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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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Lst8p is a peripheral membrane protein that cofractionates with Golgi and endosomal compartments and with Tor1p. (A) A cleared cell lysate from a strain with integrated HA-LST8 (CKY784) was fractionated by centrifugation at 13,000 g, then at 100,000 g. Compartment markers are as follows: Pep12p, endosome; Pma1p, plasma membrane; Dpm1p, ER; and Pgk1p, cytosol. The exposure time for the Pgk1p panel was very short relative to the other panels. (B) A cleared cell lysate from CKY784 was incubated with the treatments shown for 1 h at 4°C, then centrifuged for 1 h at 100,000 g. (C) Fractionation of membranes on a flotation gradient shows that HA-Lst8p associates with membranes. A cleared cell lysate from CKY784 was centrifuged at 100,000 g for 1 h onto a cushion of 80% (wt/vol) sucrose. The membranes were collected and loaded at the bottom of a continuous 30–50% sucrose gradient, and centrifuged at 100,000 g for 17 h. (D) GFP-Lst8p was visualized in an lst8Δ strain containing GFP-LST8 on a centromere plasmid (CKY785). (E) HA-Tor1p–containing membranes cofractionate with HA-Lst8p membranes. Membranes from a strain coexpressing HA-LST8 and HA-TOR1 (CKY800) were fractionated as in C.
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fig8: Lst8p is a peripheral membrane protein that cofractionates with Golgi and endosomal compartments and with Tor1p. (A) A cleared cell lysate from a strain with integrated HA-LST8 (CKY784) was fractionated by centrifugation at 13,000 g, then at 100,000 g. Compartment markers are as follows: Pep12p, endosome; Pma1p, plasma membrane; Dpm1p, ER; and Pgk1p, cytosol. The exposure time for the Pgk1p panel was very short relative to the other panels. (B) A cleared cell lysate from CKY784 was incubated with the treatments shown for 1 h at 4°C, then centrifuged for 1 h at 100,000 g. (C) Fractionation of membranes on a flotation gradient shows that HA-Lst8p associates with membranes. A cleared cell lysate from CKY784 was centrifuged at 100,000 g for 1 h onto a cushion of 80% (wt/vol) sucrose. The membranes were collected and loaded at the bottom of a continuous 30–50% sucrose gradient, and centrifuged at 100,000 g for 17 h. (D) GFP-Lst8p was visualized in an lst8Δ strain containing GFP-LST8 on a centromere plasmid (CKY785). (E) HA-Tor1p–containing membranes cofractionate with HA-Lst8p membranes. Membranes from a strain coexpressing HA-LST8 and HA-TOR1 (CKY800) were fractionated as in C.

Mentions: We examined the localization of Lst8p by fractionation of lysates from a strain containing an integrated, fully functional HA-LST8. By differential centrifugation, most Lst8p sedimented after centrifugation at both 13,000 g and 100,000 g, suggesting that Lst8p is membrane associated (Fig. 8 A). To determine conditions for extraction of Lst8p from the insoluble fraction, we treated cell extracts with detergent (1% Triton X-100), high pH, or chaotropic agents such as salt or urea. Lst8p was extracted to the soluble fraction very efficiently by urea and to a lesser extent by high pH or high salt, indicating that Lst8p was peripherally associated with membranes (Fig. 8 B).


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

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

Lst8p is a peripheral membrane protein that cofractionates with Golgi and endosomal compartments and with Tor1p. (A) A cleared cell lysate from a strain with integrated HA-LST8 (CKY784) was fractionated by centrifugation at 13,000 g, then at 100,000 g. Compartment markers are as follows: Pep12p, endosome; Pma1p, plasma membrane; Dpm1p, ER; and Pgk1p, cytosol. The exposure time for the Pgk1p panel was very short relative to the other panels. (B) A cleared cell lysate from CKY784 was incubated with the treatments shown for 1 h at 4°C, then centrifuged for 1 h at 100,000 g. (C) Fractionation of membranes on a flotation gradient shows that HA-Lst8p associates with membranes. A cleared cell lysate from CKY784 was centrifuged at 100,000 g for 1 h onto a cushion of 80% (wt/vol) sucrose. The membranes were collected and loaded at the bottom of a continuous 30–50% sucrose gradient, and centrifuged at 100,000 g for 17 h. (D) GFP-Lst8p was visualized in an lst8Δ strain containing GFP-LST8 on a centromere plasmid (CKY785). (E) HA-Tor1p–containing membranes cofractionate with HA-Lst8p membranes. Membranes from a strain coexpressing HA-LST8 and HA-TOR1 (CKY800) were fractionated as in C.
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Related In: Results  -  Collection

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fig8: Lst8p is a peripheral membrane protein that cofractionates with Golgi and endosomal compartments and with Tor1p. (A) A cleared cell lysate from a strain with integrated HA-LST8 (CKY784) was fractionated by centrifugation at 13,000 g, then at 100,000 g. Compartment markers are as follows: Pep12p, endosome; Pma1p, plasma membrane; Dpm1p, ER; and Pgk1p, cytosol. The exposure time for the Pgk1p panel was very short relative to the other panels. (B) A cleared cell lysate from CKY784 was incubated with the treatments shown for 1 h at 4°C, then centrifuged for 1 h at 100,000 g. (C) Fractionation of membranes on a flotation gradient shows that HA-Lst8p associates with membranes. A cleared cell lysate from CKY784 was centrifuged at 100,000 g for 1 h onto a cushion of 80% (wt/vol) sucrose. The membranes were collected and loaded at the bottom of a continuous 30–50% sucrose gradient, and centrifuged at 100,000 g for 17 h. (D) GFP-Lst8p was visualized in an lst8Δ strain containing GFP-LST8 on a centromere plasmid (CKY785). (E) HA-Tor1p–containing membranes cofractionate with HA-Lst8p membranes. Membranes from a strain coexpressing HA-LST8 and HA-TOR1 (CKY800) were fractionated as in C.
Mentions: We examined the localization of Lst8p by fractionation of lysates from a strain containing an integrated, fully functional HA-LST8. By differential centrifugation, most Lst8p sedimented after centrifugation at both 13,000 g and 100,000 g, suggesting that Lst8p is membrane associated (Fig. 8 A). To determine conditions for extraction of Lst8p from the insoluble fraction, we treated cell extracts with detergent (1% Triton X-100), high pH, or chaotropic agents such as salt or urea. Lst8p was extracted to the soluble fraction very efficiently by urea and to a lesser extent by high pH or high salt, indicating that Lst8p was peripherally associated with membranes (Fig. 8 B).

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