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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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The Gap1p sorting defect of an lst8-1 mutant is suppressed by deletion of gdh1. (A) Gap1p activity was measured by assaying the rate of [14C]citrulline uptake (white bars) of wild-type (CKY759), lst8-1 (CKY768), gdh1Δ (CKY762), or lst8-1 gdh1Δ (CKY769), all with the GAP1 locus replaced by PADH1-GAP1-HA, or of gap1Δ (CKY445) growing on ammonia medium. The rate of [14C]arginine uptake (gray bars) is shown for comparison. Uptake rates are expressed as a percentage of the uptake rate of wild-type. The data shown represent three independent assays, and the error bars represent one SD. (B) The first four strains shown in A were grown in ammonia medium at 24°C, and cell extracts were subjected to isopycnic fractionation on continuous 20–60% sucrose density gradients with EDTA. Fractions were collected from the top of the gradients and proteins were separated by SDS-PAGE. In all the strains shown, Pma1p, Dpm1p, and GDPase fractionated similarly.
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fig1: The Gap1p sorting defect of an lst8-1 mutant is suppressed by deletion of gdh1. (A) Gap1p activity was measured by assaying the rate of [14C]citrulline uptake (white bars) of wild-type (CKY759), lst8-1 (CKY768), gdh1Δ (CKY762), or lst8-1 gdh1Δ (CKY769), all with the GAP1 locus replaced by PADH1-GAP1-HA, or of gap1Δ (CKY445) growing on ammonia medium. The rate of [14C]arginine uptake (gray bars) is shown for comparison. Uptake rates are expressed as a percentage of the uptake rate of wild-type. The data shown represent three independent assays, and the error bars represent one SD. (B) The first four strains shown in A were grown in ammonia medium at 24°C, and cell extracts were subjected to isopycnic fractionation on continuous 20–60% sucrose density gradients with EDTA. Fractions were collected from the top of the gradients and proteins were separated by SDS-PAGE. In all the strains shown, Pma1p, Dpm1p, and GDPase fractionated similarly.

Mentions: An lst8-1 mutation causes Gap1p to be sorted to the vacuole under growth conditions in which Gap1p is normally sorted to the plasma membrane (Roberg et al., 1997a). Because Gap1p is both transcriptionally and post-transcriptionally regulated by the nitrogen source in the growth medium, we verified that the primary effect of lst8-1 on Gap1p activity was an effect on Gap1p sorting, using a PADH1-HA-GAP1 construct that renders GAP1 transcription insensitive to nitrogen source quality (Chen and Kaiser, 2002). Indeed, an lst8-1 mutant with PADH1-HA-GAP1 had no detectable Gap1p activity as measured by uptake of [14C]citrulline, which is transported exclusively by Gap1p (Fig. 1 A), and no Gap1p localized to the plasma membrane (Fig. 1 B). Uptake of [14C]arginine, which is transported by Gap1p and by the arginine permease Can1p, is similar in the lst8-1 and gap1Δ mutants, indicating that the effect of lst8-1 on permease sorting is specific for Gap1p, and is not the result of a general decrease in all permease activity (Fig. 1 A).


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

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

The Gap1p sorting defect of an lst8-1 mutant is suppressed by deletion of gdh1. (A) Gap1p activity was measured by assaying the rate of [14C]citrulline uptake (white bars) of wild-type (CKY759), lst8-1 (CKY768), gdh1Δ (CKY762), or lst8-1 gdh1Δ (CKY769), all with the GAP1 locus replaced by PADH1-GAP1-HA, or of gap1Δ (CKY445) growing on ammonia medium. The rate of [14C]arginine uptake (gray bars) is shown for comparison. Uptake rates are expressed as a percentage of the uptake rate of wild-type. The data shown represent three independent assays, and the error bars represent one SD. (B) The first four strains shown in A were grown in ammonia medium at 24°C, and cell extracts were subjected to isopycnic fractionation on continuous 20–60% sucrose density gradients with EDTA. Fractions were collected from the top of the gradients and proteins were separated by SDS-PAGE. In all the strains shown, Pma1p, Dpm1p, and GDPase fractionated similarly.
© Copyright Policy
Related In: Results  -  Collection

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

fig1: The Gap1p sorting defect of an lst8-1 mutant is suppressed by deletion of gdh1. (A) Gap1p activity was measured by assaying the rate of [14C]citrulline uptake (white bars) of wild-type (CKY759), lst8-1 (CKY768), gdh1Δ (CKY762), or lst8-1 gdh1Δ (CKY769), all with the GAP1 locus replaced by PADH1-GAP1-HA, or of gap1Δ (CKY445) growing on ammonia medium. The rate of [14C]arginine uptake (gray bars) is shown for comparison. Uptake rates are expressed as a percentage of the uptake rate of wild-type. The data shown represent three independent assays, and the error bars represent one SD. (B) The first four strains shown in A were grown in ammonia medium at 24°C, and cell extracts were subjected to isopycnic fractionation on continuous 20–60% sucrose density gradients with EDTA. Fractions were collected from the top of the gradients and proteins were separated by SDS-PAGE. In all the strains shown, Pma1p, Dpm1p, and GDPase fractionated similarly.
Mentions: An lst8-1 mutation causes Gap1p to be sorted to the vacuole under growth conditions in which Gap1p is normally sorted to the plasma membrane (Roberg et al., 1997a). Because Gap1p is both transcriptionally and post-transcriptionally regulated by the nitrogen source in the growth medium, we verified that the primary effect of lst8-1 on Gap1p activity was an effect on Gap1p sorting, using a PADH1-HA-GAP1 construct that renders GAP1 transcription insensitive to nitrogen source quality (Chen and Kaiser, 2002). Indeed, an lst8-1 mutant with PADH1-HA-GAP1 had no detectable Gap1p activity as measured by uptake of [14C]citrulline, which is transported exclusively by Gap1p (Fig. 1 A), and no Gap1p localized to the plasma membrane (Fig. 1 B). Uptake of [14C]arginine, which is transported by Gap1p and by the arginine permease Can1p, is similar in the lst8-1 and gap1Δ mutants, indicating that the effect of lst8-1 on permease sorting is specific for Gap1p, and is not the result of a general decrease in all permease activity (Fig. 1 A).

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