Amino Acid Availability Modulates Vacuolar H+-ATPase Assembly

Abstract

The vacuolar H(+)-ATPase (V-ATPase) is an ATP-dependent proton pump composed of a peripheral ATPase domain (V1) and a membrane-integral proton-translocating domain (V0) and is involved in many normal and disease processes. An important mechanism of regulating V-ATPase activity is reversible assembly of the V1 and V0 domains. Increased assembly in mammalian cells occurs under various conditions and has been shown to involve PI3K. The V-ATPase is necessary for amino acid-induced activation of mechanistic target of rapamycin complex 1 (mTORC1), which is important in controlling cell growth in response to nutrient availability and growth signals. The V-ATPase undergoes amino acid-dependent interactions with the Ragulator complex, which is involved in recruitment of mTORC1 to the lysosomal membrane during amino acid sensing. We hypothesized that changes in the V-ATPase/Ragulator interaction might involve amino acid-dependent changes in V-ATPase assembly. To test this, we measured V-ATPase assembly by cell fractionation in HEK293T cells treated with and without amino acids. V-ATPase assembly increases upon amino acid starvation, and this effect is reversed upon readdition of amino acids. Lysosomes from amino acid-starved cells possess greater V-ATPase-dependent proton transport, indicating that assembled pumps are catalytically active. Amino acid-dependent changes in both V-ATPase assembly and activity are independent of PI3K and mTORC1 activity, indicating the involvement of signaling pathways distinct from those implicated previously in controlling assembly. By contrast, lysosomal neutralization blocks the amino acid-dependent change in assembly and reactivation of mTORC1 after amino acid starvation. These results identify an important new stimulus for controlling V-ATPase assembly.

Keywords: amino acid; lysosomal acidification; mechanistic target of rapamycin (mTOR); nutrient sensing; proton transport; regulated assembly; vacuolar ATPase.

FIGURE 1.

Amino acids alter V-ATPase assembly. A, HEK293T cells were maintained in amino acids for 65 min (+/+), starved of amino acids for 65 min (−/−), or starved of amino acids for 50 min followed by readdition of amino acids for 15 min (−/+). Cell homogenates were prepared, separated into membrane (M) and cytosolic (C) fractions by sedimentation, and subjected to SDS-PAGE and Western blotting using antibodies against subunit A as a measure of V₁, subunit d1 as a loading control for the membrane fraction, and vinculin as a loading control for the cytosolic fraction, as described under “Experimental Procedures.” The amount of subunit A present in the membrane fraction indicates the amount of assembled V-ATPase. A representative Western blot is shown. B, Western blots performed as in A were quantified by ImageJ analysis to assess relative V-ATPase assembly. Subunit A band intensities were normalized to membrane and cytosolic loading controls, and the ratio of membrane to cytosolic subunit A was calculated. To combine results from independent experiments, values were normalized to baseline (+/+) assembly levels (defined as 1.0 for each trial). The average ratio for (+/+) conditions was 0.37 (± 0.05). p < 0.05, +/+ versus −/−. The number of independent trials is shown as n, and the error bars represent mean ± S.E.

FIGURE 2.

Amino acids alter V-ATPase-dependent proton transport, as measured by ATP-dependent fluorescence quenching of FITC-loaded lysosomes. A, HEK293T cells were allowed to take up FITC-Dextran by endocytosis, and the dye was chased to the lysosomal compartment as described under “Experimental Procedures.” Cells were lysed mechanically, and a fraction containing FITC-Dextran-loaded lysosomes was isolated by sedimentation. Fluorescence was measured over time to assess pH-dependent quenching following addition of 1 mm magnesium-ATP. The representative traces shown are for lysosomes isolated from HEK293T cells maintained in amino acids (+/+), starved of amino acids (−/−), or starved of amino acids for 50 min followed by readdition of amino acids for 15 (−/+). ATP-dependent fluorescence quenching was not observed for samples preincubated in the presence of 5 μm concanamycin A. B, the initial rate of magnesium-ATP-dependent fluorescence quenching was determined by a linear regression analysis for each condition. Rates of fluorescence quenching from independent experiments were normalized to the value observed for lysosomes isolated from cells maintained in amino acids throughout (+/+). p < 0.05, +/+ versus −/− and −/+. C, the time course of amino acid-dependent increase in V-ATPase-dependent proton transport after amino acid starvation. HEK293T cells were loaded with FITC-Dextran and starved of amino acids for 5, 15, 30, or 65 min before cell lysis and isolation of the lysosome-containing fraction as described above. p < 0.05, +/+ versus −/− (15 min) and −/− (65 min). D, HEK293T cells were subjected to amino acid starvation and readdition in the presence of 50 μm LY294002 (LY) to inhibit PI3K, 2 nm rapamycin to inhibit mTORC1, or in the absence of inhibitors, as indicated, followed by measurement of ATP-dependent fluorescence quenching as described above. p < 0.05; +/+ versus −/− and −/+; +/+ LY versus −/− LY and −/+ LY; −/− LY versus −/+ LY. In B—D, the number of independent trials is shown as n, and the error bars represent mean ± S.E.

FIGURE 3.

Control of amino acid-induced changes in V-ATPase assembly. A, quantification of V-ATPase assembly in HEK293T cells maintained in amino acids (+/+), starved of amino acids (−/−), or starved of amino acids for 50 min followed by readdition of amino acids for 15-min (−/+) was performed as described for Fig. 1 except, where indicated, with the addition of 5 μm concanamycin A to inhibit the V-ATPase, 50 μm LY294002 to inhibit PI3K, 2 nm rapamycin to inhibit mTORC1, or 100 μm chloroquine to neutralize the lysosomal pH. Ratios for each set of conditions are normalized to cells maintained in amino acids (+/+) in the absence of inhibitors. p < 0.05, +/+ versus −/− and −/+. The number of independent trials is shown as n, and the error bars represent mean ± S.E. B, 100 μm chloroquine neutralizes lysosomes in HEK293T cells, as assessed by DAMP staining. HEK293T cells were grown on glass coverslips and treated with 100 μm chloroquine or medium containing an equivalent volume of PBS for 65 min. DAMP was added to the culture medium 50 min before cells were fixed to allow accumulation in acidic compartments. DAMP was detected by anti-DNP antibodies (green fluorescence), and immunocytochemistry was performed. Nuclei were stained with DAPI (blue fluorescence). Staining was assessed by confocal fluorescence microscopy.

FIGURE 4.

Starvation of individual amino acids alters V-ATPase-dependent proton transport and assembly. A, HEK293T cells were allowed to take up FITC-Dextran by endocytosis, and the dye was chased to the lysosomal compartment as described under “Experimental Procedures.” Loaded cells were maintained in amino acids (+/+), starved of all amino acids (−/−), or starved of the indicated amino acid for 65 min. Cells were then lysed mechanically, and a fraction containing FITC-Dextran-loaded lysosomes was isolated by sedimentation. Fluorescence was measured over time to assess pH-dependent quenching following addition of 1 mm magnesium-ATP. ATP-dependent fluorescence quenching was not observed for samples preincubated in the presence of 5 μm concanamycin A. The initial rate of magnesium ATP-dependent fluorescence quenching was determined by linear regression analysis for each condition. Rates of fluorescence quenching from independent experiments were normalized to the value observed for lysosomes isolated from cells maintained in amino acids throughout the experiment (+/+). p < 0.05, +/+ versus −/−, −Glu, −Pro, −Ser, and −Thr. The number of independent trials is shown as n, and the error bars represent mean ± S.E. B, quantification of V-ATPase assembly in HEK293T cells maintained in amino acids (+/+), starved of amino acids (−/−), or starved of the indicated amino acids for 65 min was performed as described for Fig. 1. p < 0.05, +/+ versus −/−. The number of independent trials is shown as n, and the error bars represent mean ± S.E.

FIGURE 5.

Activation of mTORC1 by amino acids. A, HEK293T cells were maintained in amino acids for 65 min (+/+), starved of amino acids for 65 min (−/−), or starved of amino acids for 50 min followed by a readdition of amino acids for 15-min (−/+). Cell homogenates were prepared and subjected to SDS-PAGE, and Western blotting was performed using antibodies against S6 kinase or Thr(P)-389-S6 kinase as indicated. The level of phospho-S6 kinase is a measure of mTORC1 activity. B, HEK293T cells were maintained in amino acids (+/+) or deprived of amino acids for the indicated times, followed by measurement of phospho-S6 kinase levels as described in A. C, the experiment described in A was performed in the absence (DMSO) or presence of 5 μm concanamycin A (ConA), a specific V-ATPase inhibitor, as indicated. D, the experiment described in A was performed in the absence (DMSO) or presence of 50 μm LY294002, a PI3K inhibitor, as indicated. E, the experiment described in A was performed in the absence (DMSO) or presence of 2 nm rapamycin, an inhibitor of mTORC1, as indicated. F, the experiment described in A was performed in the absence (PBS) or presence of 100 μm chloroquine, a weak base that neutralizes lysosomes, as indicated. G, HEK293T cells were maintained in amino acids for 65 min (+/+), starved of amino acids for 65 min (−/−), or starved of individual amino acids for 65 min, and homogenates were prepared and assessed as described in A.

FIGURE 6.

Model of the regulated assembly of the V-ATPase in response to amino acids or changes in glucose concentrations. As demonstrated previously, changes in V-ATPase assembly in response to changes in glucose concentration, during dendritic cell maturation, or during influenza infection are dependent upon PI3K and, in the case of dendritic cell maturation, mTORC1 activity. By contrast, changes in assembly in response to changes in amino acid levels are independent of PI3K and mTORC1 activity but do depend upon the luminal pH and the catalytic activity of the V-ATPase. V₁ subunits are depicted in green and V₀ subunits in blue.