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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.


Related in: MedlinePlus

APs accumulate in the absence of sAC activity. (A) Representative immunoblot of the autophagic markers LC3-II and p62 in three independent cultures of WT and sAC KO MEFs. This immunoblot is a reprobing of the blot shown in Fig. 2 A; therefore, the GAPDH control showed in Fig. 2 A is also relevant for this immunoblot. (B) Densitometric analysis of LC3-II and p62, normalized to GAPDH, in WT (gray bars) and sAC KO (black bars) MEFs. n = 6. (C) Representative immunoblot of LC3-II in sAC KO MEFs grown in the presence of 500 µM Sp-8-cpt-cAMP for the indicated time. GAPDH was used as loading control. (D) LC3-II immunoblot levels in WT and KO 3T3 MEFs after 6 h of serum starvation. GAPDH is used as loading control. (E) Densitometric analysis of LC3-II immunoblot from D normalized to GAPDH. Autophagic induction is unaltered in WT and sAC KO cells. Ratio of LC-II in starvation/control is 1.2 and 1.7 for WT and sAC KO, respectively. (F) LC3 and p62 immunoblot levels in WT and KO 3T3 MEFs with and without treatment with 100 nM Bafilomycin A1 (BafA) for 6 h. Protein levels of each protein were normalized to GAPDH as an internal control. (G) Densitometric analysis of LC3-II immunoblot from F normalized to GAPDH. n = 3. (H) Densitometric analysis of p62 immunoblot from F normalized to GAPDH. n = 3. (I) LC3 immunoblot of WT and KO 3T3 MEFs treated with 20 mM NH4Cl for 6 h. Protein levels were normalized to GAPDH as an internal control. (J) Densitometric analysis of LC3-II from I. n = 3–5 per experimental group. (K) LC3 immunoblot of WT and KO 3T3 MEFs treated with and without lysosomal protease inhibitors (PI) for 6 h (20 µM Leupeptin, 20 µM Pepstatin A, and 10 µM E64D). Protein levels were normalized to GAPDH as an internal control. (L) Densitometric analysis of LC3-II from K. n = 3–5 per experimental group. All values are given as mean ± SEM. *, P < 0.05; **, P < 0.01.
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fig5: APs accumulate in the absence of sAC activity. (A) Representative immunoblot of the autophagic markers LC3-II and p62 in three independent cultures of WT and sAC KO MEFs. This immunoblot is a reprobing of the blot shown in Fig. 2 A; therefore, the GAPDH control showed in Fig. 2 A is also relevant for this immunoblot. (B) Densitometric analysis of LC3-II and p62, normalized to GAPDH, in WT (gray bars) and sAC KO (black bars) MEFs. n = 6. (C) Representative immunoblot of LC3-II in sAC KO MEFs grown in the presence of 500 µM Sp-8-cpt-cAMP for the indicated time. GAPDH was used as loading control. (D) LC3-II immunoblot levels in WT and KO 3T3 MEFs after 6 h of serum starvation. GAPDH is used as loading control. (E) Densitometric analysis of LC3-II immunoblot from D normalized to GAPDH. Autophagic induction is unaltered in WT and sAC KO cells. Ratio of LC-II in starvation/control is 1.2 and 1.7 for WT and sAC KO, respectively. (F) LC3 and p62 immunoblot levels in WT and KO 3T3 MEFs with and without treatment with 100 nM Bafilomycin A1 (BafA) for 6 h. Protein levels of each protein were normalized to GAPDH as an internal control. (G) Densitometric analysis of LC3-II immunoblot from F normalized to GAPDH. n = 3. (H) Densitometric analysis of p62 immunoblot from F normalized to GAPDH. n = 3. (I) LC3 immunoblot of WT and KO 3T3 MEFs treated with 20 mM NH4Cl for 6 h. Protein levels were normalized to GAPDH as an internal control. (J) Densitometric analysis of LC3-II from I. n = 3–5 per experimental group. (K) LC3 immunoblot of WT and KO 3T3 MEFs treated with and without lysosomal protease inhibitors (PI) for 6 h (20 µM Leupeptin, 20 µM Pepstatin A, and 10 µM E64D). Protein levels were normalized to GAPDH as an internal control. (L) Densitometric analysis of LC3-II from K. n = 3–5 per experimental group. All values are given as mean ± SEM. *, P < 0.05; **, P < 0.01.

Mentions: Macroautophagy (hereafter autophagy), the major lysosomal degradative pathway in cells, is responsible for degrading long-lived proteins, organelles, and protein aggregates (Klionsky, 2007; Mizushima, 2007). It involves sequestration of cytosolic regions into characteristic double-membrane APs that fuse with lysosomes (Yamamoto et al., 1998; Klionsky et al., 2012) to form single-membrane degradative autophagolysosomes (ALs). Because sAC KO cells have less acidic lysosomes (Fig. 1, A and B) and reduced lysosomal proteolytic capacity (Fig. 4), we hypothesized that the autophagic degradative system may also be impaired. The autophagic markers, LC3-II and p62 were more abundant in sAC KO MEFs (Fig. 5, A and B) and primary neurons from sAC KO mice (Fig. S6, A–C) compared with their WT counterparts. Similar to elevated lysosomal pH, mislocalization of V-ATPase, and decreased lysosomal proteolysis, this elevation of LC3-II in sAC KO MEFs was rescued by the addition of exogenous membrane-permeable cAMP (Fig. 5 C). And the time course of this rescue was consistent with the time course of cAMP-dependent acidification of microglia (Majumdar et al., 2007); it required >30 min and was complete after 1 h.


Soluble adenylyl cyclase is essential for proper lysosomal acidification
APs accumulate in the absence of sAC activity. (A) Representative immunoblot of the autophagic markers LC3-II and p62 in three independent cultures of WT and sAC KO MEFs. This immunoblot is a reprobing of the blot shown in Fig. 2 A; therefore, the GAPDH control showed in Fig. 2 A is also relevant for this immunoblot. (B) Densitometric analysis of LC3-II and p62, normalized to GAPDH, in WT (gray bars) and sAC KO (black bars) MEFs. n = 6. (C) Representative immunoblot of LC3-II in sAC KO MEFs grown in the presence of 500 µM Sp-8-cpt-cAMP for the indicated time. GAPDH was used as loading control. (D) LC3-II immunoblot levels in WT and KO 3T3 MEFs after 6 h of serum starvation. GAPDH is used as loading control. (E) Densitometric analysis of LC3-II immunoblot from D normalized to GAPDH. Autophagic induction is unaltered in WT and sAC KO cells. Ratio of LC-II in starvation/control is 1.2 and 1.7 for WT and sAC KO, respectively. (F) LC3 and p62 immunoblot levels in WT and KO 3T3 MEFs with and without treatment with 100 nM Bafilomycin A1 (BafA) for 6 h. Protein levels of each protein were normalized to GAPDH as an internal control. (G) Densitometric analysis of LC3-II immunoblot from F normalized to GAPDH. n = 3. (H) Densitometric analysis of p62 immunoblot from F normalized to GAPDH. n = 3. (I) LC3 immunoblot of WT and KO 3T3 MEFs treated with 20 mM NH4Cl for 6 h. Protein levels were normalized to GAPDH as an internal control. (J) Densitometric analysis of LC3-II from I. n = 3–5 per experimental group. (K) LC3 immunoblot of WT and KO 3T3 MEFs treated with and without lysosomal protease inhibitors (PI) for 6 h (20 µM Leupeptin, 20 µM Pepstatin A, and 10 µM E64D). Protein levels were normalized to GAPDH as an internal control. (L) Densitometric analysis of LC3-II from K. n = 3–5 per experimental group. All values are given as mean ± SEM. *, P < 0.05; **, P < 0.01.
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fig5: APs accumulate in the absence of sAC activity. (A) Representative immunoblot of the autophagic markers LC3-II and p62 in three independent cultures of WT and sAC KO MEFs. This immunoblot is a reprobing of the blot shown in Fig. 2 A; therefore, the GAPDH control showed in Fig. 2 A is also relevant for this immunoblot. (B) Densitometric analysis of LC3-II and p62, normalized to GAPDH, in WT (gray bars) and sAC KO (black bars) MEFs. n = 6. (C) Representative immunoblot of LC3-II in sAC KO MEFs grown in the presence of 500 µM Sp-8-cpt-cAMP for the indicated time. GAPDH was used as loading control. (D) LC3-II immunoblot levels in WT and KO 3T3 MEFs after 6 h of serum starvation. GAPDH is used as loading control. (E) Densitometric analysis of LC3-II immunoblot from D normalized to GAPDH. Autophagic induction is unaltered in WT and sAC KO cells. Ratio of LC-II in starvation/control is 1.2 and 1.7 for WT and sAC KO, respectively. (F) LC3 and p62 immunoblot levels in WT and KO 3T3 MEFs with and without treatment with 100 nM Bafilomycin A1 (BafA) for 6 h. Protein levels of each protein were normalized to GAPDH as an internal control. (G) Densitometric analysis of LC3-II immunoblot from F normalized to GAPDH. n = 3. (H) Densitometric analysis of p62 immunoblot from F normalized to GAPDH. n = 3. (I) LC3 immunoblot of WT and KO 3T3 MEFs treated with 20 mM NH4Cl for 6 h. Protein levels were normalized to GAPDH as an internal control. (J) Densitometric analysis of LC3-II from I. n = 3–5 per experimental group. (K) LC3 immunoblot of WT and KO 3T3 MEFs treated with and without lysosomal protease inhibitors (PI) for 6 h (20 µM Leupeptin, 20 µM Pepstatin A, and 10 µM E64D). Protein levels were normalized to GAPDH as an internal control. (L) Densitometric analysis of LC3-II from K. n = 3–5 per experimental group. All values are given as mean ± SEM. *, P < 0.05; **, P < 0.01.
Mentions: Macroautophagy (hereafter autophagy), the major lysosomal degradative pathway in cells, is responsible for degrading long-lived proteins, organelles, and protein aggregates (Klionsky, 2007; Mizushima, 2007). It involves sequestration of cytosolic regions into characteristic double-membrane APs that fuse with lysosomes (Yamamoto et al., 1998; Klionsky et al., 2012) to form single-membrane degradative autophagolysosomes (ALs). Because sAC KO cells have less acidic lysosomes (Fig. 1, A and B) and reduced lysosomal proteolytic capacity (Fig. 4), we hypothesized that the autophagic degradative system may also be impaired. The autophagic markers, LC3-II and p62 were more abundant in sAC KO MEFs (Fig. 5, A and B) and primary neurons from sAC KO mice (Fig. S6, A–C) compared with their WT counterparts. Similar to elevated lysosomal pH, mislocalization of V-ATPase, and decreased lysosomal proteolysis, this elevation of LC3-II in sAC KO MEFs was rescued by the addition of exogenous membrane-permeable cAMP (Fig. 5 C). And the time course of this rescue was consistent with the time course of cAMP-dependent acidification of microglia (Majumdar et al., 2007); it required >30 min and was complete after 1 h.

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