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Role of AP1 and Gadkin in the traffic of secretory endo-lysosomes.

Laulagnier K, Schieber NL, Maritzen T, Haucke V, Parton RG, Gruenberg J - Mol. Biol. Cell (2011)

Bottom Line: Strikingly, this endo-secretory process is not affected by treatments that inhibit endosome dynamics (microtubule depolymerization, cholesterol accumulation, overexpression of Rab7 or its effector Rab-interacting lysosomal protein [RILP], overexpression of Rab5 mutants), but depends on Rab27a, a GTPase involved in LRO secretion, and is controlled by F-actin.Moreover, we find that this unconventional endo-secretory pathway requires the adaptor protein complexes AP1, Gadkin (which recruits AP1 by binding to the γ1 subunit), and AP2, but not AP3.We conclude that a specific fraction of the AP2-derived endocytic pathway is dedicated to secretory purposes under the control of AP1 and Gadkin.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Geneva, Switzerland.

ABSTRACT
Whereas lysosome-related organelles (LRO) of specialized cells display both exocytic and endocytic features, lysosomes in nonspecialized cells can also acquire the property to fuse with the plasma membrane upon an acute rise in cytosolic calcium. Here, we characterize this unconventional secretory pathway in fibroblast-like cells, by monitoring the appearance of Lamp1 on the plasma membrane and the release of lysosomal enzymes into the medium. After sequential ablation of endocytic compartments in living cells, we find that donor membranes primarily derive from a late compartment, but that an early compartment is also involved. Strikingly, this endo-secretory process is not affected by treatments that inhibit endosome dynamics (microtubule depolymerization, cholesterol accumulation, overexpression of Rab7 or its effector Rab-interacting lysosomal protein [RILP], overexpression of Rab5 mutants), but depends on Rab27a, a GTPase involved in LRO secretion, and is controlled by F-actin. Moreover, we find that this unconventional endo-secretory pathway requires the adaptor protein complexes AP1, Gadkin (which recruits AP1 by binding to the γ1 subunit), and AP2, but not AP3. We conclude that a specific fraction of the AP2-derived endocytic pathway is dedicated to secretory purposes under the control of AP1 and Gadkin.

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Role of adaptor protein complexes in calcium-induced exocytosis of endosecretory compartments A431 cells were transfected with siRNAs against the μ chain of AP1, AP2, or AP3 and the KD efficiency was determined by Western blotting (A). Total amount (B) or intracellular distribution (C) of Lamp1 was analyzed in cells depleted for μ1, μ2, or μ3. No difference was observed as compared with control cells. (D) After KD of the indicated μ subunit and stimulation with ionomycin, Lamp1 on the cell surface was labeled as in Figure 1. The micrographs show large overview fields (Bar = 20 μm) obtained with laser scanning a confocal microscope. (E and F) After KD and stimulation as in (D), Lamp1 appearance on the plasma membrane after cell surface biotinylation was analyzed as in Figure 1, E and F. A representative blot is shown in (E), and the data are quantified in (F). Means ± SEM of eight experiments are shown. *p < 0.04, **p < 0.025, ***p < 0.0025. (G and H) After KD and stimulation, hexosaminidase release (G; means of four experiments ± SEM) was measured as in Figure 1A and cathepsin D release (H) was analyzed as in Figure 7C.
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Figure 7: Role of adaptor protein complexes in calcium-induced exocytosis of endosecretory compartments A431 cells were transfected with siRNAs against the μ chain of AP1, AP2, or AP3 and the KD efficiency was determined by Western blotting (A). Total amount (B) or intracellular distribution (C) of Lamp1 was analyzed in cells depleted for μ1, μ2, or μ3. No difference was observed as compared with control cells. (D) After KD of the indicated μ subunit and stimulation with ionomycin, Lamp1 on the cell surface was labeled as in Figure 1. The micrographs show large overview fields (Bar = 20 μm) obtained with laser scanning a confocal microscope. (E and F) After KD and stimulation as in (D), Lamp1 appearance on the plasma membrane after cell surface biotinylation was analyzed as in Figure 1, E and F. A representative blot is shown in (E), and the data are quantified in (F). Means ± SEM of eight experiments are shown. *p < 0.04, **p < 0.025, ***p < 0.0025. (G and H) After KD and stimulation, hexosaminidase release (G; means of four experiments ± SEM) was measured as in Figure 1A and cathepsin D release (H) was analyzed as in Figure 7C.

Mentions: Adaptor complexes, in addition to their functions in transport between secretory and endocytic organelles, are also involved in LRO biogenesis in specialized cell types. We thus investigated whether adaptors play a role in the endo-lysosomal exocytic pathway(s) we have been studying. To this end, the μ subunit of AP1, AP2, or AP3 was depleted with siRNAs to ≈80% of the amounts present in the corresponding mock-treated control (Figure 7A).


Role of AP1 and Gadkin in the traffic of secretory endo-lysosomes.

Laulagnier K, Schieber NL, Maritzen T, Haucke V, Parton RG, Gruenberg J - Mol. Biol. Cell (2011)

Role of adaptor protein complexes in calcium-induced exocytosis of endosecretory compartments A431 cells were transfected with siRNAs against the μ chain of AP1, AP2, or AP3 and the KD efficiency was determined by Western blotting (A). Total amount (B) or intracellular distribution (C) of Lamp1 was analyzed in cells depleted for μ1, μ2, or μ3. No difference was observed as compared with control cells. (D) After KD of the indicated μ subunit and stimulation with ionomycin, Lamp1 on the cell surface was labeled as in Figure 1. The micrographs show large overview fields (Bar = 20 μm) obtained with laser scanning a confocal microscope. (E and F) After KD and stimulation as in (D), Lamp1 appearance on the plasma membrane after cell surface biotinylation was analyzed as in Figure 1, E and F. A representative blot is shown in (E), and the data are quantified in (F). Means ± SEM of eight experiments are shown. *p < 0.04, **p < 0.025, ***p < 0.0025. (G and H) After KD and stimulation, hexosaminidase release (G; means of four experiments ± SEM) was measured as in Figure 1A and cathepsin D release (H) was analyzed as in Figure 7C.
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Related In: Results  -  Collection

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Figure 7: Role of adaptor protein complexes in calcium-induced exocytosis of endosecretory compartments A431 cells were transfected with siRNAs against the μ chain of AP1, AP2, or AP3 and the KD efficiency was determined by Western blotting (A). Total amount (B) or intracellular distribution (C) of Lamp1 was analyzed in cells depleted for μ1, μ2, or μ3. No difference was observed as compared with control cells. (D) After KD of the indicated μ subunit and stimulation with ionomycin, Lamp1 on the cell surface was labeled as in Figure 1. The micrographs show large overview fields (Bar = 20 μm) obtained with laser scanning a confocal microscope. (E and F) After KD and stimulation as in (D), Lamp1 appearance on the plasma membrane after cell surface biotinylation was analyzed as in Figure 1, E and F. A representative blot is shown in (E), and the data are quantified in (F). Means ± SEM of eight experiments are shown. *p < 0.04, **p < 0.025, ***p < 0.0025. (G and H) After KD and stimulation, hexosaminidase release (G; means of four experiments ± SEM) was measured as in Figure 1A and cathepsin D release (H) was analyzed as in Figure 7C.
Mentions: Adaptor complexes, in addition to their functions in transport between secretory and endocytic organelles, are also involved in LRO biogenesis in specialized cell types. We thus investigated whether adaptors play a role in the endo-lysosomal exocytic pathway(s) we have been studying. To this end, the μ subunit of AP1, AP2, or AP3 was depleted with siRNAs to ≈80% of the amounts present in the corresponding mock-treated control (Figure 7A).

Bottom Line: Strikingly, this endo-secretory process is not affected by treatments that inhibit endosome dynamics (microtubule depolymerization, cholesterol accumulation, overexpression of Rab7 or its effector Rab-interacting lysosomal protein [RILP], overexpression of Rab5 mutants), but depends on Rab27a, a GTPase involved in LRO secretion, and is controlled by F-actin.Moreover, we find that this unconventional endo-secretory pathway requires the adaptor protein complexes AP1, Gadkin (which recruits AP1 by binding to the γ1 subunit), and AP2, but not AP3.We conclude that a specific fraction of the AP2-derived endocytic pathway is dedicated to secretory purposes under the control of AP1 and Gadkin.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Geneva, Switzerland.

ABSTRACT
Whereas lysosome-related organelles (LRO) of specialized cells display both exocytic and endocytic features, lysosomes in nonspecialized cells can also acquire the property to fuse with the plasma membrane upon an acute rise in cytosolic calcium. Here, we characterize this unconventional secretory pathway in fibroblast-like cells, by monitoring the appearance of Lamp1 on the plasma membrane and the release of lysosomal enzymes into the medium. After sequential ablation of endocytic compartments in living cells, we find that donor membranes primarily derive from a late compartment, but that an early compartment is also involved. Strikingly, this endo-secretory process is not affected by treatments that inhibit endosome dynamics (microtubule depolymerization, cholesterol accumulation, overexpression of Rab7 or its effector Rab-interacting lysosomal protein [RILP], overexpression of Rab5 mutants), but depends on Rab27a, a GTPase involved in LRO secretion, and is controlled by F-actin. Moreover, we find that this unconventional endo-secretory pathway requires the adaptor protein complexes AP1, Gadkin (which recruits AP1 by binding to the γ1 subunit), and AP2, but not AP3. We conclude that a specific fraction of the AP2-derived endocytic pathway is dedicated to secretory purposes under the control of AP1 and Gadkin.

Show MeSH
Related in: MedlinePlus