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Soluble adenylyl cyclase is essential for proper lysosomal acidification

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ABSTRACT

Lysosomes are the main degradative compartment in cells and require an acidic luminal environment for correct function. Rahman et al. show that soluble adenylyl cyclase is required for localization of the V-ATPase proton pump to lysosomes and therefore lysosomal acidification and function.

No MeSH data available.


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Lysosomal proteolytic degradation defect in the absence of sAC. (A) Representative images of WT and sAC KO MEFs stained with BODIPY-FL–Pepstatin A. (B) BODIPY-FL–Pepstatin A intensity was quantified using MetaMorph in multiple cells from three independent experiments. All values are given as mean fluorescence intensity per cell area ± SEM. ***, P < 0.001. (C) Representative images of WT and sAC KO MEFs stained with 10 µg/ml DQ-BSA. (A and C) Bars, 10 µm. (D) DQ-BSA intensity was quantified using MetaMorph in multiple cells from three independent experiments. All values are given as mean ± SEM. *, P < 0.05. (E) Total Protein turnover is quantified as percent proteolysis (i.e., the percentage of acid-soluble radioactivity [amino acids and small peptides] divided by the initial acid-insoluble radioactivity [protein]) in WT and sAC KO MEFs after incorporation of [3H]leucine (n = 15, four independent experiment days). As control, proteolysis was assessed in the presence of NH4Cl in both WT (WT+NH4) and in sAC KO MEFs (KO+NH4). Error bars represent ±SEM. (F) WT (blue circles) and sAC KO (red squares) MEFs were plated in 96-well plates (5 × 103 per well) and treated with increasing concentrations of the proteasome inhibitor MG132 for 24 h. The percentage of growth was determined by MTT assay. Results are presented as percent viability relative to cells in the absence of any drug; data graphed are the mean of triplicate determinations (±SEM) of a representative experiment repeated three times.
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fig4: Lysosomal proteolytic degradation defect in the absence of sAC. (A) Representative images of WT and sAC KO MEFs stained with BODIPY-FL–Pepstatin A. (B) BODIPY-FL–Pepstatin A intensity was quantified using MetaMorph in multiple cells from three independent experiments. All values are given as mean fluorescence intensity per cell area ± SEM. ***, P < 0.001. (C) Representative images of WT and sAC KO MEFs stained with 10 µg/ml DQ-BSA. (A and C) Bars, 10 µm. (D) DQ-BSA intensity was quantified using MetaMorph in multiple cells from three independent experiments. All values are given as mean ± SEM. *, P < 0.05. (E) Total Protein turnover is quantified as percent proteolysis (i.e., the percentage of acid-soluble radioactivity [amino acids and small peptides] divided by the initial acid-insoluble radioactivity [protein]) in WT and sAC KO MEFs after incorporation of [3H]leucine (n = 15, four independent experiment days). As control, proteolysis was assessed in the presence of NH4Cl in both WT (WT+NH4) and in sAC KO MEFs (KO+NH4). Error bars represent ±SEM. (F) WT (blue circles) and sAC KO (red squares) MEFs were plated in 96-well plates (5 × 103 per well) and treated with increasing concentrations of the proteasome inhibitor MG132 for 24 h. The percentage of growth was determined by MTT assay. Results are presented as percent viability relative to cells in the absence of any drug; data graphed are the mean of triplicate determinations (±SEM) of a representative experiment repeated three times.

Mentions: The lysosomal cathepsins and hydrolases that degrade proteins, lipids, and polysaccharides are optimally active in the acidic (i.e., pH < 5) lumen of the lysosome (Pillay et al., 2002). Because fewer lysosomes achieve the acidic pH necessary for optimal degradation in sAC KO cells (Fig. 1, A and B), we asked whether protein degradation was affected in sAC KO MEFs. We first examined the consequences of elevated lysosomal pH by assessing the activity of lysosomal cathepsins, specifically CatD. Fluorescently tagged BODIPY-FL–Pepstatin A binds to CatD only when the CatD active site is in an open state under acidic conditions (i.e., ∼pH 4.5; Chen et al., 2000a). As expected, in WT control cells, BODIPY-FL–Pepstatin A fluorescence intensity (Fig. 4, A and B) and number of puncta (Fig. S5 A) were decreased when lysosomal pH was chemically elevated by incubating cells in NH4Cl (Fig. 4 B and Fig. S5 A). Loss of sAC, both in sAC KO cells (Fig. 4, A and B) and in WT cells treated with KH7 (Fig. 4 B), decreased BODIPY-FL–Pepstatin A fluorescence intensity and puncta (Fig. S5 A) relative to WT cells. Thus, in the absence of sAC, there is diminished catalytically active CatD.


Soluble adenylyl cyclase is essential for proper lysosomal acidification
Lysosomal proteolytic degradation defect in the absence of sAC. (A) Representative images of WT and sAC KO MEFs stained with BODIPY-FL–Pepstatin A. (B) BODIPY-FL–Pepstatin A intensity was quantified using MetaMorph in multiple cells from three independent experiments. All values are given as mean fluorescence intensity per cell area ± SEM. ***, P < 0.001. (C) Representative images of WT and sAC KO MEFs stained with 10 µg/ml DQ-BSA. (A and C) Bars, 10 µm. (D) DQ-BSA intensity was quantified using MetaMorph in multiple cells from three independent experiments. All values are given as mean ± SEM. *, P < 0.05. (E) Total Protein turnover is quantified as percent proteolysis (i.e., the percentage of acid-soluble radioactivity [amino acids and small peptides] divided by the initial acid-insoluble radioactivity [protein]) in WT and sAC KO MEFs after incorporation of [3H]leucine (n = 15, four independent experiment days). As control, proteolysis was assessed in the presence of NH4Cl in both WT (WT+NH4) and in sAC KO MEFs (KO+NH4). Error bars represent ±SEM. (F) WT (blue circles) and sAC KO (red squares) MEFs were plated in 96-well plates (5 × 103 per well) and treated with increasing concentrations of the proteasome inhibitor MG132 for 24 h. The percentage of growth was determined by MTT assay. Results are presented as percent viability relative to cells in the absence of any drug; data graphed are the mean of triplicate determinations (±SEM) of a representative experiment repeated three times.
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fig4: Lysosomal proteolytic degradation defect in the absence of sAC. (A) Representative images of WT and sAC KO MEFs stained with BODIPY-FL–Pepstatin A. (B) BODIPY-FL–Pepstatin A intensity was quantified using MetaMorph in multiple cells from three independent experiments. All values are given as mean fluorescence intensity per cell area ± SEM. ***, P < 0.001. (C) Representative images of WT and sAC KO MEFs stained with 10 µg/ml DQ-BSA. (A and C) Bars, 10 µm. (D) DQ-BSA intensity was quantified using MetaMorph in multiple cells from three independent experiments. All values are given as mean ± SEM. *, P < 0.05. (E) Total Protein turnover is quantified as percent proteolysis (i.e., the percentage of acid-soluble radioactivity [amino acids and small peptides] divided by the initial acid-insoluble radioactivity [protein]) in WT and sAC KO MEFs after incorporation of [3H]leucine (n = 15, four independent experiment days). As control, proteolysis was assessed in the presence of NH4Cl in both WT (WT+NH4) and in sAC KO MEFs (KO+NH4). Error bars represent ±SEM. (F) WT (blue circles) and sAC KO (red squares) MEFs were plated in 96-well plates (5 × 103 per well) and treated with increasing concentrations of the proteasome inhibitor MG132 for 24 h. The percentage of growth was determined by MTT assay. Results are presented as percent viability relative to cells in the absence of any drug; data graphed are the mean of triplicate determinations (±SEM) of a representative experiment repeated three times.
Mentions: The lysosomal cathepsins and hydrolases that degrade proteins, lipids, and polysaccharides are optimally active in the acidic (i.e., pH < 5) lumen of the lysosome (Pillay et al., 2002). Because fewer lysosomes achieve the acidic pH necessary for optimal degradation in sAC KO cells (Fig. 1, A and B), we asked whether protein degradation was affected in sAC KO MEFs. We first examined the consequences of elevated lysosomal pH by assessing the activity of lysosomal cathepsins, specifically CatD. Fluorescently tagged BODIPY-FL–Pepstatin A binds to CatD only when the CatD active site is in an open state under acidic conditions (i.e., ∼pH 4.5; Chen et al., 2000a). As expected, in WT control cells, BODIPY-FL–Pepstatin A fluorescence intensity (Fig. 4, A and B) and number of puncta (Fig. S5 A) were decreased when lysosomal pH was chemically elevated by incubating cells in NH4Cl (Fig. 4 B and Fig. S5 A). Loss of sAC, both in sAC KO cells (Fig. 4, A and B) and in WT cells treated with KH7 (Fig. 4 B), decreased BODIPY-FL–Pepstatin A fluorescence intensity and puncta (Fig. S5 A) relative to WT cells. Thus, in the absence of sAC, there is diminished catalytically active CatD.

View Article: PubMed Central - HTML - PubMed

ABSTRACT

Lysosomes are the main degradative compartment in cells and require an acidic luminal environment for correct function. Rahman et al. show that soluble adenylyl cyclase is required for localization of the V-ATPase proton pump to lysosomes and therefore lysosomal acidification and function.

No MeSH data available.


Related in: MedlinePlus