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

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.

Figure 1.

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 P _ADH1-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.

Figure 2.

Mutations in lst8 confer rapamycin hypersensitivity. Wild-type (CKY443), lst8-1 (CKY526), lst8-6 (CKY770), lst8-7 (CKY771), tor1Δ (Euroscarf), and gln3Δ (CKY778) were streaked onto YPD or YPD + 200 ng/ml rapamycin and incubated at 30°C for 2 or 4 d, respectively. The tor1Δ strain is included as a control for a rapamycin-hypersensitive strain, and the gln3Δ strain is included as an example of a rapamycin-resistant strain.

Figure 3.

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.

Figure 4.

Deletion of gln3 suppresses the temperature-sensitive growth defect of lst8-7. Wild-type (CKY443), gln3Δ (CKY778), lst8-7 (CKY771), and lst8-7 gln3Δ (CKY782) were spotted onto YPD plates and grown at 24°C or 37°C for 3 d.

Figure 5.

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.

Figure 6.

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.

Figure 7.

A sublethal concentration of rapamycin causes a defect in Gap1p sorting to the plasma membrane. Strains were grown for 18 h to exponential phase in ammonia medium with empty drug vehicle or with 5 ng/ml rapamycin. (A) Gap1p activity was measured by assaying the rate of [¹⁴C]citrulline uptake in wild-type (CKY759) and gdh1Δ (CKY762), both containing P _ADH1-GAP1-HA. The absolute Gap1p activity of wild-type with no rapamycin is 2,864 pmol/min/OD₆₀₀. (B) The wild-type strain in A was harvested, and cell extracts were subjected to isopycnic fractionation on 20–60% sucrose density gradients. Pma1p, Dpm1p, and GDPase fractionated in a similar manner in the presence or absence of rapamycin.

Figure 8.

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.

Figure 8.

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.

Figure 9.

A model of how the Tor1/2 and Lst8 proteins affect amino acid biosynthesis and Gap1p sorting. Tor1/2p and Lst8p negatively regulate the activity of the Rtg1p and Rtg3p transcription factors, decreasing the expression of enzymes responsible for α-ketoglutarate synthesis and limiting the synthesis of α-ketoglutarate, glutamate, glutamine, and the other amino acids. Tor1/2p and Lst8p lso negatively regulate the activity of the Gln3p transcription factor, decreasing the expression of GDH1 and GLN1, further limiting amino acid biosynthesis (Mitchell and Magasanik, 1984; Daugherty et al., 1993). Thus, mutation of lst8 or other impairment of Tor1/2p activity (such as treatment with a sublethal concentration of rapamycin) causes increased amino acid levels, which act as a signal for sorting Gap1p to the vacuole.