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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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Tor1p and Tor2p associate with Lst8p. A wild-type strain with integrated LST8-myc from its own promoter (CKY783; lanes 1, 3, 4, and 6) or untagged LST8 (CKY8; lanes 2 and 5) was transformed with pRS316 containing HA-TOR1 (pEC267; lanes 1 and 2), TOR1 (pEC262; lane 3), HA-TOR2 (pEC268; lanes 4 and 5), or TOR2 (pEC263; lane 6). Strains were grown in SMM-uracil at 30°C. Anti-myc (rabbit 9E10) immunoprecipitates (A) or cell lysates (B) were subjected to SDS-PAGE and Western blotting with either HA (12CA5) or myc (monoclonal 9E10) antibody.
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fig6: Tor1p and Tor2p associate with Lst8p. A wild-type strain with integrated LST8-myc from its own promoter (CKY783; lanes 1, 3, 4, and 6) or untagged LST8 (CKY8; lanes 2 and 5) was transformed with pRS316 containing HA-TOR1 (pEC267; lanes 1 and 2), TOR1 (pEC262; lane 3), HA-TOR2 (pEC268; lanes 4 and 5), or TOR2 (pEC263; lane 6). Strains were grown in SMM-uracil at 30°C. Anti-myc (rabbit 9E10) immunoprecipitates (A) or cell lysates (B) were subjected to SDS-PAGE and Western blotting with either HA (12CA5) or myc (monoclonal 9E10) antibody.

Mentions: We tested whether Lst8p associates with Tor1p or Tor2p, given the evidence presented thus far that mutation of lst8 causes effects similar to the mutation of tor2 and to the inactivation of TOR pathway function with rapamycin. In mammalian cells, mLst8 was found to be associated with mTOR (Kim, D.H., and D.M. Sabatini, personal communication). HA-tagged versions of Tor1p and Tor2p were reported to be functional (Fiorentino and Crabtree, 1997; Kunz et al., 2000), and we verified the function of our constructs by complementation of the rapamycin sensitivity of a tor1Δ strain (for HA-TOR1) and complementation of the lethality of a tor2Δ strain (for HA-TOR2; unpublished data). We expressed HA-TOR1 or HA-TOR2 from their own promoters on CEN vectors in strains with LST8-6xmyc integrated at the LST8 locus. Using myc antibody to precipitate Lst8p-myc–containing complexes and immunoblots to detect HA-Tor1p or HA-Tor2p, we found that HA-Tor1p and HA-Tor2p are present in complex with Lst8p-myc (Fig. 6) . We also looked at the dependence of this interaction on the nitrogen source, but found that the association of HA-Tor1p and HA-Tor2p with Lst8p-myc is not significantly influenced by growth in nitrogen-free medium or in YPD with 0.3% glutamine (unpublished data). Together with the lst8 mutant phenotypes, this result indicates that Lst8p physically associates with Tor1p and Tor2p to effect TOR pathway function.


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

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

Tor1p and Tor2p associate with Lst8p. A wild-type strain with integrated LST8-myc from its own promoter (CKY783; lanes 1, 3, 4, and 6) or untagged LST8 (CKY8; lanes 2 and 5) was transformed with pRS316 containing HA-TOR1 (pEC267; lanes 1 and 2), TOR1 (pEC262; lane 3), HA-TOR2 (pEC268; lanes 4 and 5), or TOR2 (pEC263; lane 6). Strains were grown in SMM-uracil at 30°C. Anti-myc (rabbit 9E10) immunoprecipitates (A) or cell lysates (B) were subjected to SDS-PAGE and Western blotting with either HA (12CA5) or myc (monoclonal 9E10) antibody.
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Related In: Results  -  Collection

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

fig6: Tor1p and Tor2p associate with Lst8p. A wild-type strain with integrated LST8-myc from its own promoter (CKY783; lanes 1, 3, 4, and 6) or untagged LST8 (CKY8; lanes 2 and 5) was transformed with pRS316 containing HA-TOR1 (pEC267; lanes 1 and 2), TOR1 (pEC262; lane 3), HA-TOR2 (pEC268; lanes 4 and 5), or TOR2 (pEC263; lane 6). Strains were grown in SMM-uracil at 30°C. Anti-myc (rabbit 9E10) immunoprecipitates (A) or cell lysates (B) were subjected to SDS-PAGE and Western blotting with either HA (12CA5) or myc (monoclonal 9E10) antibody.
Mentions: We tested whether Lst8p associates with Tor1p or Tor2p, given the evidence presented thus far that mutation of lst8 causes effects similar to the mutation of tor2 and to the inactivation of TOR pathway function with rapamycin. In mammalian cells, mLst8 was found to be associated with mTOR (Kim, D.H., and D.M. Sabatini, personal communication). HA-tagged versions of Tor1p and Tor2p were reported to be functional (Fiorentino and Crabtree, 1997; Kunz et al., 2000), and we verified the function of our constructs by complementation of the rapamycin sensitivity of a tor1Δ strain (for HA-TOR1) and complementation of the lethality of a tor2Δ strain (for HA-TOR2; unpublished data). We expressed HA-TOR1 or HA-TOR2 from their own promoters on CEN vectors in strains with LST8-6xmyc integrated at the LST8 locus. Using myc antibody to precipitate Lst8p-myc–containing complexes and immunoblots to detect HA-Tor1p or HA-Tor2p, we found that HA-Tor1p and HA-Tor2p are present in complex with Lst8p-myc (Fig. 6) . We also looked at the dependence of this interaction on the nitrogen source, but found that the association of HA-Tor1p and HA-Tor2p with Lst8p-myc is not significantly influenced by growth in nitrogen-free medium or in YPD with 0.3% glutamine (unpublished data). Together with the lst8 mutant phenotypes, this result indicates that Lst8p physically associates with Tor1p and Tor2p to effect TOR pathway function.

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