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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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sAC regulates V-ATPase localization to lysosomes. (A) Representative immunoblot of the V-ATPase subunit V1B2 along with GAPDH, which was used for loading control. Shown are whole cell extracts from three independently grown cultures of WT and sAC KO MEFs. n = 3. (B) Double-immunofluorescence labeling of V-ATPase V1B2 subunit (red) and LAMP2 (green) in WT, sAC KO, and sAC KO MEFs treated with 500 µM sp-8-cpt-cAMP for 1 h. Note the diffused cytosolic staining of V1B2 in KO cells. Bar, 10 µm. (C) Quantification of the colocalization between V1B2- and LAMP2-positive lysosomes. All values are given as mean percentage ± SEM. *, P < 0.05; **, P < 0.01.
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fig2: sAC regulates V-ATPase localization to lysosomes. (A) Representative immunoblot of the V-ATPase subunit V1B2 along with GAPDH, which was used for loading control. Shown are whole cell extracts from three independently grown cultures of WT and sAC KO MEFs. n = 3. (B) Double-immunofluorescence labeling of V-ATPase V1B2 subunit (red) and LAMP2 (green) in WT, sAC KO, and sAC KO MEFs treated with 500 µM sp-8-cpt-cAMP for 1 h. Note the diffused cytosolic staining of V1B2 in KO cells. Bar, 10 µm. (C) Quantification of the colocalization between V1B2- and LAMP2-positive lysosomes. All values are given as mean percentage ± SEM. *, P < 0.05; **, P < 0.01.

Mentions: In pH-sensing epithelia, sAC-generated cAMP is responsible for translocation of the proton-pumping V-ATPase (Pastor-Soler et al., 2003; Păunescu et al., 2008b, 2010) via PKA (Pastor-Soler et al., 2008; Păunescu et al., 2010). Therefore, we asked whether sAC-dependent lysosomal acidification is dependent on proper localization of the V-ATPase. The V-ATPase is comprised of multiple subunits, and we assessed V-ATPase localization on lysosomes by double-immunofluorescence labeling of the lysosomal marker LAMP2 with either of two different cytoplasmic (V1) domain V-ATPase subunits, V1B2 or V1D. Total protein levels of both V1B2 (Fig. 2 A) and V1D (Fig. S3 A) were unchanged in WT versus sAC KO cells; thus, loss of sAC did not affect the amount of V-ATPase in cells. In WT cells, the majority of both V1B2 (Fig. 2, B and C) and V1D (Fig. S3 B) colocalized with LAMP2-positive vesicles. In contrast, in sAC KO cells, fewer LAMP2-positive vesicles were costained with the V-ATPase subunit antibodies (Fig. 2 C and Fig. S3 B), and both subunits were more diffusely distributed throughout the cytoplasm (Fig. 2 B and Fig. S3 B). To determine whether this altered staining pattern correlated with the acidification defect, we assessed V-ATPase subunit localization under conditions that rescued the lysosomal acidification in sAC KO cells. Addition of membrane-permeable cAMP for 1 h, which rescued the phenotypic defect in sAC KO cells (Fig. 1, C and E), restored LAMP2 colocalization with both V-ATPase subunits (Fig. 2, B and C; and Fig. S3 B). Thus, sAC-generated cAMP is responsible for both proper localization of V-ATPase subunits and acidification of lysosomes.


Soluble adenylyl cyclase is essential for proper lysosomal acidification
sAC regulates V-ATPase localization to lysosomes. (A) Representative immunoblot of the V-ATPase subunit V1B2 along with GAPDH, which was used for loading control. Shown are whole cell extracts from three independently grown cultures of WT and sAC KO MEFs. n = 3. (B) Double-immunofluorescence labeling of V-ATPase V1B2 subunit (red) and LAMP2 (green) in WT, sAC KO, and sAC KO MEFs treated with 500 µM sp-8-cpt-cAMP for 1 h. Note the diffused cytosolic staining of V1B2 in KO cells. Bar, 10 µm. (C) Quantification of the colocalization between V1B2- and LAMP2-positive lysosomes. All values are given as mean percentage ± SEM. *, P < 0.05; **, P < 0.01.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC5037342&req=5

fig2: sAC regulates V-ATPase localization to lysosomes. (A) Representative immunoblot of the V-ATPase subunit V1B2 along with GAPDH, which was used for loading control. Shown are whole cell extracts from three independently grown cultures of WT and sAC KO MEFs. n = 3. (B) Double-immunofluorescence labeling of V-ATPase V1B2 subunit (red) and LAMP2 (green) in WT, sAC KO, and sAC KO MEFs treated with 500 µM sp-8-cpt-cAMP for 1 h. Note the diffused cytosolic staining of V1B2 in KO cells. Bar, 10 µm. (C) Quantification of the colocalization between V1B2- and LAMP2-positive lysosomes. All values are given as mean percentage ± SEM. *, P < 0.05; **, P < 0.01.
Mentions: In pH-sensing epithelia, sAC-generated cAMP is responsible for translocation of the proton-pumping V-ATPase (Pastor-Soler et al., 2003; Păunescu et al., 2008b, 2010) via PKA (Pastor-Soler et al., 2008; Păunescu et al., 2010). Therefore, we asked whether sAC-dependent lysosomal acidification is dependent on proper localization of the V-ATPase. The V-ATPase is comprised of multiple subunits, and we assessed V-ATPase localization on lysosomes by double-immunofluorescence labeling of the lysosomal marker LAMP2 with either of two different cytoplasmic (V1) domain V-ATPase subunits, V1B2 or V1D. Total protein levels of both V1B2 (Fig. 2 A) and V1D (Fig. S3 A) were unchanged in WT versus sAC KO cells; thus, loss of sAC did not affect the amount of V-ATPase in cells. In WT cells, the majority of both V1B2 (Fig. 2, B and C) and V1D (Fig. S3 B) colocalized with LAMP2-positive vesicles. In contrast, in sAC KO cells, fewer LAMP2-positive vesicles were costained with the V-ATPase subunit antibodies (Fig. 2 C and Fig. S3 B), and both subunits were more diffusely distributed throughout the cytoplasm (Fig. 2 B and Fig. S3 B). To determine whether this altered staining pattern correlated with the acidification defect, we assessed V-ATPase subunit localization under conditions that rescued the lysosomal acidification in sAC KO cells. Addition of membrane-permeable cAMP for 1 h, which rescued the phenotypic defect in sAC KO cells (Fig. 1, C and E), restored LAMP2 colocalization with both V-ATPase subunits (Fig. 2, B and C; and Fig. S3 B). Thus, sAC-generated cAMP is responsible for both proper localization of V-ATPase subunits and acidification of lysosomes.

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