mutations in lst8 cause nuclear localization of Gln3p during growth on glutamine. Wild-type (CKY779), lst8-7 (CKY781), and lst8-1 (CKY780), all with integrated GLN3-myc, were grown in glutamine medium at 24°C. Rapamycin was added to the indicated sample for 30 min, and one lst8-7 sample was incubated at 37°C for 2 h before fixation of cells for immunofluorescence. All images are shown at the same magnification.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2172900&req=5

fig3: Mutations in lst8 cause nuclear localization of Gln3p during growth on glutamine. Wild-type (CKY779), lst8-7 (CKY781), and lst8-1 (CKY780), all with integrated GLN3-myc, were grown in glutamine medium at 24°C. Rapamycin was added to the indicated sample for 30 min, and one lst8-7 sample was incubated at 37°C for 2 h before fixation of cells for immunofluorescence. All images are shown at the same magnification.

Mentions: Derepression of GAP1 transcription by rapamycin treatment has been shown to be accompanied by relocalization of the GATA-type transcription factor Gln3p from the cytoplasm to the nucleus (Beck and Hall, 1999). By immunofluorescence we found that, like rapamycin treatment of wild-type cells, mutation of lst8 caused the inappropriate nuclear localization of Gln3p-myc in many cells growing in glutamine medium (Fig. 3) . In our strain background growing on glutamine, we found that rapamycin treatment for 30 min caused nuclear localization in 28% of the cells, whereas the lst8-7 and lst8-1 mutations resulted in the nuclear localization of Gln3p in 8–19% of the cells (Table IV). Deletion of gln3 has been shown to confer rapamycin resistance (Chan et al., 2000). We found that deletion of gln3 could partially rescue the temperature-sensitive growth defect of lst8-7 (Fig. 4) . Together, these data show that mutation of lst8 produces effects similar to those observed on rapamycin treatment.


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

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

Mutations in lst8 cause nuclear localization of Gln3p during growth on glutamine. Wild-type (CKY779), lst8-7 (CKY781), and lst8-1 (CKY780), all with integrated GLN3-myc, were grown in glutamine medium at 24°C. Rapamycin was added to the indicated sample for 30 min, and one lst8-7 sample was incubated at 37°C for 2 h before fixation of cells for immunofluorescence. All images are shown at the same magnification.
© Copyright Policy
Related In: Results  -  Collection

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

fig3: Mutations in lst8 cause nuclear localization of Gln3p during growth on glutamine. Wild-type (CKY779), lst8-7 (CKY781), and lst8-1 (CKY780), all with integrated GLN3-myc, were grown in glutamine medium at 24°C. Rapamycin was added to the indicated sample for 30 min, and one lst8-7 sample was incubated at 37°C for 2 h before fixation of cells for immunofluorescence. All images are shown at the same magnification.
Mentions: Derepression of GAP1 transcription by rapamycin treatment has been shown to be accompanied by relocalization of the GATA-type transcription factor Gln3p from the cytoplasm to the nucleus (Beck and Hall, 1999). By immunofluorescence we found that, like rapamycin treatment of wild-type cells, mutation of lst8 caused the inappropriate nuclear localization of Gln3p-myc in many cells growing in glutamine medium (Fig. 3) . In our strain background growing on glutamine, we found that rapamycin treatment for 30 min caused nuclear localization in 28% of the cells, whereas the lst8-7 and lst8-1 mutations resulted in the nuclear localization of Gln3p in 8–19% of the cells (Table IV). Deletion of gln3 has been shown to confer rapamycin resistance (Chan et al., 2000). We found that deletion of gln3 could partially rescue the temperature-sensitive growth defect of lst8-7 (Fig. 4) . Together, these data show that mutation of lst8 produces effects similar to those observed on rapamycin treatment.

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